Combination therapy for diseases or disorders associated with PI3k

Abstract

To provide a method for treating diseases or disorders associated with PI3K signaling.SOLUTION: A method for inhibiting cell proliferation includes the steps of: bringing a cell in contact with an effective dose of a regulating substance of glucose metabolism; and bringing the cell in contact with an effective dose of at least one inhibitory substance of at least one kinase in an insulin receptor / PI3K / AKT / mTOR pathway, by which cell proliferation is inhibited.SELECTED DRAWING: Figure 10A

Description

[Technical Field]

[0001] This application claims the benefit of priority to the filing date of U.S. Provisional Patent Application No. 62 / 679,329, filed June 1, 2018, the entire contents of which are specifically incorporated herein by reference.

[0002] Field of the Disclosure The present disclosure relates generally to methods of treating diseases or disorders associated with insulin receptor / PI3K / AKT / mTOR pathway signaling, including hematological malignancies and solid tumors. In particular, the disclosure relates to enhancing the effects of insulin receptor / PI3K / AKT / mTOR pathway inhibitors by modulating glucose metabolism pharmacologically or dietarily. [Background technology]

[0003] background Several diseases and disorders have been linked to the insulin receptor phosphatidylinositol kinase (phosphoinositide 3-kinase (PI3K)), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) signaling pathway (termed the insulin receptor / PI3K / AKT / mTOR pathway). In cancer, mutations in PIK3CA are observed with similar frequency to mutations in KRAS (Kandoth, C. et al. Mutational landscape and significance across 12 major cancer types. Nature 502, 333-339, doi:10.1038 / nature12634 (2013) (Non-Patent Document 1); Millis, SZ, Ikeda, S., Reddy, S., Gatalica, Z. & Kurzrock, R. Landscape of Phosphatidylinositol-3-Kinase Pathway Alterations Across 19784 Diverse Solid Tumors. JAMA Oncol 2, 1565-1573, doi:10.1001 / jamaoncol.2016.0891 (2016) (Non-Patent Document 2)).

[0004] Although therapies that target this insulin receptor / PI3K / AKT / mTOR pathway are desirable, the medical community has struggled to find effective compositions and methods that target PI3K and the upstream and downstream regulators of insulin receptor / PI3K / AKT / mTOR signal transduction.Therefore, there is a long-standing unmet need for compositions and methods for treating diseases or disorders related to PI3K signal transduction.The present disclosure provides such compositions and methods, as well as others. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kandoth, C. et al. Mutational landscape and significance across 12 major cancer types. Nature 502, 333-339, doi:10.1038 / nature12634 (2013) [Non-patent document 2] Millis, SZ, Ikeda, S., Reddy, S., Gatalica, Z. & Kurzrock, R. Landscape of Phosphatidylinositol-3-Kinase Pathway Alterations Across 19784 Diverse Solid Tumors. JAMA Oncol 2, 1565-1573, doi:10.1001 / jamaoncol.2016.0891 (2016) Summary of the Invention

[0006] Summary of the Disclosure The present disclosure generally relates to compositions and methods for treating diseases or disorders associated with PI3K signaling, which may include administering a glucose metabolism regulator with or without a diet to affect the metabolic state of a subject. In some examples, the method may include administering an inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway and administering a glucose metabolism regulator. In some examples, the method includes administering the pathway inhibitor and / or glucose metabolism regulator to a subject who is following a ketogenic diet during treatment. The present disclosure further relates to pharmaceutical compositions that may include a pathway inhibitor, a glucose metabolism regulator, or a combination thereof.

[0007] The present disclosure provides a method for treating a disease or disorder associated with PI3K signaling, the method comprising administering to a subject in need thereof an effective amount of a regulator of glucose metabolism, and administering to the subject an effective amount of a pathway inhibitor of the insulin receptor / PI3K / AKT / mTOR pathway.

[0008] The present disclosure also provides a method for treating a disease or disorder associated with PI3K signaling, the method comprising administering an effective amount of a pathway inhibitor, optionally wherein the pathway inhibitor is capable of inhibiting at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway.

[0009] During any of these treatments, the subject may be on or be given a ketogenic diet.

[0010] The present disclosure also provides a pharmaceutical composition comprising a regulator of glucose metabolism and a pathway inhibitor, optionally wherein the pathway inhibitor is capable of inhibiting at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway.

[0011] The present disclosure also provides a method for inhibiting cell proliferation or suppressing a cell proliferative disorder, the method comprising administering to a subject an effective amount of a glucose uptake inhibitor and administering to the subject an effective amount of a PI3K inhibitor.

[0012] The subject may be one in need of treatment, or the treatment may be carried out to prevent the onset of a disease or condition.

[0013] The present disclosure also provides a method for inhibiting cell proliferation or suppressing a cell proliferative disorder, the method comprising administering to a subject an effective amount of a PI3K inhibitor, wherein the subject follows a ketogenic diet during treatment.

[0014] In some embodiments, the glucose metabolism regulator is a glucose uptake inhibitor. For example, such a glucose uptake inhibitor can be a sodium-glucose-linked transport protein 1 (SGLT1) inhibitor, a sodium-glucose-linked transport protein 2 (SGLT2) inhibitor, a SGLT1 / SGLT2 dual inhibitor, or a combination thereof.

[0015] In some embodiments, the glucose uptake inhibitor is selected from dapagliflozin, empagliflozin, canagliflozin, ipragliflozin, tofogliflozin, sergliflozin etabonate, remogliflozin etabonate, ertugliflozin, sotagliflozin, conagliflozin, or a combination thereof.

[0016] In some embodiments, the regulator of glucose metabolism is metformin.

[0017] In some embodiments, the modulator of glucose metabolism is an insulin receptor / insulin-like growth factor 1 (IGF1) receptor inhibitor, and optionally, the insulin receptor / IGF1 receptor inhibitor is linsitinib (OSI-906).

[0018] In some embodiments, the pathway inhibitor is selected from idelalisib, copanlisib, buparlisib (BKM120), alpelisib (BYL719), taselisib (GDC-0032), pictilisib (GDC-0941), apitolisib (GDC-0980), selavelisib (TAK-117), dactolisib, apelisib, MK2206, linsitinib (OSI-906), or a combination thereof.

[0019] In some embodiments, the PI3K inhibitor is selected from idelalisib, copanlisib, buparlisib (BKM120), alpelisib (BYL719), taselisib (GDC-0032), pictilisib (GDC-0941), apitolisib (GDC-0980), selavelisib (TAK-117), dactolisib, and apelisib.

[0020] In some embodiments, the disease or disorder associated with PI3K signaling is cancer or a cell proliferation disorder, a metabolic disorder, a neurodegenerative disease, an inflammatory disease, or a combination thereof.

[0021] In some embodiments, disruption of whole body glucose homeostasis improves the effect of pathway inhibitor treatment compared to the pathway inhibitor alone.

[0022] In some embodiments, inhibition of cell proliferation or suppression of a cell proliferative disorder is enhanced compared to administration of the PI3K inhibitor without the glucose uptake inhibitor. [The present invention 1001] A method for inhibiting cell proliferation comprising contacting a cell with an effective amount of a regulator of glucose metabolism and contacting the cell with an effective amount of at least one inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway, thereby inhibiting cell proliferation. [The present invention 1002] The method of the present invention 1001, wherein said regulator of glucose metabolism is a glucose uptake inhibitor selected from a sodium-glucose-linked transport protein 1 (SGLT1) inhibitor, a sodium-glucose-linked transport protein 2 (SGLT2) inhibitor, an SGLT1 / SGLT2 dual inhibitor, or a combination thereof. [The present invention 1003] 1001. The method of claim 1001, wherein said modulator of glucose metabolism is dapagliflozin, empagliflozin, canagliflozin, ipragliflozin, metformin, tofogliflozin, sergliflozin etabonate, remogliflozin etabonate, ertugliflozin, sotagliflozin, conagliflozin, or a combination thereof. [The present invention 1004] 1001. The method of claim 1001, wherein said regulator of glucose metabolism is a sodium-glucose cotransporter protein 2 (SGLT2) inhibitor. [The present invention 1005] 1001. The method of claim 1001, wherein said regulator of glucose metabolism is metformin. [The present invention 1006] 1001. The method of claim 1001, wherein said inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway is an inhibitor of PI3K, an inhibitor of protein kinase B (AKT), an inhibitor of target of rapamycin (mTOR), or a combination thereof. [The present invention 1007] 1001. The method of claim 1001, wherein said inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway is idelalisib, copanlisib, buparlisib (BKM120), alpelisib (BYL719), taselisib (GDC-0032), pictilisib (GDC-0941), apitolisib (GDC-0980), selavelisib (TAK-117), dactolisib, MK2206, linsitinib (OSI-906), or a combination thereof. [The present invention 1008] 1001. The method of claim 1001, wherein said inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway is buparlisib (BKM120), alpelisib (BYL719), taselisib (GDC-0032), pictilisib (GDC-0941), linsitinib (OSI-906), or a combination thereof. [The present invention 1009] 1001. The method of claim 1001, wherein said regulator of glucose metabolism is metformin or a sodium-glucose cotransporter 2 (SGLT2) inhibitor, and said inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway is buparlisib (BKM120), alpelisib (BYL719), taselisib (GDC-0032), pictilisib (GDC-0941), linsitinib (OSI-906), or a combination thereof. [The present invention 1010] A pharmaceutical composition comprising a regulator of glucose metabolism and a pathway inhibitor that inhibits at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway. [The present invention 1011] a. administering to a subject in need thereof an effective amount of at least one inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway; and b. feeding said subject a ketogenic diet and / or administering at least one modulator of glucose metabolism thereby inhibiting cell proliferation in said subject. [The present invention 1012] The method of claim 1011, wherein said inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway is an inhibitor of PI3K, an inhibitor of protein kinase B (AKT), an inhibitor of target of rapamycin (mTOR), or a combination thereof. [The present invention 1013] 1011. The method of claim 10, wherein said inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway is idelalisib, copanlisib, buparlisib (BKM120), alpelisib (BYL719), taselisib (GDC-0032), pictilisib (GDC-0941), apitolisib (GDC-0980), selavelisib (TAK-117), dactolisib, MK2206, linsitinib (OSI-906), or a combination thereof. [The present invention 1014] 1011. The method of claim 10, wherein said inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway is buparlisib (BKM120), alpelisib (BYL719), taselisib (GDC-0032), pictilisib (GDC-0941), linsitinib (OSI-906), or a combination thereof. [The present invention 1015] The method of claim 1011, wherein said subject is administered at least one modulator of glucose metabolism. [The present invention 1016] 1015. The method of claim 1015, wherein said regulator of glucose metabolism is a sodium-glucose cotransporter 1 (SGLT1) inhibitor, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, a SGLT1 / SGLT2 dual inhibitor, or a combination thereof. [The present invention 1017] 1015. The method of claim 1015, wherein said modulator of glucose metabolism is dapagliflozin, empagliflozin, canagliflozin, ipragliflozin, tofogliflozin, sergliflozin etabonate, remogliflozin etabonate, ertugliflozin, sotagliflozin, conagliflozin, or a combination thereof. [The present invention 1018] 1015. The method of claim 10, wherein said regulator of glucose metabolism is a sodium-glucose cotransporter protein 2 (SGLT2) inhibitor. [The present invention 1019] 1015. The method of claim 10, wherein said regulator of glucose metabolism is metformin. [The present invention 1020] The method of claim 1011, wherein said subject is administered said ketogenic diet. [The present invention 1021] The method of claim 1011, wherein the subject is on a ketogenic diet before, during, or before and during the administration of the at least one inhibitor of a kinase in the insulin receptor / PI3K / AKT / mTOR pathway. [The present invention 1022] 10. The method of claim 10, wherein the cell proliferative disorder is selected from the group consisting of leukemia, polycythemia vera, lymphoma, Waldenstrom's macroglobulinemia, heavy chain disease, sarcoma, carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma, and combinations thereof. [The present invention 1023] The method of claim 1011, comprising administering to the subject metformin and an inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway, thereby treating cancer in the subject, wherein the subject is on a ketogenic diet before, during, or before and during said administration. [The present invention 1024] A kit comprising at least one inhibitor of the mammalian insulin receptor / PI3K / AKT / mTOR pathway and one or more regulators of glucose metabolism. [The present invention 1025] A kit comprising at least one inhibitor of the mammalian insulin receptor / PI3K / AKT / mTOR pathway and one or more components of a ketogenic diet. [Brief explanation of the drawings]

[0023] [Figure 1A]Figures 1A-1D show that treatment with PI3K inhibitors results in systemic feedback that increases blood glucose and insulin. Figure 1A graphically depicts blood glucose levels measured over time in mice treated with the indicated PI3K inhibitor compounds (N = 5 / arm, p-value < 0.0001 by two-way ANOVA for all blood glucose curves of mice in the treatment cohort compared to vehicle). To mimic clinical treatment, the mice were not starved in this assay. Figure 1B graphically depicts insulin levels measured over time in mice treated with the indicated PI3K inhibitor compounds (N = 5 / arm, p-value < 0.0001 by two-way ANOVA for all blood glucose curves of mice in the treatment cohort compared to vehicle). To mimic clinical treatment, the mice were not starved in this assay. Figure 1C graphically displays c-peptide levels, as a representative of the area under the insulin release curve, assessed from serum samples collected from the same animals used in Figures 1A-1B at 240 min, the end of the time course. The p-values ​​for t-tests comparing vehicle treatment with buparlisib (BKM120), alpelisib (BYL719), and taselisib (GDC-0032) were 0.017, <0.0001, and 0.007, respectively. Figure 1D-1 graphically displays the amount of fluorodeoxyglucose tracer detected via fluorodeoxyglucose positron emission tomography (FDG-PET) scanning of orthotopically implanted Kras-Tp53-Pdx-Cre (KPC) tumors (shown in Figure 1D-2) imaged 90 min after a single treatment with buparlisib (BKM120) (N=4 / arm). These results indicate that in the acute setting after a single dose of BKM120, the PI3K inhibitor-induced spike in insulin increased glucose uptake in the tumors of these animals (p-value = 0.0002 by t-test). [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 2A] Figures 2A-2C show the effects of insulin feedback levels on cell proliferation, signaling, and survival. Figure 2A shows Western blots of proteins from orthotopic Kras-Tp53-Pdx-Cre (KPC) cell lines K8484 and K8082 treated with or without the PI3K inhibitor BKM120 (1 μM) and / or insulin (10 ng / ml), further demonstrating the physiological response to insulin observed in Figures 1A-1D. Figure 2B graphically depicts the results of a proliferation assay of KPC cell line K8484 grown in the presence or absence of insulin (10 ng / ml) and BKM120 (1 μM). p-values ​​determined by ANOVA comparing + / - insulin conditions are shown. Figure 2C graphically depicts the results of a proliferation assay of KPC cell line K8082 grown in the presence or absence of insulin (10 ng / ml) and BKM120 (1 μM). p-values ​​determined by ANOVA comparing the + / - insulin conditions are shown. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 3A]Figures 3A-3G show that targeting PI3K inhibitors induces glucose / insulin feedback in vivo. Figure 3A graphically depicts blood glucose levels over time in wild-type C57 / b16 mice bearing syngeneic K8484 KPC allograft tumors after metformin pretreatment, SGLT2 inhibitor (SGLT2i) pretreatment, or treatment with a single dose of BKM120 along with a ketogenic diet (N = 4 / arm). The p-values ​​calculated by two-way repeated measures ANOVA for metformin, SGLT2i, and ketogenic diet were 0.2136 (not significant), <0.0001, and 0.007, respectively. Figure 3B graphically depicts blood levels of c-peptide measured 180 minutes after BKM120 treatment in the same mice as in Figure 3A. The p-values ​​calculated by unpaired t-test for metformin, SGLT2i, and ketogenic diet were 0.7566 (not significant), 0.0386, and 0.0117, respectively. Figure 3C shows immunohistochemistry images showing pS6 (ser-235) expression to observe the level of active PI3K signaling in these tumors. Figure 3D graphically displays the quantification of the staining observed in Figure 3C, shown as the number of positive cells per high-power field (20 fields / arm acquired as five high-power images averaged for each of four mice). The p-values ​​comparing pS6-positive cells in BKM120-only treated tumors with those treated with BKM120 in combination with metformin, SGLT2i, or ketogenic diet were 0.6186, <0.0001, and <0.0001, respectively. Figures 3E-1 to 3E-4 show IVIS images of luciferase reporter luminescence in KPC K8484 tumor-bearing mice after 12 days of treatment with the PI3K α-specific inhibitor BYL-719 alone or in combination with other agents. Figure 3E-1 shows IVIS images of luciferase reporter luminescence in KPC K8484 tumor-bearing mice after 12 days of treatment with the PI3K α-specific inhibitor BYL-719 alone.Figure 3E-2 shows IVIS images of luciferase reporter luminescence in KPC K8484 tumor-bearing mice after 12 days of treatment with the PI3K α-specific inhibitor BYL-719 in combination with metformin for 10 days prior to BYL-719 treatment (N = 10 tumors / arm). Figure 3E-3 shows IVIS images of luciferase reporter luminescence in KPC K8484 tumor-bearing mice after 12 days of treatment with the PI3K α-specific inhibitor BYL-719 in combination with a ketogenic diet for 10 days prior to BYL-719 treatment (N = 10 tumors / arm). Figure 3E-4 shows IVIS images of luciferase reporter luminescence in KPC K8484 tumor-bearing mice after 12 days of treatment with the PI3K α-specific inhibitor BYL-719 in combination with canagliflozin (SGLT2i) for 10 days prior to BYL-719 treatment (N = 10 tumors / arm). The dietary intervention in this study began at the time of tumor implantation, and BYL-719 treatment began 9 days after implantation. Figure 3F graphically displays the quantification of luminescence from images of these tumors. Figure 3G graphically displays the survival rates of these animals, demonstrating that the addition of either an SGLT2 inhibitor or a ketogenic diet to BYL-719 treatment increased the overall survival rate of these animals as determined by the log-rank (Mantel-Cox) test, p-values ​​= 0.0019 and < 0.0001, respectively. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E-1] See legend to Figure 3A. [Figure 3E-2] See legend to Figure 3A. [Figure 3E-3] See legend to Figure 3A. [Figure 3E-4] See legend to Figure 3A. [Figure 3F] See legend to Figure 3A. [Figure 3G] See legend to Figure 3A. [Figure 4A]Figures 4A–4E show the effect of circumventing on-target glucose / insulin feedback by PI3K inhibitors on tumor growth. Figure 4A graphically depicts K8484 KPC tumor volume (tumor cells express doxycycline-inducible hairpins targeting Renilla (ShRenilla) or the insulin receptor (ShIR)) in mice treated with doxycycline and the PI3K inhibitor BYL-719 and / or a ketogenic diet. In this experiment, tumors were allowed to grow for 14 days to reach an average size of more than 1 cm, at which point diet, doxycycline induction of hairpin expression, and PI3K inhibitor treatment were initiated (N>5 tumors / arm, as indicated). Figure 4B graphically depicts ES272 Pik3ca mutant breast cancer allograft tumor volume treated with BYL-719 and / or insulin along with a ketogenic diet (keto), as indicated. In this experiment, tumors were allowed to grow for 10 days after implantation into mice before initiation of diet and the indicated treatments. Figure 4C graphically depicts tumor volume in subject-derived endometrial xenografts (PDX) treated with BKM120 and / or on a ketogenic diet (N=5 / arm). ANOVA comparison between mice treated with BKM120 alone and mice treated with BKM120 and a ketogenic diet demonstrates that the addition of a ketogenic diet significantly enhances treatment effects in this model (p=0.0028). Figure 4D shows histology of phospho-insulin receptor (pINSR), phospho-AKT (pAKT), phospho-S6 (pS6), cleaved caspase 3 (Cl. Casp 3), and Ki67 in tumors harvested 4 hours after the last treatment with vehicle, ketogenic diet, BKM120, or the combination of ketogenic diet and BKM120 (BKM120 / keto). Figure 4E graphically depicts the quantification of phospho-insulin receptor (pINSR), phospho-AKT (pAKT), phospho-S6 (pS6), cleaved caspase 3 (Cl. Casp 3), and ki67 in tumors shown in Figure 4D harvested 4 hours after the last treatment with vehicle, ketogenic diet, BKM120, or the combination of ketogenic diet and BKM120 (BKM120 / keto).Quantitation is shown as a score per high-power field, with four images acquired for each of five mice. The p-values ​​from a t-test comparing blinded scores in BKM120-treated tumors with those treated with BKM120 on a ketogenic diet were 0.005, 0.005, 0.017, and 0.028 for pINSR, pAKT, pS6, and Cl. Casp 3, respectively. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 5A] Figures 5A-5D show blood glucose and C-peptide levels after treatment with agents targeting the PI3K pathway. Figure 5A graphically depicts blood glucose levels over time, with time 0 representing the time of treatment with the indicated inhibitor. Figure 5B graphically depicts blood glucose levels over time, with time 0 representing the time of treatment with the indicated additional inhibitor. Figure 5C graphically depicts c-peptide levels taken from the mice in Figure 5A at 240 and 180 minutes after inhibitor treatment as a surrogate for total insulin release in these animals, demonstrating that PI3K inhibitors and IGFR / INSR inhibitors dramatically increase insulin release in these animals. In all cases, compounds that caused an acute increase in blood glucose levels also increased serum insulin levels. Figure 5D graphically depicts c-peptide levels taken from the mice described in Figure 5B at 240 and 180 minutes after inhibitor treatment as a surrogate for total insulin release in these animals, demonstrating that PI3K and IGFR / INSR inhibitors dramatically increase insulin release in these animals. In all instances, compounds that caused an acute increase in blood glucose levels also increased serum insulin levels. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A-1]Figures 6A-6G show the influence of insulin feedback levels observed in Figures 1A-D on the effects of BKM120 in vitro. Figure 6A-1 graphically depicts cell proliferation of MDA-MB-468 breast cancer cells in minimal growth medium, with growth partially rescued by the addition of the observed feedback level of insulin (10 ng / ml) induced by BKM120 in mice. Figure 6A-2 graphically depicts proliferation of BT-549 breast cancer cells in minimal growth medium, with growth partially rescued by the addition of the observed feedback level of insulin (10 ng / ml) induced by BKM120 in mice. Figure 6A-3 graphically depicts proliferation of PC-3 prostate cancer cells in minimal growth medium, with growth partially rescued by the addition of the observed feedback level of insulin (10 ng / ml) induced by BKM120 in mice. Figure 6B graphically depicts cell viability assays showing the effect of these insulin feedback levels on organoid cultures (Pt A and Pt B) from two subjects (Pt) treated with a dose-response of BKM120 at 96 hours, as measured by cell titer-glo. Figure 6C graphically depicts the proliferation (%) of HCT116 neo cells in minimal growth medium partially rescued by the addition of the observed feedback levels of insulin induced by BKM120 in mice, as observed in Figures 1A-D. Figure 6D depicts the proliferation (%) of HCT116 neo cells with and without treatment with physiologically observed levels of insulin (10 ng / ml) and the clinically relevant PI3K inhibitors GDC-0032 and BYL-719. Figure 6E graphically depicts the growth (%) of HCT116 PTEN knockout (KO) cells with and without treatment with physiologically observed levels of insulin (10 ng / ml) and the clinically relevant PI3K inhibitors GDC-0032 and BYL-719. Figure 6F shows the growth (%) of DLD1-Neo cells under the indicated treatment conditions, the same as Figure 6G.Figure 6G graphically depicts the growth (%) of DLD-1 PTEN knockout cells under the same treatment conditions as Figure 6F. Note that PTEN loss in the same gene set of colon cancer lines does not uniformly alter the effect on insulin in the context of PI3K inhibition. In the context of PTEN loss, physiological levels of insulin can restore normal growth of HCT116 cells despite the presence of PI3K inhibitors. [Figure 6A-2] See legend to Figure 6A-1. [Figure 6A-3] See legend to Figure 6A-1. [Figure 6B] See legend to Figure 6A-1. [Figure 6C] See legend to Figure 6A-1. [Figure 6D] See legend to Figure 6A-1. [Figure 6E] See legend to Figure 6A-1. [Figure 6F] See legend to Figure 6A-1. [Figure 6G] See legend to Figure 6A-1. [Figure 7A]Figures 7A-7F show blood glucose, tumor volume, ketone concentrations, and triglyceride levels for KPC K8484 allografts treated with PI3K inhibitors with or without an adjunctive approach targeting systemic insulin feedback. Figure 7A graphically depicts the blood glucose curves of the mice shown in Figures 3E-3G treated with a control diet, a ketogenic diet, metformin (250 mg / kg), or canagliflozin (SGLT2i) (6 mg / kg) after a first dose of BYL-719 (45 mg / kg). Figure 7B graphically depicts the tumor volumes of mice treated with the metabolic regulators shown in Figures 3A-3G without a PI3K inhibitor. Figure 7C graphically depicts the mean tumor volume (lines) for each of the indicated treatment cohorts, along with the scatter plots (dots). Figure 7D graphs tumor volumes from an independent experiment of mice treated daily with BKM120, with or without 6 mg / kg canagliflozin administered 60 minutes before PI3K treatment so that peak SGLT2 inhibition coincided with peak blood glucose levels after PI3K inhibitor treatment. Figure 7E graphs blood ketones in the mice shown in Figures 3A-D after a single treatment with BKM120, with or without pretreatment with metformin, canagliflozin, or a ketogenic diet, as indicated. Figure 7F graphs triglyceride levels, as determined by calorimetry, in the mice shown in Figures 3A-D after a single treatment with BKM120, with or without pretreatment with metformin, canagliflozin, or a ketogenic diet, as indicated. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 7F] See legend to Figure 7A. [Figure 8A]Figures 8A-8H demonstrate the role of insulin receptor inhibition in altering the observed tumor response. Figure 8A shows a Western blot of cell lysates from K8484 cells used to generate the xenografts in Figure 4A after doxycycline treatment to induce the indicated sh-Renilla and sh-INSR hairpins. Figure 8B graphically depicts the tumor volume over time of individual mice allografted with KPC-K8484 tumors, as measured by calipers. Figure 8C graphically depicts the survival curve of the mice in Figure 8A. Figure 8D graphically depicts the blood glucose levels of the mice 240 minutes after the indicated treatments. Two of the glucose measurements in the OSI-906 and BKM120 exceeded the range of the detection device (e.g., >600). Figure 8E graphically depicts the c-peptide levels of the mice 240 minutes after the indicated treatments. Figure 8F graphically depicts the mass of the mice over the course of the indicated treatments. As previously reported, mice lose 10–20% of their mass upon initiation of a ketogenic diet. Figure 8G graphically depicts the tumor volume of the tumors in Figure 8A following treatment with OSI-906, an INSR / IGFR inhibitor, or GDC-0032, with or without a ketogenic diet. Treatment efficacy was significantly improved in PIK3CA + MYC mutant mouse breast cancer allografts (ES-278) grown in wild-type C57 / BL6 mice when combined with a ketogenic diet. Figure 8H graphically depicts the tumor volume over time, measured by calipers, of wild-type C57 / BL6 mice bearing KPC allograft tumors. Mice were treated with the indicated combination of BYL-719, a ketogenic diet, or insulin, as in Figure 4B. Mice in the ketogenic-BYL719-insulin cohort lost >20% body weight during the 1-week treatment, and the experiment was terminated on day 7. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 8F]See legend to Figure 8A. [Figure 8G] See legend to Figure 8A. [Figure 8H] See legend to Figure 8A. [Figure 9A] Figures 9A-9E show the effects of PI3K inhibitor treatment on a subject-derived xenograft model of bladder cancer and a syngeneic allograft model of PIK3CA-mutated breast cancer. Figure 9A graphically depicts tumor volume over time in subject-derived endometrial xenografts (PDXs) obtained from a subject with bladder cancer (Subject C) treated with the pan-PI3K inhibitor GDC-0941 or the PI3K-β suppressing compound GDC-0032 alone or in combination with a ketogenic diet. Lines represent the mean tumor volume for each treatment group, and dots represent individual tumor volumes over time. Tumors were allowed to grow until their diameter was greater than 0.6 cm before treatment began. Figure 9B graphically depicts tumor volumes (from Figure 9A) harvested at the day 12 harvest time point. Figure 9C graphically depicts tumor growth over time in mice bearing ES272, an orthotopic allograft of PIK3CA (H1047R) mutant mouse breast cancer, treated with BKM120 alone or in combination with a ketogenic diet, as indicated. Figure 9D graphically depicts tumor burden at harvest in mice bearing ES272, an orthotopic allograft of PIK3CA (H1047R) mutant mouse breast cancer, treated with BKM120 alone or in combination with a ketogenic diet, as indicated. Figure 9E graphically depicts mass over time in the mice shown in Figures 9C-9D. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 9E] See legend to Figure 9A. [Figure 10A]Figures 10A-10E show the effect of capanilisib, with or without a ketogenic diet, on the growth of orthotopic Kras-Tp53-Pdx-Cre (KPC) K8082 tumor models grown in the flanks of wild-type C57 / BL6 mice. Figure 10A graphs the survival rate of mice bearing KPC K8082 allografts grown in the flanks and treated with BAY 80-6946 alone as indicated and in combination with ketogenic diet pretreatment as indicated (the p-value for comparing BAY 80-6946 with the combination of BAY 80-6946 and the ketogenic diet in this study is 0.0019 by the Mantel-Cox log-rank test). Figure 10B graphs the volume of each tumor in this cohort, plotted as an individual line. Figure 10C graphically depicts a single blood glucose measurement from a sample taken from the animals in Figures 10B and 10C 240 minutes after treatment. Figure 10D graphically depicts c-peptide measurements from a sample taken from the animals in Figures 10B and 10C 240 minutes after treatment. Figure 10E graphically depicts mass over time during treatment for the animals depicted in Figures 10A-10D. Tumors were allowed to grow until their diameter was >0.6 cm before treatment began. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 10E] See legend to Figure 10A. [Figure 11A]Figures 11A-11F show the effects of the BKM120 / ketogenic combination in a syngeneic model of AML. Figure 11A shows IVIS images of AML burden (reported by mCherry) in mice during the indicated treatment periods. Figure 11B graphically depicts the survival rate of mice with a syngeneic model of AML treated with BKM120 alone or in combination with a ketogenic diet. Individual lines indicate the initiation of a ketogenic diet before (pre) or at the same time as (concurrent) the initiation of BKM120 treatment, demonstrating that the effect of BKM120 was significantly enhanced by the addition of a ketogenic diet (p=0.0316 and 0.349 for pre and concurrent, respectively). Asterisks (*) indicate mice sacrificed due to paralysis resulting from AML infiltrating the CNS, rather than the typical deaths seen in these mice due to tumor burden. It is noteworthy that mice in the BKM + ketogenic diet group were sacrificed frequently due to paralysis, which was not a frequent cause of death in the other treatment groups. Figure 11C graphically depicts disease burden in the AML models shown in Figures 4A-4E, as measured by percent AML cells in the bone marrow. Figure 11D graphically depicts spleen weight for each treatment group. Figure 11E graphically depicts AML burden in mice pretreated with BKM120 and / or a ketogenic diet, demonstrating that the effects observed in the AML studies are not the result of engraftment problems associated with these pretreatments. Figure 11F shows images of mice treated with BKM120 or a ketogenic diet, as indicated, with the diet and BKM120 treatment initiated on the same day (co-treatment). [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 11D] See legend to Figure 11A. [Figure 11E] See legend to Figure 11A. [Figure 11F] See legend to Figure 11A. [Figure 12A]Figures 12A-12C show the in vivo effects of multiple approaches simultaneously targeting glucose / insulin feedback. Figure 12A graphically depicts blood glucose levels in wild-type C57 / BL6 mice bearing syngeneic K8484 KPC allograft tumors after treatment with a single dose of BKM120 following metformin pretreatment, SGLT2 inhibitor (SGLT2i) pretreatment, a ketogenic diet alone, or a combination thereof (N=4 / arm). Figure 12B graphically depicts ketone levels in wild-type C57 / BL6 mice bearing syngeneic K8484 KPC allograft tumors after treatment with a single dose of BKM120 following metformin pretreatment, SGLT2 inhibitor (SGLT2i) pretreatment, a ketogenic diet alone, or a combination thereof (N=4 / arm). Figure 12C graphically depicts c-peptide levels in wild-type C57 / bl6 mice bearing syngeneic K8484 KPC allograft tumors after treatment with a single dose of BKM120 along with metformin pretreatment, SGLT2 inhibitor (SGLT2i) pretreatment, ketogenic diet alone, or a combination thereof (N=4 / arm). [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description The present disclosure generally relates to compositions and methods for treating diseases or disorders associated with PI3K signaling. The methods may include administering a regulator of glucose metabolism, using a diet that affects the metabolic state of a subject, or a combination thereof. In some examples, the methods include co-administration of a pathway inhibitor (e.g., an inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway) and administration of a regulator of glucose metabolism. In some examples, the treatment method includes administration of a pathway inhibitor to a subject who is following a ketogenic diet during treatment. The present disclosure further relates to pharmaceutical compositions comprising a pathway inhibitor and a regulator of glucose metabolism.

[0025] Some studies have shown that the administration of various glucose metabolism regulators alone or the administration of various inhibitors of insulin receptor / PI3K / AKT / mTOR pathways may be associated with unfavorable safety profiles and suboptimal anti-cancer activity.However, the type of combination therapy described herein can reduce or eliminate such side effects.For example, when combined with other therapeutic agents and / or ketogenic diet, the inhibitor can be administered at a lower dose or for a shorter period of time.Such combination therapy can provide reduced toxicity and avoid some of the side effects of monotherapy of single drug treatment.

[0026] The insulin receptor / PI3K / AKT / mTOR pathway has not proven to be as promising a target for drug therapy as expected in the art given its central role in cell signaling. More than 20 PI3K inhibitors have entered clinical trials, but only two (idelalisib and copanlisib) have been approved for use in cancer therapy. These agents are effective in treating lymphoma primarily by targeting the enzyme p110Δ encoded by PIK3CD rather than the more widely mutated enzyme p110α encoded by PIK3CA. Several drugs targeting p110α have entered approval trials, but their toxicity profiles have been difficult to manage, and their response has not correlated as well with PIK3CA mutations as expected. (Massacesi, C. et al. PI3K inhibitors as new cancer therapeutics: implications for clinical trial design. Onco Targets Ther 9, 203-210 (2016); Mayer, IA et al. A Phase Ib Study of Alpelisib (BYL719), a PI3Kalpha-Specific Inhibitor, with Letrozole in ER+ / HER2- Metastatic Breast Cancer. Clin Cancer Res 23, 26-34 (2017)).

[0027] As disclosed herein, pharmacological blockade of PI3K increases serum glucose and raises serum insulin. This hyperinsulinemia reactivates the PI3K and mTOR signaling pathways in tumors within 1 or 2 hours of administration, thereby weakening the effect of PI3K blockade. The present disclosure provides various interventions to reduce serum insulin. For example, the methods described herein can be used with metformin, NaCl, or other anti-inflammatory drugs. +This method may include administering glucose cotransporter inhibitors, using ketogenic diet, or a combination thereof.As shown herein, various groups of human tumor organoids and cell lines that grow as tumors in vivo and genetically engineered tumors show enhanced response to PI3K inhibitors when subjects are on ketogenic diet.This enhanced response is found in tumors that have or do not have PIK3CA mutation.These results show that treating subjects with glucose metabolism regulators and / or managing subjects with ketogenic diet can enhance the response of subjects to PI3K inhibitors in a wide range of cancers.

[0028] Modulators of glucose metabolism and ketogenic diets improve drug efficacy with pathway inhibitors, including a number of different agents that target the PI3K pathway in addition to BKM120 and BYL719, including the pan-PI3K inhibitor GDC-0941, the PI3K-β suppressor compound GDC-0032, the mTOR / PI3K dual inhibitor GDC-0980, the orally bioavailable class I PI3K α isoform inhibitor ceravelisib (TAK-117), and the recently approved PI3K-α / δ inhibitor copanlisib.

[0029] For example, the addition of a ketogenic diet to BKM120 reduced immunohistochemical markers of insulin signaling in PTEN / PIK3CA-mutated endometrial PDX tumors compared with tumors treated with BKM120 alone. In these tumors, the ketogenic diet enhanced the ability of BKM120 to reduce levels of phosphorylated insulin receptor, phosphorylated AKT, and phosphorylated S6. This reduction in signaling correlated with decreased levels of cell proliferation, as indicated by Ki67 staining, and increased levels of apoptosis, as indicated by cleaved caspase-3 staining.

[0030] In one embodiment, the present disclosure includes a method for inhibiting cell proliferation, the method comprising contacting a cell with an effective amount of a glucose metabolism regulator and contacting the cell with an effective amount of at least one inhibitor of the insulin receptor / PI3K / AKT / mTOR pathway, thereby inhibiting cell proliferation. In certain embodiments, the at least one inhibitor inhibits at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway, thereby inhibiting cell proliferation. In certain embodiments, the insulin receptor / PI3K / AKT / mTOR pathway is the insulin receptor / PI3K / AKT / mTOR pathway of a mammal, for example, a human. In various embodiments, this method can be performed in vivo, ex vivo, or in vitro.

[0031] In another embodiment, the present disclosure includes a method for treating a cell proliferative disease, the method comprising administering to a subject in need thereof an effective amount of at least one inhibitor of the insulin receptor / PI3K / AKT / mTOR pathway, and administering to the subject a ketogenic diet and / or at least one regulator of glucose metabolism, thereby inhibiting cell proliferation in the subject. In certain embodiments, the subject is administered a ketogenic diet. In certain embodiments, the subject is administered a regulator of glucose metabolism. In certain embodiments, the subject is administered both a ketogenic diet and a regulator of glucose metabolism. In certain embodiments, the at least one inhibitor inhibits at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway, thereby inhibiting cell proliferation. In certain embodiments, the insulin receptor / PI3K / AKT / mTOR pathway is a mammalian, e.g., human, insulin receptor / PI3K / AKT / mTOR pathway.

[0032] In certain embodiments of any of the methods disclosed herein, "administration" includes providing a pathway inhibitor, a modulator of glucose metabolism, and / or a ketogenic diet to a subject, or providing a prescription for a pathway inhibitor, a modulator of glucose metabolism, and / or a ketogenic diet to a subject, e.g., to be investigated or administered at a later time. In certain embodiments, "administration" of a ketogenic diet includes instructing a subject to use a ketogenic diet.

[0033] The methods and compositions disclosed herein can be used to increase the efficacy of treatment with a pathway inhibitor (e.g., an inhibitor of the insulin receptor / PI3K / AKT / mTOR signaling pathway). Accordingly, the present disclosure provides methods for increasing the efficacy or effectiveness of treatment with a pathway inhibitor (e.g., an inhibitor of the insulin receptor / PI3K / AKT / mTOR signaling pathway). Such methods can include treating a subject with an effective amount of a pathway inhibitor and, optionally, an effective amount of a glucose metabolism regulator. The present disclosure also provides methods for increasing the efficacy or effectiveness of treatment with a pathway inhibitor (e.g., an inhibitor of the insulin receptor / PI3K / AKT / mTOR signaling pathway). Such methods can include treating a subject with an effective amount of a pathway inhibitor, wherein the subject follows a ketogenic diet during treatment. The subject can be in need of such treatment. Alternatively, the treatment can be used to reduce the onset or occurrence of a disease in a subject.

[0034] Methods and compositions disclosed herein can allow the use of lower doses of pathway inhibitors.Therefore, the present disclosure provides a method for treating the disease or disorder associated with PI3K signal transduction, the method comprises administering to the subject in need thereof an effective amount of pathway inhibitor (for example, the inhibitor of insulin receptor / PI3K / AKT / mTOR signal transduction pathway) and an effective amount of glucose metabolism regulator, wherein the effective amount of pathway inhibitor is less than the effective amount when not using glucose metabolism regulator treatment.

[0035] The present disclosure also provides a method for treating a disease or disorder associated with PI3K signaling, the method comprising administering to a subject in need thereof an effective amount of a pathway inhibitor (e.g., an inhibitor of the insulin receptor / PI3K / AKT / mTOR signaling pathway), wherein the subject consumes a ketogenic diet during treatment, and the effective amount of the pathway inhibitor is less than the effective amount when the subject does not consume a ketogenic diet during treatment. In certain embodiments, the effective amount of the pathway inhibitor, when used in combination with a regulator of glucose metabolism and / or a ketogenic diet, is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% of the amount of the pathway inhibitor when used alone.

[0036] The methods and compositions disclosed herein can allow for less frequent administration of pathway inhibitors.Therefore, the present disclosure provides a method for treating diseases or disorders associated with PI3K signaling, the method comprises administering to a subject in need thereof an effective amount of a pathway inhibitor (for example, an inhibitor of insulin receptor / PI3K / AKT / mTOR signaling pathway) and an effective amount of a regulator of glucose metabolism, wherein the pathway inhibitor is administered less frequently than the effective frequency when not treated with the regulator of glucose metabolism.The present disclosure also provides a method for treating diseases or disorders associated with PI3K signaling, the method comprises administering to a subject in need thereof an effective amount of a pathway inhibitor (for example, an inhibitor of insulin receptor / PI3K / AKT / mTOR signaling pathway), wherein the subject follows a ketogenic diet during treatment, and the pathway inhibitor is administered less frequently than the effective frequency when the subject does not follow a ketogenic diet during treatment. For example, at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 10, or at least 13, or at least 15 fewer doses of pathway inhibitor and / or regulator of glucose metabolism may be administered during a treatment period than if the pathway inhibitor or regulator of glucose metabolism were administered alone.

[0037] Diseases or disorders associated with the PI3K signaling pathway The term "disease or disorder associated with PI3K signaling" is intended to be broadly construed to refer to a disease or disorder caused by a gain-of-function or loss-of-function in one or more members of the insulin receptor / PI3K / AKT / mTOR pathway. A "disease or disorder associated with the PI3K signaling pathway" is a disease or disorder in mammals, such as humans, livestock animals, zoo animals, or laboratory animals.

[0038] The term "disease or disorder associated with the PI3K signaling pathway" is not limited to a particular form of PI3K. There are multiple PI3K genes, and the phrase encompasses diseases and / or disorders associated with any of them. Human PI3K genes include at least those listed in Table 1.

[0039] (Table 1) PI3K genes TIFF2025160313000002.tif128129

[0040] The term "diseases or disorders associated with PI3K signaling" is not limited to diseases or disorders directly caused by gain-of-function or loss-of-function in PI3K, but also encompasses diseases or disorders caused by gain-of-function or loss-of-function in other genes in the insulin receptor / PI3K / AKT / mTOR pathway. Upstream and downstream regulators of PI3K signaling include many drug targets, such as signal receptors, protein kinase B (known as AKT), target of rapamycin (mTOR), and others.

[0041] The term "disease or disorder associated with PI3K signaling" is not limited to a specific disease pathology. Pathologically distinct diseases and disorders often share a common mechanistic basis. Therefore, mechanism-based treatments are traditionally defined in terms of the target mechanism, rather than by the tissue origin or pathological characteristics of the disease or disorder.

[0042] The disease or disorder associated with PI3K signaling therefore includes various types of disease or disorder.In one embodiment, the disease or disorder associated with PI3K signaling is a cell proliferation disease.In one embodiment, the disease or disorder associated with PI3K signaling is a neurodegenerative disease.In one embodiment, the disease or disorder associated with PI3K signaling is an inflammatory disease or condition.In one embodiment, the disease or disorder associated with PI3K signaling is a metabolic disease.

[0043] In some embodiments, the disease or disorder associated with PI3K signaling is one or more of leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, e.g., sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphosarcoma, lymphangioendothelial sarcoma). sarcoma), synovial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma), or a combination thereof.

[0044] In some embodiments, the disease or disorder associated with PI3K signaling is brain trauma, spinal cord trauma, trauma to the peripheral nervous system, Alzheimer's disease, Pick's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steele-Richardson syndrome), multiple system degeneration (Shy-Drager syndrome), motor neuron diseases including amyotrophic lateral sclerosis, degenerative dyspraxia, ataxias), corticobasal degeneration, ALS-Parkinson-Dementia Complex of Guam, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathies, primary progressive aphasia, striatonigral degeneration, Machado-Joseph disease / Spinocerebellar degeneration type 3 and olivopontocerebellar degeneration, Gilles de la Tourette's disease, bulbar and pseudobulbar palsy, spinal and spinal-bulbar muscular atrophy (Kennedy's disease), primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sachs disease, Sandhoff disease, familial spastic disorders, Wohlfart-Kugelberg-Welander disease, spastic paraplegia, progressive multifocal leukoencephalopathy, and prion diseases (Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, Kuhl's disease, and Kuhl's disease). neurodegenerative diseases, including, but not limited to, dementia of any type, cerebral ischemia or infarction, hereditary cerebral angiopathy, hereditary amyloid, Down's syndrome, macroglobulinemia, secondary familial Mediterranean fever, Muckle-Wells syndrome, multiple myeloma, pancreas-associated amyloidosis, cardiac-associated amyloidosis, chronic hemodialysis arthropathy, Finnish amyloidosis, Iowa amyloidosis, or a combination thereof.

[0045] In some embodiments, the disease or disorder associated with PI3K signaling is an inflammatory disorder, including but not limited to type II diabetes, insulin-resistant cardiovascular disease, arrhythmia, atherosclerosis, coronary artery disease, hypertriglyceridemia, dyslipidemia, retinopathy, nephropathy, neuropathy, obesity and macular edema, ileitis, ulcerative colitis, Barrett's syndrome, Crohn's disease, or a combination thereof.

[0046] In some embodiments, the disease or disorder associated with PI3K signaling is a metabolic disease, including but not limited to type II diabetes, insulin-resistant cardiovascular disease, arrhythmia, atherosclerosis, coronary artery disease, hypertriglyceridemia, dyslipidemia, retinopathy, nephropathy, neuropathy, obesity, macular edema, or a combination thereof.

[0047] In some embodiments of the methods of the present disclosure, the subject is treatment-naive. In some embodiments, the subject is resistant to hyperglycemia. In some embodiments, the subject is not hyperglycemic. In some embodiments, the subject is not hyperglycemic prior to treatment. In some embodiments, the subject is hypoglycemic. In some embodiments, the subject exhibits glycemic control. In some embodiments, the disease or disorder associated with PI3K signaling is associated with impaired glucose homeostasis. In some embodiments, the disease or disorder associated with PI3K signaling is associated with impaired glucose homeostasis. In some embodiments, the disease or disorder associated with PI3K signaling is associated with normal glucose homeostasis. In some instances, the subject is in need of treatment. However, in some instances, the subject is treated to reduce the onset or occurrence of a disease or disorder.

[0048] Regulators of glucose metabolism In some embodiments, the compositions and methods of the present disclosure may include a modulator of glucose metabolism. Various modulators of glucose metabolism may be used, including, but not limited to, lipids, amino acids, small molecule drugs, antibodies, proteins, nucleic acids, and gene editing systems. In some embodiments, the modulator of glucose metabolism inhibits glucose metabolism. For example, the modulator of glucose metabolism may be a glucose uptake inhibitor. In some embodiments, the glucose uptake inhibitor may be an inhibitor of a sodium-glucose transporter protein. In some embodiments, the modulator of glucose metabolism may be an inhibitor of a glucose transporter. In some embodiments, the glucose uptake inhibitor may be selected from the group consisting of a sodium-glucose cotransporter protein 1 (SGLT1) inhibitor, a sodium-glucose cotransporter protein 2 (SGLT2) inhibitor, or an SGLT1 / SGLT2 dual inhibitor.

[0049] The glucose metabolism modulator may, in some instances, be administered while the subject is on a ketogenic diet or while the subject is being fed a ketogenic diet.

[0050] Glucose is essential for energy production in living organisms, and glucose transporters play important roles in various organs. Glucose transporters are classified into two families: facilitative glucose transporters (GLUTs), which transport glucose by facilitated diffusion. Sodium-glucose-like transporters (SGLTs) cotransport sodium ions and glucose (or other substances) using an electrochemical gradient across a membrane (Harada et al. Role of sodium-glucose transporters in glucose uptake of the intestine and kidney. J Diabetes Investig. 2012 Aug 20; 3(4): 352-353.; Gallo et al. Probing SGLT2 as a therapeutic target for diabetes: Basic physiology and consequences. Diab Vasc Dis Res. 2015 Mar; 12(2): 78-89; Wright et al. Biology of Human Sodium Glucose Transporters. Physiological Reviews. 91(2):733-794 (2011)). Six SGLT proteins and gene families are listed in Table 2. In certain embodiments, the regulator of glucose metabolism inhibits one or more SGLT proteins.

[0051] Table 2. Sodium-dependent glucose transporters and tissue distribution TIFF2025160313000003.tif61137

[0052] In some embodiments, the glucose uptake inhibitor can be dapagliflozin, empagliflozin, canagliflozin, ipragliflozin, tofogliflozin, sergliflozin etabonate, remogliflozin etabonate, ertugliflozin, sotagliflozin, conagliflozin, or a combination thereof. In some embodiments, the glucose metabolism regulator can be metformin. In some embodiments, the glucose metabolism regulator can be an insulin receptor / IGF1 receptor inhibitor. In some embodiments, the glucose metabolism regulator can be linsitinib (OSI-906).

[0053] Dapagliflozin (trade name Farxiga™ in the United States, Forxiga™ in Europe and Russia) is a drug of the gliflozin class used to treat type 2 diabetes. It was developed by Bristol-Myers Squibb in collaboration with AstraZeneca. Dapagliflozin has the following chemical structure: I have TIFF2025160313000004.tif33128.

[0054] Empagliflozin (trade name Jardiance™) is a drug of the gliflozin class that was approved in 2014 for the treatment of type 2 diabetes in adults. It was developed by Boehringer Ingelheim and Eli Lilly and Company. Empagliflozin is an inhibitor of SGLT2, which causes sugar in the blood to be excreted by the kidneys and eliminated in the urine. Empagliflozin has the following chemical structure: I have TIFF2025160313000005.tif18128.

[0055] Canagliflozin (trade names Invokana™ or Sulisent™) is a drug used to treat type 2 diabetes. It is of the gliflozin class or SGLT2 inhibitor class. Canagliflozin has the following chemical structure: I have TIFF2025160313000006.tif22128.

[0056] Ipragliflozin (trade names Suglat™ and Jardiance™) is a medication for the treatment of type 2 diabetes. Co-developed by Astellas Pharma and Kotobuki Pharmaceutical, ipragliflozin was approved as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes. Ipragliflozin has the following chemical structure: I have TIFF2025160313000007.tif31128.

[0057] Tofogliflozin (code name CSG452) is an experimental drug for the treatment of diabetes, being developed by Chugai Pharma in collaboration with Kowa and Sanofi. It is an inhibitor of SGLT2. Tofogliflozin was first approved in the world for this condition in Japan, either as monotherapy or in combination with other antihyperglycemic agents. Tofogliflozin has the following chemical structure: I have TIFF2025160313000008.tif19128.

[0058] Sergliflozin etabonate (code name GW869682X) is an investigational antidiabetic drug being developed by GlaxoSmithKline. It is an SGLT2 inhibitor. Sergliflozin etabonate has the following chemical structure: I have TIFF2025160313000009.tif22128.

[0059] Remogliflozin etabonate is a drug of the gliflozin class proposed for the treatment of nonalcoholic steatohepatitis ("NASH") and type 2 diabetes. Remogliflozin is under development by Avolynt, Inc. Remogliflozin etabonate has the following chemical structure: I have TIFF2025160313000010.tif41128.

[0060] Ertugliflozin (trade name Steglatro™) is a drug for the treatment of type 2 diabetes. In the United States, it has been approved by the Food and Drug Administration for use as monotherapy and in fixed-dose combinations with either sitagliptin or metformin. Ertugliflozin has the following chemical structure: I have TIFF2025160313000011.tif23128.

[0061] Sotagliflozin, or LX4211, is an orally delivered small molecule compound currently being developed by Lexicon Pharmaceuticals for the treatment of type 1 and type 2 diabetes. Sotagliflozin inhibits SGLT1 and SGLT2. Sotagliflozin has the following chemical structure: I have TIFF2025160313000012.tif25128.

[0062] Canagliflozin (trade names Invokana or Sulisent) is a medication used for the treatment of type 2 diabetes. It is of the gliflozin class or SGLT2 inhibitor class. Canagliflozin has the following chemical structure: I have TIFF2025160313000013.tif24128.

[0063] Metformin is a first-line medication for the treatment of type 2 diabetes, especially in overweight people. Metformin has the following chemical structure: I have TIFF2025160313000014.tif26128.

[0064] Linsitinib (code name OSI-906) is an experimental drug candidate for the treatment of various types of cancer. In some instances, it can act as a regulator of glucose metabolism, a pathway inhibitor, or both. It is an inhibitor of the insulin receptor and insulin-like growth factor 1 receptor (IGF-1R) (Fassnacht et al. Linsitinib (OSI-906) versus placebo for subjects with locally advanced or metastatic adrenocortical carcinoma: a double-blind, randomized, phase 3 study. Lancet Oncology. 16(4):426-435 (2015)). Linsitinib has the following chemical structure: I have TIFF2025160313000015.tif46128.

[0065] In some instances, the glucose metabolism regulator can be administered simultaneously with the insulin receptor / PI3K / AKT / mTOR pathway inhibitor. In some instances, the glucose metabolism regulator can be administered before the insulin receptor / PI3K / AKT / mTOR pathway inhibitor. In some instances, the glucose metabolism regulator can be administered after the insulin receptor / PI3K / AKT / mTOR pathway inhibitor is administered. The ketogenic diet can be ingested or administered before, during, or after the glucose metabolism regulator is administered.

[0066] Pathway inhibitors In some embodiments, the present disclosure provides a method for treating a disease or disorder associated with PI3K signaling, comprising administering to a subject an effective amount of a pathway inhibitor. In some embodiments, the present disclosure provides a method for treating a disease or disorder associated with PI3K signaling, comprising administering to a subject an effective amount of a pathway inhibitor, wherein the subject is on a ketogenic diet during treatment. The subject may be in need of such treatment, or the subject may be treated to reduce the onset or occurrence of a disease or disorder. In some embodiments, the pathway inhibitor can inhibit at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway. In some embodiments, the pathway inhibitor can inhibit one or more targets, e.g., INSR / IGFR, PI3K, AKT, mTOR, or a combination thereof. In some embodiments, the pathway inhibitor can inhibit PI3K. In some embodiments, the light inhibitor can inhibit one or more of p110-α, p110-β, p110-γ, p110-δ, p85-α, p85-β, p55-γ, p150, p101, p87, PI3K-C2α, PI3K-C2β, PI3K-C2γ, and Vps34.

[0067] In some embodiments, the pathway inhibitor can be idelalisib, copanlisib, buparlisib (BKM120), alpelisib (BYL719), taselisib (GDC-0032), pictilisib (GDC-0941), apitolisib (GDC-0980), selavelisib (TAK-117), dactolisib, apelisib, MK2206, linsitinib (OSI-906), or a combination thereof.

[0068] Idelalisib (trade name Zydelig™, code names GS-1101 or CAL-101) is a drug used for the treatment of certain hematological malignancies. The substance acts as a phosphoinositide 3-kinase inhibitor. More specifically, it blocks p110-δ, the delta isoform of the enzyme phosphoinositide 3-kinase (PI3K). It was developed by Gilead Sciences. Idelalisib has the following chemical structure: I have TIFF2025160313000016.tif36128.

[0069] Copanlisib (trade name Aliqopa™, code name BAY 80-6946) is a kinase inhibitor developed by Bayer HealthCare Pharmaceuticals Inc. approved for the treatment of adult subjects with relapsed follicular lymphoma who have received at least two prior systemic therapies. Copanlisib has the following chemical structure: I have TIFF2025160313000017.tif22128.

[0070] Buparlisib (code name BKM120) is an orally bioavailable, specific inhibitor of the class I phosphatidylinositol 3-kinase (PI3K) family of lipid kinases with potent antineoplastic activity. Buparlisib specifically inhibits class I PIK3 in an ATP-competitive manner, thereby preventing the production of the second messenger phosphatidylinositol-3,4,5-triphosphate and activation of the PI3K signaling pathway. Buparlisib has the following chemical structure: TIFF2025160313000018.tif38128. Some studies have shown that administration of BKM120 alone may be associated with an unfavorable safety profile and minimal antitumor activity in advanced or recurrent endometrial cancer. However, the type of combination therapy described herein may reduce or eliminate such side effects. For example, BKM120 may be administered at a lower dose when combined with other therapeutic agents and / or a ketogenic diet. Such combination therapy may provide reduced toxicity and avoid side effects.

[0071] Alpelisib (code name BYL719) is an orally bioavailable phosphatidylinositol 3-kinase (PI3K) inhibitor with potent antineoplastic activity. Alpelisib specifically inhibits PIK3, thereby preventing activation of the PI3K signaling pathway. Alpelisib has the following chemical structure: I have TIFF2025160313000019.tif24128.

[0072] Taselisib (code name GDC-0032), developed by Roche, is an orally bioavailable inhibitor of class I phosphatidylinositol 3-kinase (PI3K) α isoform (PIK3CA) with potent antineoplastic activity. Taselisib selectively inhibits PIK3CA and its mutant forms, which can lead to tumor cell apoptosis and growth inhibition in PIK3CA-expressing tumor cells. Taselisib has the following chemical structure: I have TIFF2025160313000020.tif51128.

[0073] Pictilisib (code name GDC-0941), developed by Roche, is a potent inhibitor of PI3Kα / δ with an IC50 of 3 nM in cell-free assays, and also shows moderate selectivity for p110β (11-fold) and p110γ (25-fold). Pictilisib has the following chemical structure: I have TIFF2025160313000021.tif35128.

[0074] Apitolisib (code name GDC-0980, RG7422) had IC values ​​of 5 nM / 27 nM / 7 nM / 14 nM, respectively, in cell-free assays. 50 Apitolisib is a potent Class I PI3K inhibitor of PI3Kα / β / δ / γ, exhibiting a K i Apitolisib is also an mTOR inhibitor with the following chemical structure: I have TIFF2025160313000022.tif37128.

[0075] Serravelisib (also known as MLN1117, INK1117, and TAK-117) is an orally bioavailable inhibitor of the class I phosphoinositide 3-kinase (PI3K) α isoform. Serravelisib selectively inhibits PI3K α kinases, including mutants of PIK3CA, in the PI3K / Akt / mTOR pathway. Serravelisib has the following structure: I have TIFF2025160313000023.tif27128.

[0076] Dactolisib (code names NVP-BEZ235 and BEZ-235) is an imidazoquinoline derivative that acts as a PI3K inhibitor. Dactolisib also inhibits mTOR. Dactolisib has the following chemical structure: I have TIFF2025160313000024.tif39128.

[0077] MK2206 is an orally bioavailable allosteric inhibitor of the serine / threonine protein kinase Akt (protein kinase B) with potent antineoplastic activity. MK2206 binds to and inhibits the activity of Akt in a non-ATP-competitive manner. MK2206 has the following chemical structure: I have TIFF2025160313000025.tif31128.

[0078] Linsitinib (code name OSI-906) is an experimental drug candidate for the treatment of various types of cancer. In some instances, it can act as a regulator of glucose metabolism, a pathway inhibitor, or both. It is an inhibitor of the insulin receptor and insulin-like growth factor 1 receptor (IGF-1R) (Fassnacht et al. Linsitinib (OSI-906) versus placebo for subjects with locally advanced or metastatic adrenocortical carcinoma: a double-blind, randomized, phase 3 study. Lancet Oncology. 16(4):426-435 (2015)). Linsitinib has the following chemical structure: I have TIFF2025160313000026.tif46128.

[0079] In some examples, a combination of inhibitors of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway can be used or administered to a subject.For example, the inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway can be a combination of two or more of buparlisib (BKM120), taselisib (GDC-0032), pictilisib (GDC-0941), or linsitinib (OSI-906).

[0080] In certain embodiments of methods that include the use of both a pathway inhibitor and a modulator of glucose metabolism, and compositions that include both a pathway inhibitor and a modulator of glucose metabolism, combinations that include any combination of agents shown in any column of Table 6 may be used.

[0081] Table 6. Combinations of glucose metabolism regulators and pathway inhibitors TIFF2025160313000027.tif90128TIFF2025160313000028.tif220123TIFF20251603130 00029.tif220123TIFF2025160313000030.tif220123TIFF2025160313000031.tif158128

[0082] In some instances, the insulin receptor / PI3K / AKT / mTOR pathway inhibitor can be administered simultaneously with the glucose metabolism regulator. In some instances, the insulin receptor / PI3K / AKT / mTOR pathway inhibitor can be administered before the glucose metabolism regulator. In some instances, the insulin receptor / PI3K / AKT / mTOR pathway inhibitor can be administered after the glucose metabolism regulator. The ketogenic diet can be ingested or administered before, during, or after the insulin receptor / PI3K / AKT / mTOR pathway inhibitor is administered.

[0083] Ketogenic diet In some embodiments, the present disclosure provides a method for treating a disease or disorder associated with PI3K signaling, wherein the subject consumes a ketogenic diet during treatment. In some embodiments, the method includes administering a ketogenic diet to the subject.

[0084] In some examples, a pathway inhibitor of the insulin receptor / PI3K / AKT / mTOR pathway can be administered to the subject while the subject is on a ketogenic diet or while the subject is receiving a ketogenic diet. In some embodiments, the method can include administering to the subject an effective amount of a regulator of glucose metabolism while the subject is receiving a ketogenic diet and / or while the subject is receiving a pathway inhibitor of the insulin receptor / PI3K / AKT / mTOR pathway.

[0085] In some embodiments, the subject consumes a ketogenic diet prior to administration of the pathway inhibitor. In some embodiments, the subject consumes a ketogenic diet after administration of the pathway inhibitor. In some embodiments, the subject consumes a ketogenic diet concurrently with administration of the pathway inhibitor.

[0086] The ketogenic diet has been used in epileptic subjects since the 1970s and has been shown to reduce blood glucose levels and increase insulin sensitivity compared to a normal Western diet (Hopkins, BD, Goncalves, MD & Cantley, LC Obesity and Cancer Mechanisms: Cancer Metabolism. J Clin Oncol 34, 4277-4283, doi:10.1200 / JCO.2016.67.9712 (2016); Sampaio, LP Ketogenic diet for epilepsy treatment. Arq Neuropsiquiatr 74, 842-848, doi:10.1590 / 0004-282X20160116 (2016)). In some embodiments, the ketogenic diet comprises a high-fat, low-carbohydrate diet. In some embodiments, the ketogenic diet is more strict than the modified Atkins diet. In some embodiments, the ketogenic diet includes consuming defined amounts of calories, fruit, and protein. Ketogenic diets are available at many major hospitals.

[0087] The classic ketogenic diet is defined by a set ratio of grams of fat to grams of carbohydrates and protein. The most common ratios are 3:1 or 4:1. In the classic ketogenic diet, approximately 90% of energy comes from fat and 10% comes from a combination of carbohydrates and protein. Calories are typically restricted to 80-90% of the daily recommended intake for an age group. In some instances, fruit restrictions are imposed on subjects following this diet.

[0088] A ketogenic diet useful for experimental purposes, in one example, is the AIN-76A purified rat and mouse diet available from ThermoFischer® or its equivalent.

[0089] In some embodiments, a ketogenic diet includes up to 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% protein, with the remainder of the diet consisting of fat, fiber, ash, and carbohydrates. In some embodiments, a ketogenic diet includes up to 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% carbohydrates, with the remainder of the diet consisting of fat, fiber, ash, and protein. In some embodiments, the ketogenic diet includes fat, measured in grams, and carbohydrates and protein, measured collectively in grams, in a fat to carbohydrate / protein ratio of 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, or 4.5 to 1. A comparison of a ketogenic diet to a normal diet is shown in Table 3 below. The ketogenic diets shown in Table 3 were used for experimental purposes, but a ketogenic diet given to or ingested by a subject according to one or more of the methods described herein may have similar ratios of fat, protein, carbohydrates, ash, and other components listed in Table 3.

[0090] In some instances, such a ketogenic diet may involve a 3:1 ratio of ketogenic to anti-ketogenic macromolecules, with approximately 85% fat, 12% protein, and 3% carbohydrates. The fat mix may vary. For example, fats may include those derived from plants, grains, and animal products. The diet may be actively managed on a specific timetable, e.g., on a weekly or monthly basis, by a nutritionist who works with the patient regarding the diet. Such diets may achieve up to 80% compliance, up to 90% compliance, up to 95% compliance, up to 96% compliance, up to 98% compliance, up to 99% compliance, or even up to 100% compliance. For example, 100% compliance over a 4-week period was achieved in an ongoing pilot study in women with endometrial cancer.

[0091] Route of administration, formulation, and dosage The disclosed treatment methods can be achieved through any mode of therapeutic administration, including systemic or local administration, such as oral, nasal, parenteral, transdermal, subcutaneous, vaginal, buccal, rectal, or topical modes of administration.

[0092] Depending on the intended mode of administration, the disclosed compositions, sometimes in unit dosage form, may be in solid, semi-solid, or liquid dosage form, in accordance with conventional pharmaceutical practice, such as injectables, tablets, suppositories, pills, sustained release capsules, elixirs, tinctures, emulsions, syrups, powders, liquids, suspensions, etc. Likewise, the compositions may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.

[0093] Exemplary pharmaceutical compositions include a pathway inhibitor (and / or a modulator of glucose metabolism) and a pharmaceutically acceptable carrier, such as (a) a diluent, such as purified water, a triglyceride oil, such as a hydrogenated or partially hydrated vegetable oil, or a mixture thereof, corn oil, olive oil, sunflower oil, safflower oil, fish oil, such as EPA or DHA, or an ester or triglyceride thereof, or a mixture thereof, an omega-3 fatty acid or a derivative thereof, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose, and / or glycine; (b) a lubricant, such as silica, talcum, stearic acid, magnesium or calcium salts thereof, sodium oleate, sodium stearate, magnesium stearate, or a mixture thereof; for tablets, also (c) binders, if desired, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, magnesium carbonate, natural sugars such as glucose or β-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, waxes and / or polyvinylpyrrolidone; (d) disintegrants, such as starch, agar, methylcellulose, bentonite, xanthan gum, alginic acid or its sodium salt, or effervescent mixtures; (e) absorbents, colorants, flavorings and sweeteners; (f) emulsifiers or dispersing agents, such as Tween 80, Labrasol, HPMC, DOSS, Caproyl 909, Labrafac, Labrafil, Peceol, Transcutol, Capmul MCM, Capmul PG-12, Captex 355, Gelucire, Vitamin E TGPS or other acceptable emulsifier; and / or (g) tablets and gelatin capsules containing an agent that enhances absorption of the compound, such as cyclodextrin, hydroxypropyl-cyclodextrin, PEG400, PEG200.

[0094] Liquid, particularly injectable, compositions can be prepared, for example, by dissolving, dispersing, etc. For example, a pathway inhibitor and / or modulator of glucose metabolism is dissolved in or mixed with a pharmaceutically acceptable solvent, such as water, saline, aqueous dextrose, glycerol, ethanol, etc., to form an injectable isotonic solution or suspension. Proteins, such as albumin, chylomicron particles, or serum proteins, can be used to solubilize the disclosed compounds.

[0095] The disclosed pharmaceutical compositions can also be formulated as suppositories which can be prepared from fatty emulsions or suspensions, using polyalkylene glycols, for example, propylene glycol, as the carrier.

[0096] The disclosed pharmaceutical compositions can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.Liposomes can be formed from various phospholipids, including cholesterol, stearylamine, or phosphatidylcholine.In some embodiments, a film of lipid components is hydrated with an aqueous solution of drug, forming a lipid layer that encapsulates pathway inhibitors and / or glucose metabolism regulators, as described in U.S. Patent No. 5,262,564, the entire contents of which are incorporated herein by reference.

[0097] The disclosed pharmaceutical compositions can also be delivered using monoclonal antibodies as independent carriers to which pathway inhibitors and / or regulators of glucose metabolism are linked. Pathway inhibitors and / or regulators of glucose metabolism can also be linked to soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, poly(hydroxypropyl)methacrylamide-phenol, poly(hydroxyethyl)-aspanamide phenol, or poly(ethylene oxide)-polylysine substituted with palmitoyl residues. Furthermore, pathway inhibitors and / or regulators of glucose metabolism can be linked to classes of biodegradable polymers useful for achieving controlled drug release, such as polylactic acid, polyepsiloncaprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and crosslinked or amphiphilic block copolymers of hydrogels. In one embodiment, the pathway inhibitor and / or modulator of glucose metabolism is not covalently attached to the polymer, eg, a polycarboxylic acid polymer or a polyacrylate.

[0098] Parenteral injection administration is generally used for subcutaneous, intramuscular, or intravenous injections and infusions. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, or in solid forms suitable for dissolving in liquid prior to injection.

[0099] The pharmaceutical compositions may be prepared according to conventional mixing, granulating, or coating methods, respectively, and the pharmaceutical compositions of the present invention may contain about 0.1% to about 99%, about 5% to about 90%, or about 1% to about 20% by weight or volume of a glucose metabolism regulator and / or kinase inhibitor.

[0100] The dosing regimen is selected according to various factors, including the type, race, age, weight, sex, and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; and the particular compound used. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the drug required to prevent, combat, or halt the progress of the disease or disorder.

[0101] In some instances, the effective dose of pathway inhibitor and / or regulator of glucose metabolism used for the indicated effect ranges from about 0.5 mg to about 5000 mg of pathway inhibitor and / or regulator of glucose metabolism required to treat a disease or disorder. Compositions for in vivo or in vitro use may contain about 0.5, about 5, about 20, about 50, about 75, about 100, about 150, about 250, about 500, about 750, about 1000, about 1250, about 2500, about 3500, or about 5000 mg of pathway inhibitor and / or regulator of glucose metabolism, or a range from one amount to another amount in the above list of doses. In one embodiment, the composition is in the form of a tablet that can be scored.

[0102] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a regulator of glucose metabolism and an inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway.

[0103] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a glucose uptake inhibitor and an inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway.

[0104] In some embodiments, the present disclosure provides a kit comprising a pharmaceutical composition comprising a glucose uptake inhibitor and a pharmaceutical composition comprising an inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway.

[0105] kit Also described herein are kits containing packaged pharmaceutical compositions for managing, preventing, or treating cell proliferative disorders. The kits of the invention can be designed to manage, prevent, or treat cell proliferation or cell proliferative disorders.

[0106] In one embodiment, the kit or container contains at least one inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway. The kit may also contain a regulator of glucose metabolism. Each inhibitor or regulator may be individually packaged. Alternatively, one or more inhibitors or regulators may be packaged or formulated together.

[0107] Here, the kit or container may include at least one inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway, at least one regulator of glucose metabolism, or a combination thereof.

[0108] The kit may also include instructions for administering at least one inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway, at least one modulator of glucose metabolism, or a combination thereof.

[0109] In another embodiment, the kit includes one or more components of a ketogenic diet. Each component of the ketogenic diet can be individually packaged. Alternatively, one or more components of the ketogenic diet can be packaged together.

[0110] In another embodiment, the kit or container includes instructions for initiating and / or maintaining a ketogenic diet, along with instructions for using at least one inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway, at least one modulator of glucose metabolism, or a combination thereof. For example, the instructions can include a method for mixing the components of a ketogenic diet, a method for obtaining supplemental components of a ketogenic diet, a timetable for the consumption of the components of a ketogenic diet, or a combination thereof.

[0111] The kits of the present invention may also include containers with tools useful for administering the compositions described herein and for maintaining the ketogenic diet described herein, including syringes, swabs, catheters, antiseptic solutions, package opening tools, forks, spoons, straws, etc.

[0112] The compositions, kits, and / or methods described herein are useful for treating cell proliferation diseases, such as cancer or cell proliferation disorders, metabolic disorders, neurodegenerative diseases, or inflammatory diseases.For example, the compositions, kits, and / or methods described herein can reduce the occurrence or progression of such diseases by 1% or more, 2% or more, 3% or more, 5% or more, 7% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, or 50% or more compared with a control.Such a control can be the initial frequency or previous progression rate of the subject's disease.The control can also be the average frequency or progression rate of the subject's disease. For example, in the case of cancer treatment, the compositions and / or methods described herein can reduce tumor volume in a treated subject by 1% or more, 2% or more, 3% or more, 5% or more, 7% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, or 50% or more compared to a control. Such a control can be the initial tumor volume. In some examples, the compositions and / or methods described herein can reduce the occurrence or progression of such diseases by at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold compared to a control. [Example]

[0113] The following examples are provided to illustrate various aspects of the present disclosure and are not intended to limit the present disclosure in any way. Those skilled in the art will recognize modifications thereto and other uses that fall within the spirit of the present disclosure, as defined by the claims. Appendix A provides further details of the experiments related to the development of the methods and compositions described herein.

[0114] Overview Gain-of-function mutations in PIK3CA, which encodes insulin-activated phosphoinositide-3-kinase (PI3K), and loss-of-function mutations in PTEN, a phosphatase that degrades phosphoinositide lipids generated by PI3K, are the most frequent events in human cancer. However, pharmacological inhibition of PI3K using various classes of inhibitors has resulted in variable clinical responses in humans, suggesting the possibility of an inherent resistance mechanism to PI3K inhibition. Here, we show that pharmacological blockade of PI3K not only elevates serum glucose but also dramatically increases serum insulin. This hyperinsulinemia reactivates the PI3K and mTOR signaling pathways in tumors within 1 or 2 hours of administration, thereby compromising the efficacy of PI3K blockade. Herein, we demonstrate that metformin, NaCl, and other anti-inflammatory drugs (anti-inflammatory drugs) are effective in treating PD-L1 / ... + This study demonstrates that various interventions that reduce serum insulin, including glucose cotransporter inhibitors and ketogenic diets, reduce serum insulin.The inventors have found that a diverse group of human tumor organoids and cell lines that are grown as tumors in mice, and genetically engineered mouse tumors, show enhanced response to PI3K inhibitors when mice are fed a ketogenic diet.This enhanced response is found in tumors that have or do not have PIK3CA mutation.These results show that managing patients with a ketogenic diet or administering glucose metabolism regulators (such as serum insulin-reducing treatments) can enhance the response of patients to PI3K inhibitors in a wide range of cancers.

[0115] The PI3K pathway is one of the most frequently mutated pathways in human cancer, and mutations in PIK3CA are observed at a frequency similar to that of KRAS. More than 20 PI3K inhibitors have entered clinical trials, but only two (idelalisib and copanlisib) have been approved for use in cancer therapy. These agents are effective in treating lymphoma primarily by targeting the enzyme p110Δ encoded by PIK3CD rather than the more widely mutated enzyme p110α encoded by PIK3CA. Several drugs targeting p110α have entered approval trials, but their toxicity profiles, including hyperglycemia, have been difficult to manage, and response has not correlated as well with PIK3CA mutations as expected. Because p110α mediates virtually all cellular responses to insulin, hyperglycemia is an expected on-target effect of p110α inhibitors. Blocking insulin signaling promotes glucose release from the liver and prevents glucose uptake into skeletal muscle and fat. The resulting acute hyperglycemia varies from patient to patient, but in most cases resolves within a few hours of drug administration due to compensatory insulin release from the pancreas.Many patients with borderline insulin resistance must discontinue treatment because they cannot endogenously maintain serum glucose levels using glucose-lowering drugs, such as metformin, during treatment.Experimental subjects who are subjected to long-term PI3K inhibition show reduced glucose tolerance and increased insulin resistance.

[0116] Here, we demonstrate that systemic glucose-insulin feedback, resulting from targeted inhibition of this pathway, activates PI3K signaling in a variety of tumors, even in the presence of PI3K inhibitors. The feedback hyperinsulinemia disclosed herein can be prevented using dietary or pharmaceutical approaches, significantly improving the efficacy / toxicity ratio of insulin receptor / PI3K / AKT / mTOR pathway inhibitors. These findings have direct clinical relevance for the numerous p110α inhibitors currently in clinical trials, providing a means to significantly increase treatment efficacy for a wide variety of patients.

[0117] Example 1: Disruption of Whole-Body Glucose Homeostasis Using Therapeutic Doses of Compounds Targeting Various Kinases in the Insulin Receptor / PI3K / mTOR Pathway Hyperglycemia is often treated as a treatment-related complication that requires management only in a subset of patients whose hyperglycemia becomes persistent.Due to the body's normal blood glucose regulation, patients treated with these agents experience a degree of systemic hyperinsulinemia as the pancreas attempts to normalize serum glucose levels.Because insulin is a potent stimulator of PI3K signal transduction in tumors and can have a significant effect on cancer progression, the inventors hypothesized that treatment-induced hyperinsulinemia limits the therapeutic effect of agents that target the PI3K pathway.

[0118] Wild-type mice were treated with therapeutic doses of compounds targeting various kinases in the insulin receptor / PI3K / mTOR pathway, including inhibitors of INSR / IGFR, PI3K, AKT, and mTOR, and their blood glucose levels were monitored over time after treatment (Figures 1A, 5A, and 5B). We observed that many of these agents significantly increased blood glucose levels. Importantly, we found that this hyperglycemia resolved after only a few hours without additional intervention, suggesting that PI3K signaling was reactivated in muscle and liver even in the presence of the drug. For each of these drugs that increased blood glucose, there was also a time-dependent increase in insulin released into serum, as measured by ELISA for insulin (Figure 1B), and an increase in the amount of c-peptide, a clinically used surrogate for insulin (Figures 1C, 5C, and 5D).

[0119] To assess whether the glucose and insulin elevations induced by these PI3K inhibitors affected tumors, we performed fluorodeoxyglucose positron emission tomography (FDG-PET) in mice bearing orthotopic Kras-Tp53-Pdx-Cre (KPC) tumor allografts in their pancreas. We observed increased glucose uptake in these tumors in the acute setting after PI3K inhibition compared with vehicle-treated mice, suggesting that elevated insulin may be causing a transient increase in glucose uptake in these tumors (Figure 1D).

[0120] Example 2: Insulin stimulates PI3K signaling in the context of PI3K inhibition To test whether these increases in insulin stimulated PI3K signaling in the context of PI3K inhibition, KPC cells were treated in vitro with PI3K inhibitors in the presence or absence of 10 ng / ml insulin, a level observed in mice within 15–30 min after drug administration (Figure 1D). This insulin level was sufficient to partially rescue PI3K signaling in the continued presence of PI3K inhibitors, as indicated by partial reactivation of phosphorylated AKT (pAKT) and nearly complete reactivation of phosphorylated S6 (pS6), a reporter of growth signaling through the mTORC1 complex (Figure 2A). In addition, this enhanced signaling, associated with a partial restoration of cell proliferation (Figures 2B–2C).

[0121] Similar effects of insulin stimulating proliferation in the presence of PI3K inhibitors were observed in various other tumor cell lines and in breast, endometrial, and prostate tumor-derived organoids obtained from patients (Figures 6A-6G). The amount of stimulation was not uniform across all cell lines, as expected for tumors with differential expression of insulin receptors and differential dependence on PI3K signaling for growth. These observations support the conclusion that insulin is a potent activator of PI3K signaling in certain tumors and that elevated serum insulin following administration of PI3K inhibitors reactivates PI3K signaling in both normal tissues and tumors, and potentially other PI3K-independent responses to insulin.

[0122] Example 3: Metformin, SGLT2 inhibitors, and ketogenic diet Research and treatment of diabetic patients has led to the development of numerous approaches to control blood glucose and insulin levels. Using these tools, we sought to identify approaches to enhance PI3K inhibitor therapy by bypassing acute glucose / insulin feedback. In our mouse model of cancer, we evaluated metformin and a sodium-glucose cotransporter 2 (SGLT2) inhibitor, along with a ketogenic diet.

[0123] Treatment-naive mice bearing KPC allografts were placed on a ketogenic diet or treated with metformin for 10 days prior to a single treatment with BKM120. Blood glucose was monitored during this treatment, and c-peptide (a surrogate for blood insulin) was assessed 3 hours later (Figures 3A-3B). In some mice, tumors were harvested at 90 minutes and stained for pS6 (Figures 3C-3D). These results indicated that pretreatment with metformin had minimal impact on PI3K inhibitor-induced elevations in blood glucose and insulin levels or growth signaling through mTORC1. In contrast, both the SGLT2 inhibitor and the ketogenic diet approach reduced the hyperglycemia and total insulin released in response to BKM120 treatment, effects correlated with reduced signaling through mTORC1 in the tumors. A similar effect was seen in mice treated with the p110α-specific inhibitor BYL-719, where we observed an enhanced response of KPC allografts to BYL-719 in a manner that corresponded to the relative ability of each treatment to reduce serum insulin levels (Figures 3E-G, 7A-D).

[0124] Example 4: Knockdown or inhibition of the insulin receptor Various hormones and metabolites can reactivate growth in the context of PI3K inhibition. To test whether enhanced tumor signaling and growth was directly insulin-mediated, we generated a doxycycline-inducible shRNA targeting the insulin receptor in KPC tumors (Figure 4A). Induction of this hairpin in the absence of a PI3K inhibitor had little effect on tumor growth. However, induction of this hairpin at the start of BYL719 treatment resulted in tumor shrinkage that was nearly as effective as a ketogenic diet (Figure 4A). This result indicates that the insulin receptor does not play a major role in tumor growth until supraphysiological amounts of insulin are released after PI3K inhibitor treatment. The specificity of this effect was further supported by the fact that combining the PI3K inhibitor BKM120 with the insulin receptor / IGF1 receptor inhibitor OSI-906 resulted in a more effective response to KPC allograft growth than either drug alone (Figures 8B–8G).

[0125] Example 5: Exogenous insulin To further test whether the improved response to PI3K inhibitors during ketogenic dieting was the result of reduced blood insulin levels, we attempted to "rescue" PI3K reactivation with exogenous insulin. Cohorts of mice bearing Pik3ca mutant breast allografts were treated with a combination of a ketogenic diet and BYL-719, followed by 0.4 mU of insulin 15 minutes after each dose of PI3K inhibitor (Figure 4B). The addition of insulin dramatically reduced the therapeutic benefit of supplementing PI3K inhibitor treatment with a ketogenic diet, and insulin addition also rescued tumor growth in allografted KPC tumors (Figure 8H). Note that the combination of a ketogenic diet, insulin, and BYL-719 was not well tolerated in young mice, and therefore the ethical endpoint was reached by the reduction in KPC tumor weight after only 1 week of treatment.

[0126] Collectively, these data indicate that modulation of glucose metabolism improves response to PI3K inhibitors by reducing circulating insulin and, consequently, its ability to activate insulin receptors in tumors. As shown herein, modulation of glucose metabolism improves response to PI3K inhibitors in tumors with a wide range of genetic abnormalities. Therapeutic benefit was observed in patient-derived xenografts of late-stage endometrial adenocarcinoma (with PTEN deletion and PIK3CA mutation) and bladder cancer (with FGFR amplification), as well as in syngeneic allografts of Pik3ca-mutated breast cancer and MLL-AF9-induced acute myeloid leukemia (Figure 4C, Figures 9A-9E, Figures 10A-10E, Figures 11A-11E).

[0127] Modulation of glucose metabolism improved the efficacy of numerous different agents targeting the PI3K pathway, including the pan-PI3K inhibitor GDC-0941, the PI3K-β inhibitor GDC-0032, the dual mTOR / PI3K inhibitor GDC-0980, and the recently approved PI3K-α / δ inhibitor copanlisib, in addition to BKM120 and BYL719 (Figure 5). In some instances, the ketogenic diet alone had variable effects in different tumor models, indicating that this dietary modification alone was insufficient to produce the tumor responses observed across mouse models. In some instances, such as in AML models, the ketogenic diet alone promoted disease progression, suggesting that this diet may be harmful to some cancer patients when used independently.

[0128] The data presented herein demonstrate that insulin feedback limits the effects of PI3K inhibition in a variety of hematological malignancies and solid tumors. By reducing systemic insulin response, the addition of BKM120 to a ketogenic diet reduced immunohistochemical markers of insulin signaling in PTEN / PIK3CA mutant endometrial PDX tumors compared with tumors from mice treated with BKM120 alone. In these exemplary tumors, the ketogenic diet enhanced the ability of BKM120 to reduce levels of phosphorylated insulin receptor, phosphorylated AKT, and phosphorylated S6. This reduction in signaling correlated with decreased levels of proliferation, as indicated by Ki67 staining, and increased levels of apoptosis, as indicated by cleaved caspase 3 staining (Figure 4D-E).

[0129] Although these data do not exclude insulin-independent effects of combining PI3K inhibition with antihyperglycemic therapy, they do indicate that this modulation of glucose metabolism significantly enhances the therapeutic efficacy of these pathway inhibitors. In light of these results, it may also be important to consider how common clinical practices, such as IV glucose administration, the use of glucocorticoids, or providing patients with glucose-containing nutritional supplements, may affect treatment response. Therapeutics targeting this important oncogenic pathway should be combined with strategies such as modulators of glucose metabolism or the administration of a ketogenic diet to limit this self-destructive systemic feedback.

[0130] Example 6: Various approaches to targeting glucose / insulin feedback This example demonstrates the in vivo impact of multiple approaches that simultaneously target glucose / insulin feedback.

[0131] Figures 12A-12C graphically depict blood glucose, ketone, and c-peptide levels in wild-type C57 / BL6 mice bearing syngeneic K8484 KPC allograft tumors after treatment with a single dose of BKM120 along with metformin pretreatment, SGLT2 inhibitor (SGLT2i) pretreatment, a ketogenic diet alone, or a combination of such treatments (N=4 / arm). These data demonstrate that combining these approaches can have additive effects on glucose / insulin feedback control and can augment treatment efficacy by significantly enhancing the systemic metabolic response to PI3K inhibition.

[0132] Example 7: Methods Obtaining and treating mice All animal studies were conducted in accordance with IACUC-approved animal protocols at Weill Cornell Medicine (#2013-0116) and Columbia University (AC-AAAQ5405). Mice were maintained in specific pathogen-free, temperature- and humidity-controlled conditions under a 12-hour light / dark cycle and fed either regular chow (PicoLab Rodent 20 5053 lab Diet St. Louis, MO) or a ketogenic diet (Thermo-Fisher AIN-76A) with free access to drinking water. Diets were structured as shown in Table 3.

[0133] Table 3: Ketogenic diet compared to regular diet TIFF2025160313000032.tif42135

[0134] For solid tumor studies, 8-week-old nude and C57 / BL6 mice were purchased from Jackson Laboratories (Bar Harbor, ME) and were inoculated with 0.5–1 × 10 cells in a 1:1 mixture of growth medium and Matrigel (Trevigen, #3433-005-R1). 6cells were injected and tumors were allowed to grow to a minimum diameter of 0.6 cm before treatment began. Tumors that did not meet this criterion at the time treatment began were not used in the experiment.

[0135] For AML studies, 10-12 week-old male C57BL / 6J mice were used for MLL-AF9 Ds-Red AML studies (approved protocol AC-AAAQ5405). For pretreatment studies with MLL-AF9 Ds-Red cells, mice in the keto and keto / BKM groups were infused with MLL-AF9 Ds-Red cells (2 × 10 per mouse in 200 μl) via the lateral tail vein. 5 Mice were placed on a ketogenic diet for 10 days prior to the injection of 100 mg / kg of 10 ...

[0136] For parallel treatment studies with MLL-AF9 Ds-Red cells, mice were infused with MLL-AF9 Ds-Red cells (2 × 10 per mouse in 200 μl) via a lateral tail vein. 5 Mice were injected with 100 mg / kg of BKM120 (37.5 mg / kg) of IV IgG. Starting the day after the iv injection, mice were given vehicle or BKM120 (37.5 mg / kg) by oral gavage for two weeks. The keto or keto / BKM groups were switched to a ketogenic diet on the same day. The mice were euthanized after two weeks of treatment to examine bone marrow for AML progression.

[0137] To test whether keto / BKM treatment affected AML engraftment, mice in the keto and keto / BKM groups were fed a ketogenic diet for 10 days and then treated with vehicle or BKM120 by oral gavage for 2 weeks. MLL-AF9 Ds-Red cells (2 x 10 per mouse in 200 μl) were then injected into the mice via a lateral tail vein. 5Two weeks after iv injection, the mice were euthanized for bone marrow examination for AML burden.

[0138] Survival, pretreatment, and parallel studies were conducted simultaneously. Mice were treated with vehicle or BKM120 until natural death (5 out of 7 days) or euthanized if they appeared severely ill (reduced spontaneous activity, surface disorganization, and apparent dehydration), reached a weight loss of more than 20%, or showed symptoms of limb paralysis. To examine whether keto / BKM treatment affected bone marrow cell populations, C57BL / 6J mice were treated with eight doses of vehicle or BKM120 over a 9-day period. The mice were euthanized, and one femur and one tibia were removed from each mouse. Bone marrow cells were flushed with PBS (2% FBS). Red blood cells were lysed with ACK lysis buffer (Invitrogen).

[0139] Antibodies used for flow cytometry were CD34 (RAM34) from eBioscience, c-Kit (2B8), Sca-1 (D7), CD3ε (145-2C11), B220 (RA3-6B2), CD150 (TC-15-12F2.2), CD49b (DX5), and CD48 (HM48-1) from Biolegend. The "lineage cocktail" included CD3, CD4, Gr-1, Mac-1 (CD11b), B220, and Terr-119. DAPI was used to exclude dead cells.

[0140] compound GDC-0032, MK2206, BEZ235, BKM-120, GDC-0941, GDC-0980, and canagliflozin were all obtained from MedChem Express (Monmouth Junction, NJ) and administered via 100 μl oral gavage. Metformin was obtained from Sigma Aldrich (St. Louis, MO). Bay-80 6946 and OSI-906 were obtained from Selleck Chem colorimeters S2802 and S1091, respectively. Target information for these compounds is shown in Table 4. IC50 data were obtained from the Selleck Chem website (Selleckchem.com). Canagliflozin was administered 60 minutes before the PI3K pathway inhibitor to ensure its maximal effect coincided with peak glucose levels. Metformin-treated mice were pretreated for 10 days before BKM120 treatment. The ketogenic diet was initiated at the time of initial PI3K inhibitor treatment unless otherwise stated. Doxycycline was obtained from Sigma (St. Louis, Missouri) catalog number D3072-1ML and was administered via intraperitoneal injection once daily at a dose of 3 mg / kg.

[0141] Table 4: Exemplary pathway inhibitors TIFF2025160313000033.tif105134

[0142] cell line Mouse pancreatic cell lines were a kind gift from Dr. Kenneth Olive of Columbia University. Mouse mammary lines were a kind gift from Dr. Ramon Parsons of Mount Sinai School of Medicine. PDX models were generated by the Englander Institute of Precision Medicine. Cell lines HEK293, HCC-38, MDA-MB-468, PC-3, and BT-549 were purchased from ATCC and grown in DMEM supplemented with 10% FBS and 1% Pen / Strep. HCT-116 and DLD-1 isogenic lines with or without PTEN deletion were kindly provided by the Laboratory of Todd Waldman. A list of cells / organoids used is provided in Table 5, along with known oncogenic modifications described in the publications cited above or available from the ATCC (see website atcc.org / ~ / media / PDFs / Culture%20Guides / Cell_Lines_by_Gene_Mutation.ashx).

[0143] Table 5: Exemplary cell lines TIFF2025160313000034.tif234135

[0144] Signaling assays For signaling assays, cells were washed once in PBS, plated in starvation medium (-FBS) for 6–18 h, depending on the cell line, and treated with PI3K inhibitors alone or in combination with insulin 10 min prior to harvest, as indicated, 1 h prior to harvest. Three-dimensional culture and dose-response experiments of patient-derived organoids were performed as previously described. Briefly, approximately 1,000 cells were plated in 10 μl of 1:1 Matrigel:culture medium in a 96-well angiogenesis plate and allowed to solidify for 30 min at 37°C before adding 70 μl of culture medium. Organoids were then treated in triplicate for a logarithmic dose response, and the CellTiter-Glo assay (Promega) was performed for 96 h to determine IC. 50Values ​​were determined. Proliferation assays in two-dimensional cultures were performed as described in the figure legends. Knockdown of the insulin receptor was achieved using a doxycycline-inducible shRNA strategy. To generate miR-E shRNAs, 97-mer oligonucleotides encoding shRNAs predicted using the siRNA prediction tool Splash RNA (see the website splashrna.mskcc.org / ) were purchased (IDT Ultramers).

[0145] The oligonucleotides are TIFF2025160313000035.tif24128. The PCR product was purified, and both the PCR product and the LT3GEPIR vector (Fellmann, C. et al. An optimized microRNA backbone for effective single-copy RNAi. Cell Rep 5, 1704-1713) were double-digested with EcoRI-HF and XhoHI. The PCR product and vector backbone were ligated, transformed into Stbl3 competent cells, and grown overnight at 32°C. Colonies were identified using the primers Screening was performed using TIFF2025160313000036.tif4128. TIFF2025160313000037.tif50128

[0146] Immunoblotting Cell lysates were prepared in 1x CST Cell Lysis Buffer #9803 (Danvers, MA). Total protein concentration was assessed using a BCA kit (Pierce) 23227. The lysates were run on 4-20% Tris-Glycine gels (ThermoFisher, Carlsbad, CA). Primary antibodies against pAKT473, pAKT308, pS6, pTYR, AKT, and S6 were obtained from Cell Signaling (Danvers, MA) and used overnight at 1:1000 in 5% bovine serum albumin. Actin and tubulin antibodies were obtained from Sigma-Aldrich and used at 1:5,000 in 5% milk. All of these antibodies were visualized with HRP-conjugated secondary antibodies from Jackson Immuno at 1:5000 in 5% milk.

[0147] immunohistochemistry Tumor sections (3 μm) were subjected to antigen retrieval using 10 mmol / L citric acid, 0.05% Tween 20, pH 6.0, and incubated with the indicated antibodies: Ki67 (Abcam, ab16667) 1:500; cleaved caspase 3 (Asp175; 5A1E; Cell Signaling Technology, 9664) 1:200; phospho-INSR (Tyr 1162; Thermo Fisher #AHR0271) 1:100; phospho-AKT (Ser473; Cell Signaling Technology, 8101) 1:20; and phospho-S6 ribosomal protein (Ser235 / 236; Cell Signaling Technology, 2211) 1:300.

[0148] Blood measurements For blood glucose assessment, 10 μl of blood was collected from the tail of mice using a OneTouch Ultra Glucometer before treatment (time 0) and again at the indicated time points (15, 30, 60, 90, 120, and 180 min). At the endpoint, >100 μl of blood was drawn from the mice into EDTA tubes (Sarstedt #16.444). Blood was centrifuged (10,000 × g, 10 min, 4°C), and plasma was stored at -20°C. Plasma β-hydroxybutyrate, triglycerides (Stanbio Laboratory, Boerne, TX), and serum insulin and c-peptide (APLCO Diagnostics, Salem, NH) levels were quantified by ELISA.

[0149] FDG-PET Male C57 / BL6 mice (n = 4 / arm) bearing orthotopic pancreatic adenocarcinoma allografts were injected with 200–250 μCi [ 89 [Zr] liposomes (3-4 μmol lipid) were injected. At the time of peak blood insulin feedback, 90 min after BKM120 injection, animals were anesthetized and then scanned using an Inveon PET / CT scanner (Siemens Healthcare Global). Whole-body PET scans recording a minimum of 50 million coincidence events were performed over a 10-min period. The energy and coincidence timing window was 350-750 keV and 6 ns. The data were normalized to correct for PET response non-uniformity, dead-time omissions, positron branching ratios, and physical attenuation up to the time of injection. Count rates in reconstructed images were calculated using the following formula: 89 Radioactivity concentrations were converted to percent injected dose per gram of tissue (%ID) using system calibration coefficients derived from images of the Zr-containing phantom. Images were analyzed using ASIPro VM™ software (Concorde Microsystems). Radioactivity concentrations were quantified by averaging the maximum values ​​in at least five regions of interest (ROIs) on adjacent pancreatic tumor sections.

[0150] Metabolomics Metabolites were extracted from cells or tissues using 80% methanol. Each sample was transferred to a pre-chilled (dry ice) 2 mL homogenization tube containing one stainless steel bead (5 mm). Pre-chilled 80% methanol (1 mL) was added to each sample, and homogenization was performed using a Qiagen TissueLyser II. The samples were then centrifuged at 14,000 rpm for 15 minutes at 4°C. The supernatant was extracted and normalized based on tissue weight. Targeted LC / MS analysis was performed on a Q Exactive Orbitrap mass spectrometer (Thermo Fisher) connected to a Vanquish UPLC system (Thermo Scientific). The Q Exactive was operated in polarity-switching mode. A Sequant ZIC-HILIC column (2.1 mm id × 150 mm, Merck) was used for metabolite separation. The flow rate was 150 μL / min. Buffers consisted of 100% acetonitrile for A and 0.1% NHOH / 20 mM CHCOONH in water for B. The gradient was 85% to 30% A over 20 min, followed by a wash at 30% A and re-equilibration at 85% A. Metabolites were identified based on accurate mass within 5 ppm and standard retention time. Relative metabolite quantification was performed based on the peak area of ​​each metabolite. All data analysis was performed using in-house written scripts.

[0151] References: TIFF2025160313000038.tif57135TIFF2025160313000039.tif204134TIFF2025160313000040.tif204135TIFF2025160313000041.tif191135

[0152] All patents and publications referenced or mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each such referenced patent or publication is individually incorporated by reference herein in its entirety or to the same extent as if fully set forth herein. Applicant reserves the right to physically incorporate into this specification any and all materials and information from any such cited patent or publication.

[0153] The following presentation is intended to describe and summarize various aspects of the present invention in accordance with the detailed description set forth hereinabove.

[0154] Presentation: 1. administering to a subject in need thereof an effective amount of a regulator of glucose metabolism, and administering to the subject an effective amount of a pathway inhibitor of the insulin receptor / PI3K / AKT / mTOR pathway. 10. A method for treating a disease or disorder associated with PI3K signaling, comprising: 2. The method of claim 1, wherein the modulator of glucose metabolism is a glucose uptake inhibitor optionally selected from the group consisting of a sodium-glucose cotransporter protein 1 (SGLT1) inhibitor, a sodium-glucose cotransporter protein 2 (SGLT2) inhibitor, or an SGLT1 / SGLT2 dual inhibitor. 3. The method of claim 2, wherein the glucose uptake inhibitor is selected from the group consisting of dapagliflozin, empagliflozin, canagliflozin, ipragliflozin, tofogliflozin, sergliflozin etabonate, remogliflozin etabonate, ertugliflozin, sotagliflozin, and conagliflozin. 4. The method of claim 1, wherein said regulator of glucose metabolism is metformin. 5. The method of claim 1, wherein said regulator of glucose metabolism is an insulin receptor / IGF1 receptor inhibitor, and optionally, said insulin receptor / IGF1 receptor inhibitor is linsitinib (OSI-906). 6. The method of any of presentations 1-5, wherein said pathway inhibitor is capable of inhibiting one or more kinases selected from the group consisting of INSR / IGFR, PI3K, AKT, and mTOR. 7. The method of claim 6, wherein the pathway inhibitor is selected from the group consisting of idelalisib, copanlisib, buparlisib (BKM120), alpelisib (BYL719), taselisib (GDC-0032), pictilisib (GDC-0941), apitolisib (GDC-0980), selavelisib (TAK-117), dactolisib, apelisib, MK2206, and linsitinib (OSI-906). 8. The method of any of claims 1 to 7, wherein said disease or disorder associated with PI3K signaling is cancer or a cell proliferation disorder, a metabolic disorder, a neurodegenerative disease, or an inflammatory disease. 9. The disease or disorder associated with PI3K signaling is a neurodegenerative disease, optionally brain trauma, spinal cord trauma, trauma to the peripheral nervous system, Alzheimer's disease, Pick's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steele-Richardson syndrome), multiple system degeneration (Shy-Drager syndrome), motor neuron disease including amyotrophic lateral sclerosis, ataxia, corticobasal degeneration, ALS-Parkinson-Dementia Complex of Guam, subacute sclerosing panencephalitis, Huntington's disease, Disease, Parkinson's disease, Synucleinopathies, Primary Progressive Aphasia, Striatonigral Degeneration, Machado-Joseph Disease / Spinocerebellar Degeneration Type 3 and Olivopontocerebellar Degeneration, Gilles de la Tourette's Disease, Bulbar and Pseudobulbar Palsy, Spinal and Spinobulbar Muscular Atrophy (Kennedy's Disease), Primary Lateral Sclerosis, Familial Spastic Paraplegia, Werdnig-Hoffmann Disease, Kugelberg-Welander Disease, Tay-Sachs Disease, Sandhoff Disease, Familial Spastic Disorders, Wolfahrt-Kugelbe Disease Lug-Welander disease, spastic paraplegia, progressive multifocal leukoencephalopathy, and prion diseases (including Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, kuru, and fatal familial insomnia), age-related dementia, vascular dementia, diffuse white matter disease (Binswanger disease), dementia of endocrine or metabolic origin, dementia of head trauma and diffuse brain injury, dementia pugilistica or frontal lobe dementia, cerebral ischemia or infarction, including embolic and thrombotic occlusion Any of the methods presented in 1 to 8 is a neurodegenerative disorder resulting from any type of intracranial hemorrhage, intracranial and intraspinal lesions, hereditary cerebral angiopathy, hereditary amyloid, Down syndrome, macroglobulinemia, secondary familial Mediterranean fever, Muckle-Wells syndrome, multiple myeloma, pancreas-associated amyloidosis, cardiac-associated amyloidosis, chronic dialysis hemoarthropathy, Finnish-type amyloidosis, Iowa-type amyloidosis, or a combination thereof. 10. The method of any of claims 1 to 8, wherein said disease or disorder associated with PI3K signaling is an inflammatory disorder, optionally type II diabetes, insulin-resistant cardiovascular disease, arrhythmia, atherosclerosis, coronary artery disease, hypertriglyceridemia, dyslipidemia, retinopathy, nephropathy, neuropathy, obesity and macular edema, ileitis, ulcerative colitis, Barrett's syndrome or Crohn's disease. 11. Any of the methods of claims 1-8, wherein the disease or disorder associated with PI3K signaling is a metabolic disease, optionally type II diabetes, insulin-resistant cardiovascular disease, arrhythmia, atherosclerosis, coronary artery disease, hypertriglyceridemia, dyslipidemia, retinopathy, nephropathy, neuropathy, obesity, or macular edema. 12. The method of any of claims 1-10 or 11, wherein the subject consumes or is provided with a ketogenic diet during treatment. 13. Administering an effective amount of at least one pathway inhibitor of at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway, wherein the subject follows or is provided with a ketogenic diet during treatment. 10. A method for treating a disease or disorder associated with PI3K signaling, comprising: 14. Any of the above-listed methods that disrupt whole-body glucose homeostasis and improve the effect of pathway inhibitor treatment compared to the pathway inhibitor alone. 15. Use of a regulator of glucose metabolism and / or an inhibitor of the insulin receptor / PI3K / AKT / mTOR pathway to treat a disease or disorder associated with PI3K signaling in a subject. 16. The use of presentation 15 in combination with the use of a ketogenic diet by said subject. 17. A pharmaceutical composition comprising a regulator of glucose metabolism and a pathway inhibitor that inhibits at least one kinase in the insulin receptor / PI3K / AKT / mTOR pathway. 18. The pharmaceutical composition of claim 17, wherein the modulator of glucose metabolism is a glucose uptake inhibitor, a sodium-glucose cotransporter protein 1 (SGLT1) inhibitor, a sodium-glucose cotransporter protein 2 (SGLT2) inhibitor, or a dual SGLT1 / SGLT2 inhibitor. 19. The pharmaceutical composition of claim 17 or 18, wherein the glucose uptake inhibitor is selected from the group consisting of dapagliflozin, empagliflozin, canagliflozin, ipragliflozin, tofogliflozin, sergliflozin etabonate, remogliflozin etabonate, ertugliflozin, sotagliflozin, and conagliflozin. 20. The pharmaceutical composition of claim 17, 18, or 19, wherein said regulator of glucose metabolism is metformin. 21. The pharmaceutical composition of claims 17-19 or 20, wherein said regulator of glucose metabolism is an insulin receptor / IGF1 receptor inhibitor, and optionally, said insulin receptor / IGF1 receptor inhibitor is linsitinib (OSI-906). 22. The pharmaceutical composition of any of claims 17-20 or 21, wherein the pathway inhibitor is capable of inhibiting one or more kinases selected from the group consisting of INSR / IGFR, PI3K, AKT, and mTOR. 23. The pharmaceutical composition of claims 17-21 or 22, wherein the pathway inhibitor is selected from the group consisting of idelalisib, copanlisib, buparlisib (BKM120), alpelisib (BYL719), taselisib (GDC-0032), pictilisib (GDC-0941), apitolisib (GDC-0980), selavelisib (TAK-117), dactolisib, apelisib, MK2206, and linsitinib (OSI-906). 24. Administering an effective amount of a glucose uptake inhibitor to a subject in need thereof, and administering an effective amount of a PI3K inhibitor to the subject. 20. A method for inhibiting cell proliferation or suppressing a cell proliferative disorder, comprising: 25. The method of claim 24, wherein the glucose uptake inhibitor is selected from the group consisting of dapagliflozin, empagliflozin, canagliflozin, ipragliflozin, tofogliflozin, sergliflozin etabonate, remogliflozin etabonate, ertugliflozin, sotagliflozin, and conagliflozin. 26. The method of claim 24 or 25, wherein the PI3K inhibitor is selected from the group consisting of idelalisib, copanlisib, buparlisib (BKM120), alpelisib (BYL719), taselisib (GDC-0032), pictilisib (GDC-0941), apitolisib (GDC-0980), selavelisib (TAK-117), dactolisib, MK2206, linsitinib (OSI-906), and apelisib. 27. Any of the methods of claims 24-25 or 26, wherein the inhibition of cell proliferation or suppression of a cell proliferative disorder is enhanced compared to administration of a PI3K inhibitor without the glucose uptake inhibitor.

[0155] The specific compositions and methods described herein are representative and illustrative and are not intended to be limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon reading this specification, and are encompassed within the spirit of the invention as defined by the claims. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The terms and expressions used are used in a descriptive rather than a limiting sense, and the use of such terms and expressions is not intended to exclude equivalents of the features shown and described or portions thereof, but it is understood that various modifications are possible within the scope of the invention as defined by the claims. Thus, while the invention has been specifically disclosed by embodiments and optional features, it will be understood that modifications and derivations of the technical concepts disclosed herein may be recognized by those skilled in the art, and that such modifications and derivations are considered to be within the scope of the invention as defined by the appended claims and the present invention.

[0156] The inventions illustratively described herein may be practiced in the absence of any element or elements or limitation or limitations not specifically disclosed herein as essential. The methods and processes illustratively described herein may be practiced in different orders of steps, and are not necessarily limited to the orders of steps set forth herein or in the claims.

[0157] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" or "a drug" or "an inhibitor" includes a plurality of such compounds or drugs or inhibitors, etc. As used herein, the term "or" is not exclusive; unless otherwise indicated, "A or B" includes "A but not B," "B but not A," and "A and B."

[0158] This patent should not be construed under any circumstances as being limited to the specific examples or aspects or methods thereof specifically disclosed herein. This patent should not be construed under any circumstances as being limited by any reference made by the examiner or other employee or employee of the Patent and Trademark Office unless expressly acknowledged separately and without condition or reservation in a written response by the applicant.

[0159] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings encompassed by this generic disclosure also form part of the invention. This includes generic descriptions of the invention with provisos or negative limitations removing any object from that class, regardless of whether the carved-out object is specifically set forth herein. In addition, when features or aspects of the invention are described in terms of Markush groups, those skilled in the art will understand that the invention is also described in terms of any individual member or subgroup of members of that Markush group.

[0160] The Abstract is provided to comply with 37 CFR §1.72(b) to allow the reader to quickly ascertain the nature and gist of the technical disclosure. The Abstract is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the invention.

[0161] Sequence information SEQUENCE LISTING <110> Cornell University THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK <120> COMBINATION THERAPY FOR PI3K-ASSOCIATED DISEASE OR DISORDER <150> US 62 / 679,329 <151> 2018-06-01 <160> 5 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide primer <400> 1 tgaactcgag aaggtatatt gctgttgaca gtgagcg 37 <210> 2 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide primer <400> 2 tgaactcgag aaggtatatt gctgttgaca gtgagcg 37 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide primer <400> 3 tgtttgaatg aggcttcagt ac 22 <210> 4 <211> 97 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide primer <400> 4 tgctgttgac agtgagcgca ggaattataa tgcttatcta tagtgaagcc acagatgtat 60 agataagcat tataattcct atgcctactg cctcgga 97 <210> 5 <211> 97 <212> DNA <213> Artificial Sequence <220> <223> A synthetic oligonucleotide primer <400> 5 tgctgttgac agtgagcgcg gggttcatgc tgttctacaa tagtgaagcc acagatgtat 60 tgtagaacag catgaacccc atgcctactg cctcgga 97

Claims

1. 1. A pharmaceutical composition for use in a method for improving the efficacy of a PI3K inhibitor in treating a patient having cancer, the cancer being pancreatic cancer, breast cancer, bladder cancer, leukemia, lung cancer, liver cancer, ovarian cancer, cervical cancer, uterine cancer, colon cancer, prostate cancer, glioma, melanoma, or renal cell cancer, wherein the pharmaceutical composition comprises a PI3K inhibitor that inhibits PI3K alpha; The method comprises administering to the patient the PI3K inhibitor in combination with a ketogenic diet that reduces serum insulin levels, thereby improving the efficacy of the PI3K inhibitor.

2. The pharmaceutical composition of claim 1, wherein the PI3K inhibitor further inhibits one or more of p110-β, p110-γ, p110-δ, p85-α, p85-β, p55-γ, p150, p101, p87, PI3K-C2α, PI3K-C2β, PI3K-C2γ, and Vps34.

3. The pharmaceutical composition of claim 1 , wherein the PI3K inhibitor selectively inhibits PI3K α.

4. 10. The pharmaceutical composition of claim 1, wherein the method further comprises administering at least one regulator of glucose metabolism.

5. 5. The pharmaceutical composition of claim 4, wherein the at least one regulator of glucose metabolism is a sodium-glucose cotransporter 1 (SGLT1) inhibitor, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, or a dual SGLT1 / SGLT2 inhibitor.

6. 5. The pharmaceutical composition of claim 4, wherein the at least one modulator of glucose metabolism is dapagliflozin, empagliflozin, canagliflozin, ipragliflozin, tofogliflozin, sergliflozin etabonate, remogliflozin etabonate, ertugliflozin, or sotagliflozin.

7. 5. The pharmaceutical composition of claim 4, wherein the at least one regulator of glucose metabolism is a sodium glucose cotransporter protein 2 (SGLT2) inhibitor.

8. 5. The pharmaceutical composition of claim 4, wherein the at least one regulator of glucose metabolism is metformin.

9. 10. The pharmaceutical composition of claim 1, wherein the patient is on the ketogenic diet before and during administration of the PI3K inhibitor.

10. 10. The pharmaceutical composition of claim 1, wherein the patient is on the ketogenic diet prior to administration of the PI3K inhibitor.

11. 10. The pharmaceutical composition of claim 1, wherein the patient is on the ketogenic diet during administration of the PI3K inhibitor.

12. 10. The pharmaceutical composition of claim 1, wherein the ketogenic diet comprises a ratio of about 2.5:1 to about 3:1 grams of fat to grams of carbohydrates and protein combined.

13. 10. The pharmaceutical composition of claim 1, wherein the ketogenic diet comprises up to 10% carbohydrates.

14. 10. The pharmaceutical composition of claim 1, wherein the ketogenic diet comprises about 2% to about 5% carbohydrates.

15. 10. The pharmaceutical composition of claim 1, wherein the ketogenic diet comprises about 90% calories from fat and about 10% calories from a combination of carbohydrates and protein.

16. The pharmaceutical composition of claim 1, wherein the cancer is lung cancer.

17. The pharmaceutical composition of claim 1, wherein the cancer is liver cancer.

18. 2. The pharmaceutical composition of claim 1, wherein the cancer is pancreatic cancer.

19. The pharmaceutical composition of claim 1, wherein the uterine cancer is endometrial cancer.

20. The pharmaceutical composition of claim 1, wherein the cancer is bladder cancer.

21. 10. The pharmaceutical composition of claim 1, wherein the cancer is leukemia.

22. The pharmaceutical composition of claim 1, wherein the cancer is breast cancer.

23. 2. The pharmaceutical composition of claim 1, wherein the cancer is ovarian cancer, cervical cancer, uterine cancer, colon cancer, or prostate cancer.

24. 10. The pharmaceutical composition of claim 1, wherein the cancer is glioma, melanoma, or renal cell carcinoma.

25. The pharmaceutical composition of claim 1, wherein the cancer comprises one or more of a PIK3CA mutation and a PIK3R1 mutation.

26. The pharmaceutical composition of claim 1, wherein the cancer comprises a PTEN alteration.

27. 10. The pharmaceutical composition of claim 1, wherein the cancer comprises a PTEN deletion.

28. The pharmaceutical composition of claim 1, wherein the method further comprises administering an mTOR inhibitor.

29. The pharmaceutical composition of claim 1, wherein improving the efficacy of the PI3K inhibitor comprises reducing the level of PI3K signaling.

30. The pharmaceutical composition of claim 1, wherein improving the efficacy of the PI3K inhibitor comprises one or more of reducing the level of phosphorylated AKT (pAKT) and reducing the level of phosphorylated S6 (pS6).

31. The PI3K inhibitor has a PI3Kα IC 50 2. The pharmaceutical composition of claim 1, comprising:

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