Methods and products for screening and treating glioblastoma multiforme and other cancers, including breast cancer, using a combination of PI3 kinase inhibitors and checkpoint inhibitors

Abstract

The present invention recognizes that there is a long-standing need for methods of treating cancer. A first aspect of the invention generally relates to a method of screening for cancer treatments. A second aspect of the invention generally relates to a compound for treating cancer. A third aspect of the invention generally relates to a pharmaceutical composition for treating cancer. A fourth aspect of the invention generally relates to a method of treating cancer. A fifth aspect of the invention generally relates to a PI3K inhibitor in combination with a checkpoint inhibitor for treating cancer. A sixth aspect of the invention generally relates to a method of treating cancer in combination with a PI3K inhibitor and a checkpoint inhibitor or other immunotherapy. A seventh aspect of the invention generally relates to a method of treating glioblastoma, breast cancer, and / or triple-negative breast cancer in combination with a PI3K inhibitor and a checkpoint inhibitor or other immunotherapy.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of priority to U.S. Provisional Application No. 63 / 329,096, filed April 8, 2022, now expired, and to U.S. Provisional Application No. 63 / 368,499, filed July 15, 2022, now pending, each of which is incorporated by reference in its entirety herein.

[0002] The present invention relates generally to the field of cancer treatment. [Background technology]

[0003] Glioblastoma multiforme (GBM) is a severe and aggressive cancer occurring in the central nervous system with no reported cure. Unlike breast and lung cancers, survival rates have not improved significantly over the past 40 years. GBM can progress from pre-existing astrocytomas or develop de novo. Approximately 90% of GBMs arise as primary tumors in patients aged an average of 64 years, but they can occur at any age. Current treatments include resection, when possible, and combination chemotherapy, radiation therapy, steroid therapy, and virotherapy. Despite treatment, median survival is 9-22 months.

[0004] One of the most promising new strategies in the treatment of many cancers is the development of therapies that target the body's immune system or its components to fight the disease. Promising approaches in immunotherapy include those using drugs that target "immune checkpoint" molecules. For example, cancer-free survival rates for advanced melanoma patients treated with immune checkpoint inhibitors (ICIs) range from 30% to 40%. While this has been a positive outcome for some melanoma patients, the question remains as to why a large proportion of melanoma patients, approximately 60% to 70%, do not respond well to otherwise blockbuster ICI drugs. Similarly, the question remains as to why treatment of GBM patients with ICI drugs does not lead to improved outcomes compared to GBM patients treated with temozolomide (TMZ) or Avastin (TM), the currently used drugs for the treatment of GBM, despite the fact that ICI drugs are somewhat effective in treating certain cancers.

[0005] The phosphatidylinositol 3-kinase (PI3K) / AKT / mTOR pathway is a key intracellular signaling pathway involved in numerous biological processes, including proliferation, apoptosis, angiogenesis, and glucose metabolism. This pathway is initiated by the binding of extracellular growth factors to receptor tyrosine kinases (RTKs), which activates PI3K and subsequently serine / threonine protein kinase B (PKB or AKT), leading to phosphorylation of a range of downstream substrates that mediate cellular responses. Hyperactivation of the PI3K / Akt pathway is frequently seen in cancers, including glioblastoma and breast cancer, where this pathway plays a central role in controlling tumor cell survival, growth, angiogenesis, and metabolism. Numerous PI3K inhibitors, including pan-PI3K inhibitors, isoform-selective inhibitors, and dual PI3K / mTOR inhibitors, have shown favorable preclinical results and are in clinical trials for a variety of malignancies. There are currently several PI3K inhibitors in phase I / II clinical trials for breast cancer.

[0006] A central issue that has limited the use of PI3K inhibitors for breast cancer, glioblastoma multiforme, and diffuse intrinsic cavernous glioma (DIPG) is the potential toxicity of systemic administration of these agents. One potentially advantageous treatment approach would be to administer high concentrations of drugs locally only at the site of tumor tissue, avoiding toxicity in normal tissues. Many cancer researchers have proposed the concept of prodrugs, focusing on potential activators such as tumor-specific enzymes, light, and heat, but multiple problems have arisen. For example, enzymes are often distributed unevenly, difficult to access, and often not completely tumor-specific. It is difficult to precisely target heat or light deep into tissue structures, and blood flow can scatter and dissipate the energy deposition.

[0007] Recent studies on tumor cell expression of major histocompatibility complex class II (MHCII) molecules have revealed at least part of the reason why ICI drugs have not been effective in treating some melanoma patients and may also explain why ICI drugs have been ineffective in the treatment of GBM so far. In particular, recent studies have demonstrated that tumors in patients who respond to ICI therapy are those that express MHC class II molecules on the cell surface. Expression of MHCII on the cell surface is well known to provide the first (and probably necessary) signal for T cell activation (a key cellular event in antitumor immune responses) and, under appropriate conditions, can trigger cell death of MHCII-expressing cells. These MHCII-related studies provide important implications for improving the potential efficacy of ICI drugs in the treatment of various cancers and can generally support the hypothesis that MHC II expression on GBM cells may provide the missing element necessary to increase the frequency of patients who respond to ICI drugs via effective antitumor immunity, in combination with immune checkpoint blockade. In the paper "Melanoma-specific MHC-II expression represents a tumor-autonomous phenotype and predicts response to anti-PD-1 / PD-L1 therapy," the authors identified melanoma cell lines that expressed or could induce MHC II expression. They found that HLA-DR expression correlated with a good response to immune checkpoint therapy targeting PD-1. They also found a positive correlation between HLA-DR expression and the percentage of CD4+ T-cell infiltration. They hypothesized that CD4+ helper cells are involved in this process but did not identify any subsets of CD4+ cells, including the involvement of unwanted T regulatory cells that suppress antitumor T-cell responses.

[0008] MHC II-encoded molecules are usually present on professional antigen-presenting cells (APCs), such as macrophages, dendritic cells, and B cells, but many cells can be induced to express the molecule and may act as APCs. In humans, MHC class II molecules are known as human leukocyte antigen gene complexes (HLA Dr). In their grooves, MHC II molecules present antigens (Ag) to T cell receptors (TCRs) expressed on T lymphocytes. T cell activation also requires a second co-signal molecule, provided by the interaction of the presenting cell with the T cell. Some co-stimulatory signals activate T cells. Other co-stimulatory signals, likely mediated by the interaction of PD1 with PDL-1, appear to suppress conventional T cell activation, whereas proliferating CD4+ T cells are regulatory. Summary of the Invention [Means for solving the problem]

[0009] The present invention recognizes that there is a long-felt need for methods of treating cancer.

[0010] A first aspect of the invention relates generally to methods for screening for cancer therapeutics.

[0011] A second aspect of the invention relates generally to compounds for treating cancer.

[0012] A third aspect of the invention relates generally to pharmaceutical compositions for treating cancer.

[0013] A fourth aspect of the invention relates generally to methods of treating cancer.

[0014] A fifth aspect of the invention relates generally to pharmaceutical compositions comprising a PI3K inhibitor in combination with a checkpoint inhibitor or other immunotherapeutic agent.

[0015] A sixth aspect of the invention relates generally to methods of treating cancer using PI3K inhibitors in combination with checkpoint inhibitors or other immunotherapies.

[0016] A seventh aspect of the invention relates generally to methods of treating glioblastoma, breast cancer, and / or triple-negative breast cancer using a PI3K inhibitor in combination with a checkpoint inhibitor or other immunotherapy. [Brief description of the drawings]

[0017] [Figure 1] Gl261 mouse GBM cells were cultured in the presence of GCT.GLIO.1 at the indicated concentrations for 24, 48, and 72 hours, after which cells were harvested and counted. [Diagram 2] GL261 cells were cultured with or without GCT.GLIO.1 for 72 hours, then harvested and stained with anti-PD-L1. The histograms show increased cell surface expression of PD-L1 corresponding to increasing doses of GCT.GLIO.1, indicating that increased PD-L1 can be targeted by immune checkpoint inhibitor therapy. [Diagram 3] GL261 cells were cultured with or without GCT.GLIO.1 at 1 for 7 days as indicated, after which cells were harvested and stained for changes in cell surface expression of PD-L1 and MHC class II, or expression of LC3, a marker of autophagy, as indicated. Increases in MHC class II and PD-L1 increase the likelihood that the agent will be recognized by CD4 T cells, suggesting that increased PD-L1 may be a target for immune checkpoint inhibitor therapy. [Figure 4] A and B show the effect of GCT.GLIO.1 on the radiosensitivity and cell cycle arrest of GL261. In the next experiments, we sought to identify the optimal radiation dose after treatment with GCT.Glio.1. In these studies, increasing doses of radiation were administered after 24 h of GCT.Glio.1: 2 Gy, 4 Gy, and 8 Gy. As a result, retreatment with GCT.Glio.1 significantly amplified the response to radiation and caused a significant increase in cells arrested at the G2 cell cycle checkpoint (see Figures 5-6). [Diagram 5]Panels A and B show human GBM cell line U251 treated with GCT.Glio.1, harvested, counted, and stained for changes in cell surface PD-L1 (left panel) and MHC class II (right panel). The results showed that GCT.Glio.1 induced growth arrest and increased cell surface PD-L1 and MHC class II (expressed as mean fluorescence intensity (MFI)), potentially making these cells better targets for immunotherapy. [Figure 6] In A and B, human U251 cell line was treated with GCT.Glio.1, harvested, counted, and assessed for changes in cell number and cell death. The results show that GCT.Glio.1 induced growth arrest, which increased with increasing doses of compound in the left panel, and with increasing cell death in the right panel. [Figure 7] Figure 1 shows cell viability of human MCF7 breast cancer cells. Human MCF7 cells were treated with 1 uM apelisib (Alp), 1 uM GCT.BC.1 (GCT), or DMSO control for 48 hours. Viability was determined by cleaved caspase-7 and measured using the Caspase Glo assay (Promega) as described by the manufacturer. [Figure 8] Figure 1 shows cell viability of murine TC11 breast cancer cells. TC11 cells were treated with 100 nM paclitaxel (Pac), 100 nM paclitaxel + 1 uM alpelisib (Alp), 0.50 uM GCT.BC.1 (GCT), 0.50 uM GCT.Glio.1 + 100 nM paclitaxel, 0.50 uM GCT.BC.1 + 7.5 nM fulvestrant, or DMSO control for 48 hours as indicated. Viability was determined by cleaved caspase-7 and measured using the Caspase Glo assay (Promega) as described by the manufacturer. [Figure 9] Figure 1 shows proliferation of 66CL4 mouse breast cancer cells. 66CL4 breast cancer cells in mice were treated with the indicated doses of GCT.BC.01. Proliferation was measured using an Agilent Biotek Biospa system and Biospa software. [Figure 10]Luciferase-labeled GL261 tumor cells were implanted into the caudate nucleus of C57Bl6 mice on day 0. Treatment began on day 9. Treatment included no treatment, oral GCT alone, anti-PD-1 + DMSO vehicle, or oral GCT followed by checkpoint inhibitor anti-PD1 24 hours later as indicated. Tumor growth was monitored by MRI. Kaplan-Meier survival curves. Black lines indicate survival time of animals implanted with GL261, red lines indicate survival time of animals treated with GCT.1. Blue lines indicate animals cycled with GCT.1 followed by anti-PD1 24 hours later. As of day 28, three animals in the combination group are still alive. [Figure 11] Luciferase-labeled GL261 tumor cells were implanted into the caudate nucleus of C57B16 mice on day 0. Treatment began on day 9. Treatment included no treatment, oral GCT alone, anti-PD-1 + DMSO vehicle, or oral GCT followed by checkpoint inhibitor anti-PD-1 24 hours later as indicated. GL261 tumors were measured using bioluminescence and observed daily. In the top panel, bioluminescence quantification shows that tumor growth was significantly delayed when animals were treated with the checkpoint inhibitor anti-PD-1 after GCT.1 administration. [Figure 12A] A, B, and C show the structures of various compounds mentioned in the examples. (R)-NPT520-232 refers to GCT.2 (GCT.Glio.2), (-)-NPT520-337 refers to GCT.1 (GCT.Glio.1), and (-)-NPT520-338 refers to GCT.Glio.3. [Figure 13] A, B, and C. SEMA7A-overexpressing (OE) cells are poorly responsive to endocrine therapy but become sensitive upon addition of a Pl3K inhibitor. MCF7EV and SEMA7A OE cells were treated with (A) fulvestrant (7.5 nM), (B) alpelisib (1 μM), or (C) their combination for 48 h. [Figure 14]In A, B, C, D, and E, cells expressing SEMA7A are responsive to both alpelisib and a novel Pl3K inhibitor (GCT.BR.1). (A) MCF7 cells treated with vehicle, 1 μM alpelisib, and 1 μM GCT.BR.1. Scale bar = 10 microns. (B) Quantification of A. (C) MCF7 S7 OE spheroids in suspension were treated with vehicle (DMSO), 1 uM alpelisib, or 0.5 uM GCT and stained with calcein green viability dye 48 hours later. (D) Tumor burden in MCF7 SEMA7 A OE-bearing NCG mice treated with fulvestrant (arrows) every 5 days starting on day 30. (E) Size-matched graph. [Figure 15] SK-BR-3 is a human breast cancer cell line that overexpresses the Her2 (Neu / ErbB-2) gene product. These cells display epithelial morphology in tissue culture and can form poorly differentiated tumors in immunodeficient mice. MDA-MB-231 is a highly aggressive, invasive, and poorly differentiated triple-negative breast cancer (TNBC) cell line that lacks expression of estrogen receptor (ER) and progesterone receptor (PR) as well as amplification of HER2 (human epidermal growth factor receptor 2). [Figure 16] SKBR3 cells treated with GCT.BR.1 at the indicated doses. Histogram showing changes in HER2 expression analyzed by flow cytometry. Treatment with GCT007 dose-dependently increased HER2 expression in triple-negative SKRB3 breast cancer cells. [Figure 17] 231 cells treated with GCT.BR.1 (GCT007) at the indicated doses. Histogram showing changes in HER2 expression analyzed by flow cytometry. Treatment with GCT007 increased HER2 expression in breast cancer 231 cells in a dose-dependent manner. [Figure 18] Quantification of median fluorescence intensity of HER-2 expression in 231 cells is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description of the Invention definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In general, the nomenclature used herein and the experimental procedures in cell culture, chemistry, microbiology, molecular biology, cell science, flow cytometry, cell culture described below are well known and commonly employed in the art. Conventional methods are used for these medical procedures, such as those generally known in the art and as provided in the art and various general references. When a term is described in the singular form, the inventors also contemplate the plural. The nomenclature used herein and the experimental procedures described below are well known and commonly employed in the art. As used throughout this disclosure, the following terms shall be understood to have the following meanings, unless otherwise indicated:

[0019] "Directly" refers to a direct causal relationship to a process without the need for intermediate steps.

[0020] "Indirectly" refers to an indirect causal relationship that requires an intermediate step.

[0021] "Subject" refers to a vertebrate mammal, including but not limited to a human or a primate, such as a dog, cat, horse, goat, and monkey. Thus, the present invention can be used to treat cancer in non-human subjects. Preferably, the subject is a human.

[0022] The terms "treat," "treated," or "treating," when used in reference to a disease, refer to prophylactic treatment that increases a subject's resistance to developing the disease, in other words, reduces the likelihood that the subject will develop the disease, as well as treatment to combat the disease after the subject has developed the disease, to prevent the disease from worsening, or to slow the progression of the disease compared to when no treatment is administered.

[0023] Other technical terms used herein have their ordinary meaning in the art in which they are used, as exemplified in various technical dictionaries.

[0024] Introduction The present invention recognizes that there is a long-felt need for methods of treating cancer.

[0025] To introduce the scope of the present invention in a non-limiting manner, the present invention includes several general and useful aspects as follows. 1) screening methods for cancer treatment, 2) compounds for treating cancer; 3) a pharmaceutical composition for treating cancer; 4) a method for treating cancer; 5) A pharmaceutical composition comprising a PI3K inhibitor together with a checkpoint inhibitor or other immunotherapeutic agent; 6) Methods for treating cancer using PI3K inhibitors in combination with checkpoint inhibitors or other immunotherapies; and 7) Methods of treating glioblastoma, breast cancer, and / or triple-negative breast cancer using a PI3K inhibitor in combination with a checkpoint inhibitor or other immunotherapy.

[0026] These aspects of the invention, as well as other aspects described herein, can be achieved by using the methods, articles of manufacture, and compositions described herein. To fully appreciate the scope of the invention, it will be further recognized that various aspects of the invention can be combined to create desirable embodiments of the invention.

[0027] I. Screening method for cancer treatment The present invention includes a method for screening for cancer treatments.

[0028] A first aspect of the invention includes a method of screening at least one test compound, at least one procedure, or a combination thereof, for an ability to render at least one cancer cell more susceptible to one or more treatments for reducing the number of cancer cells in a cell population, the method comprising: a. providing one or more cancer cells expressing one or more markers; b. providing one or more of the at least one test compound, at least one procedure, or a combination thereof; c. contacting the one or more cancer cells with the one or more test compounds; d. determining a change in expression of the one or more markers by the one or more cancer cells in the presence or absence of the one or more test compounds; wherein an alteration in expression of said one or more markers in said one or more cancer cells in the presence of said one or more test compounds compared to the absence of said one or more test compounds reasonably predicts that said one or more cancer cells will be more sensitive to said one or more treatments for reducing cancer cell number.

[0029] The activity of the compounds and combinations thereof used according to the present invention can be determined by any method known in the art or as shown in the Examples. In one embodiment of the present invention, the activity of the inhibitors or antagonists is determined using various experimental animal models, including, but not limited to, cancer animal models such as immunocompetent wild-type animals implanted with genetically matched tumors, SCID mouse models, or nude mice implanted with human tumor xenografts.

[0030] The purification process for the inhibitors or antagonists employs a variety of in vitro and in vivo assays to test the activity of the inhibitors or antagonists. The protocols and compositions of the invention are also preferably tested in vitro and then in vivo for the desired therapeutic or prophylactic activity prior to use in humans.

[0031] Compounds for use in treatment may be tested in appropriate and acceptable or recognized animal model systems, including, but not limited to, rats, mice, chickens, cows, monkeys, rabbits, etc., prior to testing in humans.

[0032] The toxicity and efficacy of the prophylactic and / or therapeutic protocols of the invention can be evaluated, for example, by LD 50 (the dose lethal to 50% of the population) and ED 50 The LD50 dose can be determined by standard pharmaceutical procedures in cell cultures or experimental animals to determine the dose that is therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the ratio LD50. 50 / ED 50 The therapeutic index can be expressed as: Prophylactic and / or therapeutic agents that exhibit large therapeutic indices are preferred. Prophylactic and / or therapeutic agents that exhibit toxic side effects can also be used, although care should be taken to design administration systems that target such agents to the site of affected tissue in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0033] The data obtained from cell culture assays and animal studies can be used in formulating a range of prophylactic and / or therapeutic dosages for use in humans. The dosages of such agents may be adjusted to induce ED with little or no toxicity. 50 Preferably, the therapeutically effective dose will be within a circulating concentration range including the IC 2 , IC 3 , IC 4 , IC 5 , IC 6 , IC 7 , IC 8 , IC 9 , IC 10 , IC 11 , IC 12 , IC 13 , IC 14 , IC 15 , IC 16 , IC 17 , IC 18 , IC 19 , IC 20 , IC 21 , IC 22 , IC 34 , IC 15 , IC 25 , IC 35 , IC 16 , IC 26 , IC 36 , IC 17 , IC 27 , IC 38 , IC 18 , IC 27 , IC 39 , IC 19 , IC 20 , IC 34 , IC 15 , IC 25 , IC 35 , IC 16 50 The compound may be formulated in animal models to achieve a circulating plasma concentration range that includes (e.g., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0034] In certain embodiments of the invention, a pharmaceutical composition may contain, for example, at least about 0.1% of an active compound. In other embodiments of the invention, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.

[0035] The compounds and compositions described herein can be used in the detection and treatment methods of the invention, either alone or in conjugates with other molecules, such as detection agents or cytotoxic agents, as described in more detail herein.

[0036] Typically, one of the components may contain or be bound or conjugated to a detectable label for use in a particular binding-type assay, as known in the art. A detectable label is a moiety whose presence can be ascertained directly or indirectly. In some cases, detection of the label involves the emission of energy by the label. A label can be directly detected by its ability to emit and / or absorb photons or other atomic particles of a particular wavelength (e.g., radioactivity, luminescence, optical density or electron density, etc.). A label can be indirectly detected by its ability to bind, recruit, and in some cases cleave, another moiety that can itself emit or absorb light of a particular wavelength (e.g., an epitope tag such as the FLAG epitope, an enzyme tag such as horseradish peroxidase, etc.). An example of indirect detection is the use of a first enzyme label that cleaves a substrate into a visible product. The label can be chemical, peptide, or nucleic acid molecular in nature, but is not so limited. Other detectable labels include radioisotopes such as P32 and H3, luminescent markers such as fluorochromes, optical and electron-dense markers, and the like, epitope tags such as FLAG epitope and HA epitope, and enzyme tags such as biotin, avidin, horseradish peroxidase, and β-galactosidase. The labels can be attached during or after the synthesis of the peptide. Many different labeling and display methods are known to those of skill in the art. Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Those of skill in the art will know of other suitable labels for the peptides described herein, or will be able to ascertain such labels using routine experimentation. Furthermore, coupling or conjugation of these labels to the peptides of the present invention can be accomplished using standard techniques common to those of skill in the art.

[0037] Different categories of detectable labels include, for example, for magnetic resonance imaging (MRI): Gd(DOTA); for nuclear medicine: 201Tl, the gamma-emitting radionuclide 99mTc; for positron emission tomography (PET): positron-emitting isotopes, (18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride, copper-64, gadodiamide, and 203 Pb(II) radioisotopes, such as Pb; 111In diagnostic and imaging labels (commonly referred to as in vivo detectable labels);

[0038] The conjugations or modifications described herein use routine chemistry, which does not form part of the present invention, and which is well known to those skilled in the art of chemistry. The use of protecting groups and known linkers, such as mono- and heterobifunctional linkers, are well described in the literature and will not be repeated here.

[0039] As used herein, "conjugated" refers to two entities stably bound to each other by any physicochemical means. It is important that the nature of the bond is such that it does not substantially impair the effectiveness of either entity. With these parameters in mind, covalent or non-covalent linkages known to those of skill in the art may be employed. In some aspects of the invention, covalent bonds are preferred. Non-covalent bonds include hydrophobic interactions, ionic interactions, high affinity interactions such as biotin-avidin complexes or biotin-streptavidin complexes, and other affinity interactions. Such attachment means and methods are well known to those of skill in the art.

[0040] A variety of methods can be used to detect the label, depending on the nature of the label and other assay components. For example, the label can be detected while bound to the solid substrate or after separation from the solid substrate. The label can be detected directly, through optical or electron density, radioactive emission, non-radioactive energy transfer, etc., or indirectly, using antibody conjugation, streptavidin-biotin conjugation, etc. Methods for detecting the label are known in the art.

[0041] A. Test Compounds, Test Procedures, or Combinations Thereof In one embodiment of the present invention, such test compounds, test procedures, or combinations thereof include at least one chemical compound, at least one chemotherapy, at least one immunotherapy, at least one radiation therapy, at least one cryotherapy, at least one hyperthermia, at least one laser therapy, at least one surgery, at least one nanoparticle delivery system, or combinations thereof.

[0042] Another aspect of the invention includes the nanoparticle delivery system including at least one scintillator.

[0043] A further aspect of the invention includes that such nanoparticle delivery systems include at least one cleavable linker.

[0044] Further aspects of the invention include where such a cleavable linker is between the drug and the nanoparticle.

[0045] B. Cancer cells One aspect of the invention includes cancer cells derived from endoderm, mesoderm, ectoderm, or a combination thereof.

[0046] Another aspect of the invention is where the cancer cells comprise carcinoma cells, sarcoma cells, leukemia cells, lymphoma cells, myeloma cells, or a combination thereof.

[0047] A further aspect of the invention is that the cancer cells comprise skin cells, melanoma cells, non-melanoma cells, brain cells, prostate cells, breast cells, colon cells, kidney cells, bladder cells, non-Hodgkin's lymphoma cells, thyroid cells, endometrial cells, head cells, cervical cells, brain cells, glioblastoma cells, or combinations thereof.

[0048] C. More sensitive treatments Further aspects of the invention include that such cancer cells are more susceptible to such treatments due to cell death, a slower proliferation rate, an increased doubling time, being more susceptible to DNA damage, having a reduced ability to repair DNA damage, being more susceptible to recognition by immune cells, or a combination thereof.

[0049] D. Treatment to reduce the number of cancer cells One aspect of the present invention includes that the treatment for reducing the number of cancer cells includes at least one chemotherapy, at least one immunotherapy, at least one radiation therapy, at least one cryotherapy, at least one hyperthermia, at least one laser therapy, at least one surgery, at least one nanoparticle delivery system, or a combination thereof.

[0050] E. Cell populations Another aspect of the invention is where the cell population comprises a pure culture, a mixed culture, a biopsy, in vivo, in situ, or a combination thereof.

[0051] F. Marker Further aspects of the invention include one or more of the following markers: i.PD-L1, ii. MHC Class II; iii.CD80, iv.CD86, v.CD40, vi CD95, vii. MHC Class I, viii.Lag-3, ix. Vista, x.LC3, xi. One or more of the following: CD95, CD44, CD47, TRA-1-60, SSEA-1, EpCam, ALDH1A1, Lgr5, CD13, CD19, CD20, CD24, CD26, CD27, CD34, CD38, CD44, CD45, CD47, CD49f, CD66c, CD90, CD166, TNFRSF16, CD105, CD133, CD117 / c-kit, CD138, CD151, and CD166; and xii. Combinations of these

[0052] G. Measurement of Expression Changes Further aspects of the invention include where measuring expression changes comprises immunological methods, nucleic acid methods, proteomic methods, flow cytometric methods, genomic methods, or a combination thereof, pure cultures, mixed cultures, biopsies, or a combination thereof.

[0053] H. Changes in expression One aspect of the invention includes that the change in expression comprises an increase, decrease, or a combination thereof from a pure culture, a mixed culture, a biopsy, or a combination thereof.

[0054] I. Reasonable Expectations Another aspect of the invention includes a reasonable expectation is a statistically significant difference with a p-value of less than 0.10, less than 0.05, or acceptable to one of skill in the art.

[0055] J. Further Testing Further aspects of the invention include further comprising testing the cancer cells with one or more test compounds in conjunction with one or more therapeutic approaches for the reduction of cancer cell numbers.

[0056] II. Compounds for the Treatment of Cancer The present invention also includes compounds for treating cancer. A second aspect of the invention includes compounds identified by the method of aspect I of the invention.

[0057] III. Pharmaceutical Compositions for Treating Cancer The present invention includes pharmaceutical compositions for treating cancer.

[0058] A third aspect of the invention includes a pharmaceutical composition, which comprises a pharma- ceutical effective amount of a compound of the invention in a pharma- ceutical acceptable carrier.

[0059] An embodiment of the present invention includes a pharmaceutical composition of the present invention further comprising at least one additional cancer therapeutic agent.

[0060] Another aspect of the present invention includes the pharmaceutical composition of the present invention, wherein such treatment comprises at least one chemotherapy, at least one immunotherapy, at least one radiation therapy, at least one cryotherapy, at least one hyperthermia, at least one laser therapy, at least one surgery, at least one nanoparticle administration system, or a combination thereof.

[0061] Further aspects of the invention include pharmaceutical compositions comprising: at least one pan-PI3K inhibitor, [ka] TIFF2025511969000003.tif205161 Or a combination thereof.

[0062] IV Methods for Treating Cancer The present invention also includes a method of treating cancer.

[0063] A fourth aspect of the invention includes a method of treating a subject with cancer, comprising: a. providing a subject in need of treatment for at least one cancer; b. providing at least one compound or pharmaceutical composition of the invention or a combination thereof; c. administering to the subject a pharma- tically effective amount of said at least one compound; wherein the subject is treated for said at least one cancer.

[0064] One embodiment of the invention further comprises administering to the subject at least one treatment.

[0065] Another aspect of the invention is where the treatment comprises at least one chemotherapy, at least one immunotherapy, at least one radiation, at least one cryotherapy, at least one hyperthermia, at least one laser, at least one surgery, at least one nanoparticle administration system, or a combination thereof.

[0066] V. PI3K INHIBITORS IN COMBINATION WITH CHECKPOINT INHIBITORS OR OTHER IMMUNOTHERAPY FOR TREATING CANCER - PHARMACEUTICAL COMPOSITIONS The present invention includes PI3K inhibitors and checkpoint inhibitors or other immunotherapies for the treatment of cancer. The present invention can also include other components as described herein, such as, but not limited to, CAR-T cells and antibodies.

[0067] A fifth aspect of the invention includes a pharmaceutical composition comprising: a) a pharma- tically effective amount of at least one PI3K inhibitor in a pharma- tically acceptable carrier, and b) a pharma- tically effective amount of at least one checkpoint inhibitor or other immunotherapy, including CAR T cells or antibodies.

[0068] One embodiment of the invention includes that the at least one PI3K inhibitor and the checkpoint inhibitor are provided together or separately.

[0069] A. PI3K Inhibitors and Agents In some embodiments of the present invention, a PI3K inhibitor may be administered alone or in combination with other components of the present invention.

[0070] Any PI3K inhibitor can be used in the present invention. Many are disclosed herein, and additional PI3K inhibitors are disclosed in U.S. Published Patent Application No. 2021 / 0163462 A1 to Stocking et al., published June 3, 2021, U.S. Patent No. 11,492,348 to Stocking et al., published November 8, 2022, WO 2009 / 066087 to Hoffman-La Roche et al., published May 28, 2009, WO 2007 / 084786 to Novartis, published July 26, 2007, and U.S. Patent No. 8,921,361 to Cmiljanovic et al., published December 30, 2014, each of which is incorporated herein by reference in its entirety.

[0071] Preferred PI3K inhibitors are disclosed in U.S. Pat. No. 11,492,348 to Stocking et al., issued Nov. 8, 2022, and include, but are not limited to, the following:

[0072] In one aspect, the present disclosure provides a compound of formula (I): [ka] A compound of the formula: During the ceremony, R 1 is -(CR a R b ) m -aryl, -CH=CH-aryl, -(CR c R d ) n -heteroaryl, -(CR e R f ) o -heterocycloalkyl, or -(CR g R h ) p -cycloalkyl, m, n, o, and p are each independently 0, 1, or 2; R a , R b , Rc , R d , R e , R f , R g , and R h are each independently H, halo, or C 1-4 Is it an alkyl group? Or R a and R b together with the carbons to which they are attached form a cycloalkyl ring, Or R a and R b taken together to form =CH2 or =O, R 1 Each aryl, heteroaryl, heterocycloalkyl, or cycloalkyl occurring in is unsubstituted or contains one or two R x is substituted with a substituent, Here, each R x The substituents are independently halo, C 1-4 Alkyl, cycloalkyl, -C 1-2 -Haloalkyl, -OH, -OC 1-4 Alkyl, -OC 1-2 -Haloalkyl, cyano, -C(O)C 1-4 Alkyl, -C(O)NR i R j , -SO2C 1-4 Alkyl, -SO2NR k R l , -NR q R r , -C(O)-cycloalkyl, -C(O)-aryl (optionally substituted with methyl or halo), -COC 1-4 alkyl, -COaryl, -C(O)CH-aryl (optionally substituted with methyl or halo), -CH-aryl (optionally substituted with methyl or halo), or monocyclic heterocycloalkyl (methyl, -C(O)C 1-4 Alkyl or -CO2C 1-4 optionally substituted with alkyl; Here, R i , R j , R k and R l are each independently H, C 1-4Alkyl, -C 1-4 Alkyl-OH or -C 1-4 Alkyl-OC 1-4 is alkyl, Here, R q and R r are each independently H, C 1-4 Alkyl, -C 1-4 Alkyl-OH, -C 1-4 Alkyl-OC 1-4 Alkyl, -C(O)C 1-4 Alkyl, -CO2C 1-4 Alkyl or -SO2C 1-4 is alkyl, L is absent, -S(O)2-, -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or -CR s R t - where R s and R t are independently H or alkyl, or R s and R t together with the carbon atom to which they are attached form a cycloalkyl ring, X is O, S, NH, N(COC 1-4 Alkyl), N(SO2C 1-4 N(alkyl), N(SOcycloalkyl), or CH; Y1, Y2, and Y3 are each independently CH or N, where when L is other than -S(O)2-, then Y2 and Y3 are each CH; G2 is N or CR 2 and G3 is N or CR 3 and G4 is N, NR 4b or CR 4a and G5 is N or CR 5 and G6 is N or CR 6 and Here, R 2 , R 3 , R 4a , R 5 and R 6are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NR u R v and Or, R 4b But R 6 and together with the atom to which they are attached form a heteroaryl or heterocyclic ring, where R 4b and R 6 A heteroaryl ring containing up to one N and optionally substituted with alkyl, R 4b and R 6 is optionally substituted with oxo; R u is H or C 1-4 is alkyl, R v , H, C 1-4 Alkyl, monocyclic cycloalkyl, -C(O)C 1-4 Alkyl, or -C(O)NR w R y and Here, R v Each alkyl present in is unsubstituted or is selected from the group consisting of -OH, -NH, -NH(C 1-4 alkyl), or -N(C 1-4 alkyl)2; R w and R y are each independently H or C 1-4 is alkyl, where [ka] but not unsubstituted phenyl, R 7 and R 8 are each independently hydrogen or C 1-4 is alkyl, Or, R 7 and R 8 together to form -CH2CH2- A compound or a pharma- ceutically acceptable salt thereof is provided.

[0073] In certain embodiments, the compound of formula (I) is a compound selected from those species described or exemplified in the detailed description herein.

[0074] In some embodiments of Formula (I) or any variation thereof, R 1 is -(CR a R b ) m -aryl. In some embodiments, R 1 (CR c R d ) n -heteroaryl. In some embodiments, R 1 is (CR e R f )o-heterocycloalkyl or (CR g R h ) p -cycloalkyl.

[0075] In some embodiments of Formula (I) or any variation thereof, L is -S(O)-. In some embodiments, L is -C(O)-, -CH-, -CF-, C(CH), -C(=CH)-, or -CR s R t In other embodiments, L is -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or -CR s R t In some embodiments, L is absent.

[0076] In another embodiment, the compound of formula (II) [ka] A compound of the formula: During the ceremony, X is O, S, NH, N(COC 1-4 Alkyl), N(SO2C 1-4 N(alkyl), N(SOcycloalkyl), or CH; Y1, Y2 and Y3 are each independently CH or N; G2 is N or CR 2 and G3 is N or CR 3 and G4 is N, NR 4b or CR 4a and G5 is N or CR 5 and G6 is N or CR 6 and Here, R 2 , R 3 , R 4a , R 5 and R 6 are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NR u R v and Or, R 4b is R 6 and together with the atom to which they are attached form a heteroaryl or heterocyclic ring, where R 4b and R 6 Heteroaryl rings containing up to one N and optionally substituted with alkyl, R 4b and R 6 is optionally substituted with oxo; R u is H or C 1-4 is alkyl, R v , H, C 1-4 Alkyl, monocyclic cycloalkyl, -C(O)C 1-4 Alkyl, or -C(O)NR w R y and Here, R v Each alkyl present in is unsubstituted or is selected from the group consisting of -OH, -NH, -NH(C 1-4 alkyl), or -N(C 1-4 alkyl)2; R w and R y are each independently H or C 1-4 is alkyl, Where: [ka] is not an unsubstituted phenyl, R 7 and R 8 are each independently hydrogen or C 1-4 is alkyl, Or, R 7 and R 8 together to form -CH2CH2- R 9a and R 9b are each independently hydrogen or halogen; R 10 , R 11 , R 12 , R 13 and R 14 are each independently hydrogen, halogen, -OH, -CN, -alkyl, -Oalkyl, -haloalkyl, heterocycloalkyl, -O-haloalkyl, -SO2C 1-4 Alkyl, or -NR aa R bb and R aa is hydrogen, C 1-4 Alkyl or -C 1-4 alkyl-OH, R bb is hydrogen or C 1-4 is alkyl, Or, R 9a But R 10 and together with the intervening atoms form a heteroaryl or heterocyclic ring, Or R 11 But R 12 and together with the atom to which they are attached form a heteroaryl or heterocyclic ring; A compound or a pharma- ceutically acceptable salt thereof is provided.

[0077] In another embodiment, the compound of formula (III) [ka] A compound of the formula: During the ceremony, R 1 is -(CR a R b ) m -aryl, -CH=CH-aryl, (CR c R d ) n Heteroaryl, (CR e R f ) o -heterocycloalkyl, or (CR g R h ) p -cycloalkyl, where L is SO, R 1 Each heteroaryl and heterocycloalkyl present in is monocyclic; m is 0 or 2; n, o, and p are each independently 0, 1, or 2; R a , R b , R c , R d , R e , R f , R g , and R h are each independently H, halo, or C 1-4 Is it an alkyl group? Or R a and R b together with the carbons to which they are attached form a cycloalkyl ring, Or R a and R b taken together to form =CH2 or =O, R 1 Each aryl, heteroaryl, heterocycloalkyl, or cycloalkyl occurring in is unsubstituted or contains one or two R x is substituted with a substituent, Here, each R x The substituents are independently halo, C 1-4 Alkyl, cycloalkyl, -C 1-2 -Haloalkyl, -OH, -OC 1-4 Alkyl, -OC 1-2 -Haloalkyl, cyano, -C(O)C1-4 Alkyl, -C(O)NR i R j , -SO2C 1-4 Alkyl, -SO2NR k R l , -NR q R r , -C(O)-cycloalkyl, -C(O)-aryl (optionally substituted with methyl or halo), -COC 1-4 alkyl, -COaryl, -C(O)CH-aryl (optionally substituted with methyl or halo), -CH-aryl (optionally substituted with methyl or halo), or monocyclic heterocycloalkyl (optionally substituted with methyl, -C(O)C 1-4 Alkyl or -CO2C 1-4 substituted with alkyl; Here, R i , R j , R k and R l are each independently H, C 1-4 Alkyl, -C 1-4 Alkyl-OH or -C 1-4 Alkyl-OC 1-4 is alkyl, Here, R q and R r are each independently H, C 1-4 Alkyl, -C 1-4 Alkyl-OH, -C 1-4 Alkyl-OC 1-4 Alkyl, -C(O)C 1-4 Alkyl, -CO2C 1-4 Alkyl or -SO2C 1-4 is alkyl, Or, R 1 but, [ka] and During the ceremony, R 10 , R 11 , R 12 , R 13 and R 14are each independently hydrogen, halogen, -OH, -CN, -alkyl, -Oalkyl, -haloalkyl, heterocycloalkyl, -O-haloalkyl, -SO2C 1-4 Alkyl, or -NR aa R bb and R aa is hydrogen, C 1-4 Alkyl or -C 1-4 alkyl-OH, R bb is hydrogen or C 1-4 is alkyl, Or, R 10 But R 11 and together with the atom to which they are attached form a heteroaryl or heterocyclic ring, Or R 11 But R 12 and together with the atom to which they are attached form a heteroaryl or heterocyclic ring, L is absent, -S(O)2-, -C(O)-, -O-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, or -CR s R t - where R s and R t are independently H or alkyl, or R s and R t together with the carbon atom to which they are attached form a cycloalkyl ring, X is O, S, NH, N(COC 1-4 Alkyl), N(SO2C 1-4 N(alkyl), N(SOcycloalkyl), or CH; Y1, Y2, and Y3 are each independently CH or N, where when L is other than -S(O)2-, then Y2 and Y3 are each CH; G2 is N or CR 2 and G3 is N or CR 3 and G4 is N, NR 4b or CR 4a and G5 is N or CR 5and G6 is N or CR 6 and Here, R 2 , R 3 , R 4a , R 5 and R 6 are each independently hydrogen, halogen, -OH, -alkyl, -Oalkyl, -haloalkyl, -O-haloalkyl, or -NR u R v and Or R 4b But R 6 and together with the atom to which they are attached form a heteroaryl or heterocyclic ring, where R 4b and R 6 A heteroaryl ring containing up to one N and optionally substituted with alkyl, R 4b and R 6 is optionally substituted with oxo; R u is H or C 1-4 is alkyl, R v , H, C 1-4 Alkyl, monocyclic cycloalkyl, -C(O)C 1-4 Alkyl, or -C(O)NR w R y and Here, R v Each alkyl present in is unsubstituted or is selected from the group consisting of -OH, -NH, -NH(C 1-4 alkyl), or -N(C 1-4 alkyl)2; R w and R y are independently H or C 1-4 is alkyl, where [ka] but not unsubstituted phenyl, R 7 and R 8 are each independently hydrogen or C 1-4 is alkyl, Or, R 7 and R 8 together to form -CH2CH2- A compound or a pharma- ceutically acceptable salt thereof is provided.

[0078] In some embodiments of any of the compounds of formula (I), (II), or (III), Y1, Y2, and Y3 are each CH. In some embodiments, Y1 is N, and Y2 and Y3 are each CH. In some embodiments, Y2 is N, and Y1 and Y3 are each CH. In some embodiments, Y3 is N, and Y1 and Y2 are each CH.

[0079] In some embodiments of any of the compounds of Formula (I), (II), or (III), X is O. In some embodiments, X is NH, N(COC 1-4 Alkyl), N(SO2C 1-4 alkyl), or N(SO2 cyclo-alkyl).

[0080] In some embodiments of any of the compounds of Formula (I), (II), or (III), G2 and G4 are each N and G6 is CR 6 In some embodiments of any of the compounds of Formula (I), (II), or (III), G3 is CR 3 and G5 is CR 5 In some embodiments of any of the compounds of Formula (I), (II), or (III), one of G2 and G4 is N.

[0081] In some embodiments of any of the compounds of Formula (I), (II), or (III), R 6 -NR u R v In some embodiments of any of the compounds of formula (I), (II), or (III), R 4b is R 6 and together with the atom(s) to which they are attached form a heteroaryl or heterocyclic ring.

[0082] In a further aspect, the present disclosure provides a pharmaceutical composition comprising at least one compound of formula (I), (II), or (III), or a pharma- ceutically acceptable salt thereof. The pharmaceutical composition according to the embodiment may further comprise a pharma- ceutically acceptable excipient. In some embodiments, a pharmaceutical composition is provided comprising (a) at least one compound of formula (I), (II), or (III), or a pharma- ceutically acceptable salt thereof, and (b) a pharma- ceutically acceptable excipient.

[0083] The PI3K inhibitor can be administered in combination with other anti-cancer agents, such as checkpoint inhibitors, CAR T cells, and antibodies. The PI3K inhibitor can be administered in a suitable formulation, dosage, route of administration, and administration regime. The PI3K inhibitor of the present invention is preferably administered locally, but can be administered by any suitable route of administration. Other agents can be administered by a suitable route of administration, such as systemic administration or local administration.

[0084] PI3K antagonists as well as PI3K inhibitors can be used alone or in combination with other components of the present invention. Any PI3K agonist can be used in the present invention. Many are disclosed herein, and additional PI3K agonists are disclosed in U.S. Published PCT Patent Application No. WO2019 / 199865 and U.S. Patent No. 11,492,348, each of which is incorporated herein by reference in its entirety.

[0085] B. Checkpoint Inhibitors In some aspects of the invention, checkpoint inhibitors may be administered alone or in combination with other components of the invention.

[0086] Any checkpoint inhibitor can be used in the present invention, many of which are disclosed herein, and additional checkpoint inhibitors are disclosed, for example, in PMID:27054314 and https: / / doi.org / 10.3389 / fimmu.2020.01508, each of which is incorporated by reference in its entirety.

[0087] Examples of inhibitory checkpoint molecules include, but are not limited to, PD-1, PD-L1, PD-L2, TIM-3, VISTA, A2AR, B7-H3, B7-H4, B7-H6, BTLA, CTLA-4, IDO, KIR, and LAG3. CTLA-4, PD-1, and their ligands are members of the CD28-B7 family of co-signaling molecules and play important roles in all stages of T cell function and other cell functions. CTLA-4, cytotoxic T lymphocyte-associated protein 4 (CD152), is involved in the regulation of T cell proliferation.

[0088] PD-1 receptor is expressed on the surface of activated T cells (and B cells) and normally binds to its own ligands (PD-L1 and PD-L2) expressed on the surface of antigen-presenting cells such as dendritic cells and macrophages. This interaction sends a signal to the T cells to inhibit them. Cancer cells exploit this system by overexpressing PD-L1 on their surface. This allows the cancer cells to control the PD-1 pathway, switching off PD-1-expressing T cells that can enter the tumor microenvironment and suppressing the anti-cancer immune response. Pembrolizumab (formerly MK-3475 and lambrolizumab, trade name KEYTRUDA®) is a human antibody used in cancer immunotherapy that targets the PD-1 receptor.

[0089] Some aspects of the invention further include administering a checkpoint inhibitor to the subject. In some aspects of the invention, the checkpoint inhibitor can be an antibody selected from an anti-CTLA4 antibody or antigen-binding fragment thereof that specifically binds to CTLA4, an anti-PD1 antibody or antigen-binding fragment thereof that specifically binds to PD1, an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds to PD-L1, an anti-VISTA antibody or antigen-binding fragment thereof that specifically binds to VISTA, or a combination thereof. In other aspects of the invention, the checkpoint inhibitor can be an anti-PD-L1 antibody selected from atezolizumab, avelumab, durvalumab, or a combination thereof. In other aspects of the invention, the checkpoint inhibitor is an anti-CTLA-4 antibody that can be selected from tremelimumab or ipilimumab, or a combination thereof. In yet other embodiments of the invention, the checkpoint inhibitor can be an anti-PD1 antibody that can be selected from nivolumab or pembrolizumab, or a combination thereof.

[0090] The checkpoint inhibitor, in some aspects of the invention, is a molecule such as a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, a small molecule, or a combination thereof. For example, the checkpoint inhibitor can inhibit a checkpoint protein, which can be CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, B7-H6, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, or a combination thereof. Ligands for checkpoint proteins include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands, or combinations thereof. In some aspects of the invention, the anti-PD-1 antibody can be BMS-936558 (nivolumab). In other aspects of the invention, the anti-CTLA-4 antibody can be ipilimumab (trade name Yervoy®, formerly MDX-010 and MDX-101). In another aspect of the invention, the checkpoint inhibitor can be J43 (anti-PD1 antibody), RMP1-14 (anti-PD1 antibody), atezolizumab (TECENTRIQ®; anti-PDL1 antibody), or combinations thereof. In yet another aspect of the invention, the checkpoint inhibitor can be pembrolizumab.

[0091] Pembrolizumab is a potent humanized immunoglobulin G4 monoclonal antibody with high specificity of binding to the PD-1 receptor, thus inhibiting its interaction with PD-L1 and programmed cell death 1 ligand 2. Preclinical in vitro data show that pembrolizumab has high affinity and potent receptor blocking activity for PD-1. Pembrolizumab has had an acceptable safety profile in preclinical studies and is in clinical development as an intravenous immunotherapy for advanced malignancies. KEYTRUDA® (pembrolizumab) is approved for the treatment of patients with several indications. Pembrolizumab has been approved for several cancer types and is in several stages of clinical development for many more cancer types.

[0092] Preferred checkpoint inhibitors include, but are not limited to, at least one anti-CTLA-4 inhibitor, at least one PD-1 inhibitor, at least one anti-PD-L1 inhibitor, or a combination thereof. Preferred anti-CTLA-4 inhibitors include, but are not limited to, ipilimumab. Preferred PD-1 inhibitors include, but are not limited to, pembrolizumab, nivolumab, cemipillumab (trade name LIBTAYO®), dostallimab (trade name JEMPERLI®), or a combination thereof. Preferred anti-PD-L1 inhibitors include, but are not limited to, atezolizumab (trade name TECENTRIQ®), durvalumab (trade name IMFINZI®), and avelumab (trade name BAVENCIO®), or a combination thereof.

[0093] In some aspects of the invention, the invention comprises administering at least one immune checkpoint inhibitor, as described herein or as otherwise known, In some aspects of the invention, immune checkpoint inhibitors are administered in combination.

[0094] C.CAR-T cells In some embodiments of the invention, cellular therapies such as CAR-T cells may also be administered alone or in combination with other components of the invention.

[0095] Any suitable CAR-T cell may be used in the present invention, many of which are disclosed herein and many more are known in the literature.

[0096] Preferably, a PI3K inhibitor can be administered with such CAR-T cells. As used herein, CAR-T cells refer to T cells that have been introduced with a chimeric antigen receptor, and the specificity of the receptor is directed to a selected antigen. Such receptors contain an ectodomain that recognizes antigens independent of MHC restriction, in combination with a cytoplasmic signaling domain. As the ectodomain of the chimeric antigen receptor, various proteins can be introduced into T cells. For example, they include, but are not limited to, nanobodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, human antibodies, antibody fragments, or combinations thereof.

[0097] A CAR-T cell can be a cell (e.g., a T cell) engineered to express a CAR, and the CAR-T cell preferably exhibits antitumor properties. The CAR can be engineered to include an ectodomain peptide fused to an intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). When expressed in a T cell, the CAR can redirect antigen recognition based on antigen binding specificity. The antigen-binding peptide is preferably fused to one or more intracellular domains of a costimulatory molecule and a zeta chain. In some embodiments of the invention, the antigen-binding peptide is fused to one or more intracellular domains selected from the group consisting of CD137 (4-1BB) signal domain, CD28 signal domain, CD3 Zeta signal domain, and combinations thereof.

[0098] In some aspects of the invention, the invention involves administering antigen-specific CAR-T cells, with or without a PI3 kinase inhibitor, to a subject with cancer. The CAR-T cells can, for example, have a modified PI3 kinase receptor in an amount effective to treat the cancer.

[0099] D. Antibodies against cancer cells Another form of anti-cancer drug therapy is the administration of antibodies specific for cell surface antigens, for example, of cancer cells.

[0100] In some aspects of the invention, antibodies against cancer cells may also be administered alone or in combination with other components of the invention.

[0101] Any suitable antibody to cancer cells can be used in the present invention, many of which are disclosed herein, and additional antibodies to cancer cells are known in the art.

[0102] In one embodiment of the invention, the antibody is selected from the group consisting of: Libtaxin, Herceptin, Rituximab, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncorhythm, SMART M195, ATRAGEN, Ovalex, Vexar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22, OV103, 3622W94, Anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, BABS, Anti-FLK-2, MDX-260, ANA Ab, SMART The antibodies may be 1D10 Ab, SMART ABL 364 Ab, ImmuRAIT-CEA, and combinations thereof. Other antibodies include, but are not limited to, anti-CD20 antibodies, anti-CD40 antibodies, anti-CD19 antibodies, anti-CD22 antibodies, anti-HLA-DR antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-CD54 antibodies, anti-CD69 antibodies, and combinations thereof. These antibodies are commercially available or can be synthesized de novo.

[0103] Other cancer therapeutic agents and their dosages, routes of administration, and recommended methods of use are known in the art and described in such literature.

[0104] VI Combining PI3K inhibitors with checkpoint inhibitors or other immunotherapies, including CAR-T cells and antibodies, for the treatment of cancer The present invention also includes a method for treating cancer.

[0105] A sixth aspect of the present invention includes a method of treating a subject having cancer, the method comprising: a) providing a subject in need of treatment for at least one cancer; b) providing at least one compound or pharmaceutical composition of the present invention, or a combination thereof; and c) administering a pharmacologic effective amount of at least one compound to the subject, wherein the subject is treated for the at least one cancer.

[0106] In some aspects, the present invention provides a method for treating cancer (including improving one or more symptoms thereof) in a subject refractory to existing monotherapy for such cancer, comprising administering to the subject a composition comprising a therapeutically effective amount of an inhibitor or antagonist and one or more therapeutic agents other than the inhibitor or antagonist. The present invention also provides a method for treating cancer by administering a composition comprising an inhibitor or antagonist in combination with any other anti-cancer treatment (e.g., checkpoint inhibitor therapy, CAR-T cell therapy, radiation therapy, chemotherapy, or surgery) to a patient who has proven refractory to other treatments or who may benefit from adjuvant therapy. The present invention also provides a method for treating a patient who is suffering from cancer and is immunosuppressed due to having previously undergone one or more other cancer treatments. The present invention also provides an alternative method for the treatment of cancer where chemotherapy, radiation therapy, hormonal therapy, and / or biological therapy / immunotherapy have proven or may prove to be too toxic for the subject being treated, e.g., resulting in unacceptable or intolerable side effects.

[0107] A. Eligibility The subject may be any suitable subject, including, but not limited to, humans and non-human primates. Companion animals, such as, but not limited to, dogs and cats, are also of interest. Test animals, ranging from mice to humans, are also considered subjects in the present invention.

[0108] B. Types of cancer to be treated Cancers treatable by the present invention include, but are not limited to, neoplasia, malignancy, metastasis, or any disease or disorder characterized by uncontrolled cell proliferation such that it is considered to be cancerous. The cancer may be a primary or metastatic cancer. Such cancers further include brain cancer, including glioblastoma and medulloblastoma; biliary tract cancer; bladder cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms, including acute lymphocytic leukemia and myeloid leukemia; multiple myeloma, AIDS-related leukemia, and adult T-cell leukemia-lymphoma; intraepithelial neoplasia, including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphoma; neuroblastoma; oral cancer, including squamous cell carcinoma; epithelial cell, stromal cell, and leukemia. Cancers that may be used include, but are not limited to, ovarian cancer, including those arising from cysts, germ cells, and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer, including melanoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma; testicular cancer, including germinal tumors such as seminoma, non-seminoma, teratoma, and choriocarcinoma; interstitial tumors and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma; and kidney cancer, including adenocarcinoma and Wilms' tumor. Commonly encountered cancers include breast cancer, prostate cancer, lung cancer, colon cancer, and brain cancer. In some embodiments, the cancer is not gastric or gastrointestinal cancer.

[0109] Preferred cancers to treat include, but are not limited to, glioblastoma, breast cancer, triple-negative breast cancer, and combinations thereof.

[0110] Glioblastoma is the most common type of primary brain tumor. The cancer begins in the brain's astrocytes and grows very rapidly. Brain tumor cells can also suppress the immune system, making it less effective against cancer. As a result, patients with glioblastoma and a variant of the disease called liposarcoma have poor long-term outcomes.

[0111] Breast cancer is a disease in which cells in the breast grow uncontrollably and form tumors. There are many different types of breast cancer. When cancer cells spread to other parts of the body, the disease is called metastatic breast cancer. Approximately 12% of women in the United States, or 1 in 8 women, will develop breast cancer during their lifetime. It is the second most common cancer in American women after skin cancer. Breast cancer occurs primarily in women over the age of 50, but men can also develop it. Approximately 245,000 cases of breast cancer are diagnosed in women and 2,200 cases in men in the United States each year. Although many risk factors have been identified that increase a woman's risk of developing breast cancer, it is still unclear what causes normal cells to become cancerous. It is generally believed that breast cancer is caused by a combination of genetic, hormonal, and environmental factors.

[0112] Triple-negative breast cancer is a type of breast cancer that does not have the receptors commonly found in breast cancer: the first for female hormones, the second for female hormones, and the third for a protein called human epidermal growth factor 2 (HER2).

[0113] The compounds and compositions of the present invention may also be administered to prevent progression to a neoplastic or malignant condition. Such prophylactic use is recommended in the event of a condition known or suspected to precede progression to a tumor or cancer, particularly a non-neoplastic cell proliferation consisting of hyperplasia, metaplasia, or especially dysplasia. Hyperplasia is a form of controlled cell proliferation involving an increase in the number of cells in a tissue or organ, proceeding without significant changes in structure or function. Intrauterine hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell proliferation in which one type of adult cell or fully differentiated cell replaces another type of adult cell. Metaplasia can occur in epithelial cells or connective tissue cells. A typical metaplasia involves a somewhat disorganized transformed epithelium. Dysplasia is often a precursor to cancer and is found primarily in epithelium. Dysplasia is the most disorganized form of non-neoplastic cell proliferation, involving loss of uniformity of individual cells and structural orientation of cells. Dysplastic cells often have abnormally large, deeply stained, and pleomorphic nuclei. Dysplasia is characterized by occurring in areas of chronic irritation or inflammation, and is commonly found in the cervix, respiratory tract, oral cavity, and gallbladder.

[0114] Alternatively, or in addition to the presence of abnormal cell proliferation characterized as hyperplasia, metaplasia, or dysplasia, the presence of one or more characteristics of a transformed or malignant phenotype exhibited in vivo or in vitro by a cell sample from a patient can indicate the desirability of prophylactic / therapeutic administration of a composition of the invention, such as altered morphology, loosening of attachment to substrates, loss of contact inhibition, loss of anchorage dependence, release of proteases, increased sugar transport, decreased serum requirement, expression of fetal antigens, etc.

[0115] In another embodiment of the invention, a patient exhibiting one or more predisposing factors for malignant tumors is treated by administering an effective amount of a composition of the invention. Examples of predisposing factors include chromosomal translocations associated with malignancies (e.g., Philadelphia chromosome t(9:22) in chronic myeloid leukemia and t(14;18) in follicular lymphoma), familial polyposis and Gardner syndrome (which may be a precursor to colon cancer), benign monoclonal gamma diseases (which may be a precursor to multiple myeloma), first-degree relatives with cancer or precancerous diseases that show Mendelian (genetic) inheritance patterns (e.g., familial polyposis of the colon, Gardner syndrome, hereditary exostoses, polyendocrine adenomatosis, amyloid-producing medullary thyroid carcinoma and pheochromocytoma, Peutz-Jeghers syndrome, von Recklinghausen neurofibromatosis, retinoblastoma, carotid body tumors, cutaneous melanoma, intraocular melanoma, xeroderma pigmentosum, ataxia-telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi anemia, Bloom syndrome; Robbins and These include causes of cancer, such as cancer-causing factors (see Angell, 1976, Basic Pathology, 2nd ed., WB Saunders Co., Philadelphia, pp. 112-113), or exposure to carcinogens (e.g., smoking, inhalation of or contact with certain chemicals).

[0116] In one set of aspects, the invention includes a method of treating a subject susceptible to or exhibiting symptoms of cancer. The cancer may be primary, metastatic, recurrent, or multi-drug resistant. In some cases, the cancer may be drug resistant or multi-drug resistant. As used herein, a "drug resistant cancer" refers to a cancer that is resistant to conventional, commonly known cancer treatments. Examples of conventional cancer treatments include treatment of cancer with drugs such as methotrexate, trimetrexate, adriamycin, taxotere, doxorubicin, 5-flurouracil, vincristine, vinblastine, disodium pamidronate, anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab, megestrol, tamoxifen, paclitaxel, docetaxel, capecitabine, and goserelin acetate. A "multidrug resistant cancer" is a cancer that is resistant to more than one type or class of anti-cancer drug; for example, a cancer can be resistant to a first drug having a first mechanism of action and a second drug having a second mechanism of action.

[0117] C. Dosage of Pharmaceutical Composition The active agents of the present invention can be administered to a subject in an amount effective to treat a disease, such as cancer.

[0118] For example, a "pharmaceutical effective amount" is an amount necessary or sufficient to achieve a desired biological effect. An "effective amount for an autoimmune disease" can be an amount sufficient to prevent or suppress the decrease of TH cells compared to when the peptide is not administered. According to some aspects of the present invention, an effective amount refers to an amount of the compound of the present invention, when administered alone or in combination with other pharmaceutical agents, in combination or co-administration, or when administered alone, that results in a therapeutic response to the disease, either in the prevention or treatment of the disease. The biological effect can be the improvement or complete elimination of symptoms caused by the disease. In another embodiment, the biological effect is the complete disappearance of the disease, as evidenced, for example, by the absence of symptoms of the disease.

[0119] The pharmacologic effective amount of the compounds of the present invention in treating the diseases described herein may vary depending on the specific compound used, the mode of administration of the compound, and whether it is used alone or in combination. The effective amount for a particular application may also vary depending on factors such as the disease being treated, the specific compound being administered, the size of the subject, the severity of the disease or condition, etc. Those skilled in the art can empirically determine the effective amount of a particular molecule of the present invention without undue experimentation. By selecting from among the various active compounds, combined with the teachings provided herein, and considering factors such as potency, relative bioavailability, patient weight, severity of side effects, and preferred mode of administration, one can design an effective prophylactic or therapeutic treatment regimen that does not cause substantial toxicity, yet is completely effective in treating a particular subject.

[0120] A pharma- ceutically effective dose should preferably result in a serum concentration of the PI3 kinase antagonist and / or inhibitor, for example, from about 0.1 ng / ml to about 50-100 μg / ml. The pharmaceutical composition should typically provide from about 0.001 mg to about 2000 mg of the PI3 kinase antagonist and / or inhibitor per kg of body weight per day, for example, from about 0.01 mg to about 200 mg of the PI3 kinase antagonist and / or inhibitor per kg of body weight per day, or from about 0.1 mg to about 20 mg of the PI3 kinase antagonist and / or inhibitor per kg of body weight per day, or from about 1 mg to about 10 mg of the PI3 kinase antagonist and / or inhibitor per kg of body weight per day, or from about 1 mg to about 5 mg of the PI3 kinase antagonist and / or inhibitor per kg of body weight per day. Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 1000 mg, for example from about 10 mg to about 500 mg of the active agent or combination of agents per dosage unit form.

[0121] A therapeutically effective dose preferably results in a serum concentration of the PI3 kinase antagonist and / or inhibitor of, for example, about 0.1 ng / ml to about 50-100 μg / ml. The pharmaceutical composition should typically provide about 0.001 mg to about 2000 mg of the PI3 kinase antagonist and / or inhibitor per kg of body weight per day, for example, about 0.01 mg to about 200 mg of the PI3 kinase antagonist and / or inhibitor per kg of body weight per day, or about 0.1 mg to about 20 mg of the PI3 kinase antagonist and / or inhibitor per kg of body weight per day, or about 1 mg to about 10 mg of the PI3 kinase antagonist and / or inhibitor per kg of body weight per day, or about 1 mg to about 5 mg of the PI3 kinase antagonist and / or inhibitor per kg of body weight per day. Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 1000 mg, for example from about 10 mg to about 500 mg of the active agent or combination of agents per dosage unit form.

[0122] Dosage amounts of the compounds described herein typically range from about 0.1 μg to 10,000 mg, more typically from about 1 μg / day to 8,000 mg, and most typically from about 10 μg to 100 μg. In terms of subject body weight, typical dosages range from about 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 100 μg / kg / body weight, about 200 μg / kg / body weight, about 350 μg / kg / body weight, about 500 μg / kg / body weight per administration, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight per administration, to about 1000 mg / kg / body weight or more, and any range derivable therein. Non-limiting examples of ranges derivable from the numerical values ​​recited herein include ranges of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc., based on the numerical values ​​above. The absolute amount will depend on a variety of factors, including concurrent treatments, number of doses, and individual patient parameters such as age, physical condition, size, and weight. These are factors well known to those of skill in the art and can be addressed with no more than routine experimentation. It is generally preferred to use the maximum dose, i.e., the safest dose based on sound medical judgment. Multiple doses of the compounds of the invention are also contemplated.

[0123] When used in combination with the treatment of the present invention, the dosage of the known treatment may optionally be reduced to avoid side effects.

[0124] In some aspects of the invention, the invention provides a method of treating cancer, comprising administering a therapeutically effective amount of a composition comprising an inhibitor or antagonist to a subject desiring such treatment. The compositions of the invention can be used, for example, as a first-line, second-line, third-line, or fourth-line cancer treatment. In some aspects of the invention, the invention provides a method for treating cancer (including ameliorating symptoms) in a subject refractory to one or more conventional therapies for such cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising an inhibitor or antagonist. A cancer is determined to be refractory to a treatment if at least a significant portion of the cancer cells do not die or stop dividing in response to the treatment. Such a determination can be made by any method known in the art for assessing the effectiveness of a treatment on cancer cells, either in vivo or in vitro, using the art-accepted meaning of "refractory" in this context. In a specific aspect of the invention, a cancer is refractory when the number of cancer cells is not significantly reduced or is increasing.

[0125] D. Routes of Administration of Pharmaceutical Compositions According to the methods of the invention, the compounds and compositions can be administered as pharmaceutical compositions. Generally, a pharmaceutical composition comprises a compound of the invention and a pharma- ceutically acceptable carrier.

[0126] "Local administration" or "regional administration" refers to administration of a pharmaceutical agent to or near a target tissue, muscle or subcutaneously by a non-systemic route (e.g., by subcutaneous, intramuscular, subcutaneous, intraperitoneal, intraorgan (e.g., injection into cancer tissue) or transdermal routes), or other routes of administration. Thus, local administration excludes systemic (e.g., into the blood circulation) routes of administration, such as intravenous or oral administration. Peripheral administration means administration to the periphery (e.g., on or within the face, limbs, trunk, or head of a patient), as opposed to visceral or gastrointestinal administration (e.g., administration into the internal organs). In some embodiments, local administration is administration to the site of a tumor, for example, by intratumoral injection.

[0127] "Systemic administration" or "systemic administration" refers to the administration of a pharmaceutical agent to or near central tissues, such as the blood, lungs, or intestines, away from the local tissue in need of treatment (e.g., intravenous administration, inhalation administration, oral administration, etc.). Thus, systemic administration does not include local administration to diseased or damaged tissue. In some aspects of the invention, systemic administration is administration to the blood circulatory system.

[0128] The compounds of the present invention can be administered directly to tissue. Direct administration to tissue can be accomplished by other acceptable means or methods, including, but not limited to, direct injection, electroporation, direct contact with tissue or organs, etc. The compounds can be administered once or in multiple doses. When administered multiple times, the compounds can be administered by different routes. For example, the first (or first few) doses can be administered directly to the affected tissue, and subsequent doses can be administered systemically.

[0129] Other drugs when used in combination with the inhibitors of the present invention can be administered systemically.

[0130] Injectables are available for local and systemic administration. Typically, a therapeutically effective dose is formulated to include a concentration of at least about 0.1% w / w up to about 90% w / w or more, e.g., greater than 1% w / w, of the PI3K kinase antagonist and / or inhibitor in the treated tissue(s). The PI3K kinase antagonist and / or inhibitor can be administered all at once or divided into several smaller doses spaced apart over time. It is understood that the exact dosage and duration of treatment will depend on the tissue being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It should be noted that concentration and dosage values ​​will also vary with the age of the person being treated. It should be further understood that for any particular subject, a particular dosage regimen will need to be adjusted over time based on the individual needs and professional judgment of the person prescribing or administering the treatment, and the concentration ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions.

[0131] Injectables are available for local and systemic administration. Typically, a therapeutically effective dose is formulated to include a concentration of at least about 0.1% w / w up to about 90% w / w or more, e.g., greater than 1% w / w, of the PI3K kinase antagonist and / or inhibitor in the treated tissue(s). The PI3K kinase antagonist and / or inhibitor can be administered all at once or divided into several smaller doses spaced apart over time. It is understood that the exact dosage and duration of treatment will depend on the tissue being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It should be noted that concentration and dosage values ​​will also vary with the age of the person being treated. It should be further understood that for any particular subject, a particular dosage regimen will need to be adjusted over time based on the individual needs and professional judgment of the person prescribing or administering the treatment, and the concentration ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions.

[0132] A. Pharmaceutical Composition Regimen The pharmaceutical compositions of the present invention, including but not limited to PI3 kinase antagonists and / or inhibitors, can be administered at once or divided into several smaller doses to be administered at time intervals. It is understood that the exact dosage and duration of treatment will depend on the disease state being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It should also be noted that concentration and dosage values ​​may vary depending on the severity of the condition being alleviated. It should further be understood that dosage regimens must be adjusted over time for a particular subject based on the individual needs and the professional judgment of the person administering or supervising the composition, and that the concentration ranges set forth herein are merely examples and are not intended to limit the scope or practice of the invention.

[0133] The pharmaceutical composition of the present invention can be administered at once or divided into several smaller doses to be administered at intervals of time. It is understood that the exact dosage and duration of treatment will depend on the disease state being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It should also be noted that concentration and dosage values ​​may vary depending on the severity of the condition being alleviated. It should further be understood that specific dosing regimens will need to be adjusted over time for any particular subject based on the individual needs and the professional judgment of the person administering or supervising the composition, and that the concentration ranges set forth herein are merely examples and are not intended to limit the scope or practice of the methods claimed.

[0134] The compounds and compositions of the present invention can be administered directly to tissue. Direct administration to tissue can be by direct injection. The compounds and compositions can be administered once or in multiple doses. When administered multiple times, the compounds and compositions can be administered by different routes. For example, the first (or first few) doses can be administered directly to the affected tissue, and subsequent doses can be administered systemically.

[0135] B. Formulation of Pharmaceutical Compositions According to the methods of the invention, the pharmaceutical compositions of the invention can be administered to a subject in a pharmaceutical composition. Generally, a pharmaceutical composition comprises a compound of the invention and a pharma- ceutical acceptable carrier.

[0136] The formulations of the present invention are administered in pharma- ceutically acceptable solutions, which may routinely contain pharma- ceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

[0137] The pharmaceutical compositions of the present invention comprise an effective amount of one or more agents dissolved or dispersed in a pharma- ceutically acceptable carrier. The term "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals, such as humans, as appropriate. It is further understood that for administration to animals (including human and non-human subjects), preparations must meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards. The compounds and compositions of the present invention are generally suitable for administration to humans and non-humans. Preferably, the compounds or compositions are non-toxic and of sufficient purity that no additional manipulation of the compounds or compositions is required prior to administration to humans.

[0138] The pharmaceutical compositions of the present invention comprise an effective amount of one or more agents dissolved or dispersed in a pharma- ceutically acceptable carrier. The term "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals, such as humans, as appropriate. It is further understood that for administration to animals (including human and non-human subjects), preparations must meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards. Compounds are generally suitable for administration to humans and non-humans. This term requires that the compound or composition is non-toxic and of sufficient purity that no additional manipulation of the compound or composition is required prior to administration to humans.

[0139] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, coloring agents, and similar substances and combinations thereof, as would be well known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences (1990), which is incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0140] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, coloring agents, and similar substances and combinations thereof, as would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences (1990), which is incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0141] Pharmaceutically acceptable carriers include, but are not limited to, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Exemplary pharma-ceutically acceptable carriers, particularly for peptides, are described in U.S. Pat. No. 5,211,657. Such preparations may routinely include salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. When used as pharmaceuticals, the salts should be pharma-ceutically acceptable, but salts that are not pharma-ceutically acceptable may be conveniently used to prepare pharma-ceutically acceptable salts thereof and are not excluded from the scope of the present invention. Such pharmacologically and pharma-ceutically acceptable salts include, but are not limited to, those prepared from acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. Pharmaceutically acceptable salts may also be prepared as alkali metal or alkaline earth salts, such as sodium, potassium, or calcium salts.

[0142] Typically, by way of example and not limitation, the compositions of the present invention are preferably formulated for single administration from a single container or tablet, rather than multiple administrations, although such multiple administrations are within the scope of the present invention. To formulate the compositions of the present invention, a weight fraction of the PI3 kinase inhibitor and / or agent or other composition of the present invention is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is alleviated or improved. Pharmaceutical carriers or vehicles suitable for administration of compositions such as PI3 kinase inhibitors and / or agents can include any such carriers known to those skilled in the art to be suitable for a particular mode of administration.

[0143] The agent may include various types of carriers depending on whether it is administered in solid, liquid, or aerosol form, and whether it needs to be sterile for the route of administration, such as injection. The present invention may be administered intravenously, intradermally, intraarterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesically, intramucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, infusion, continuous infusion, localized irrigation to directly wash target cells, via catheter, via lavage, in a cream, in a lipid composition (e.g., liposomes), or by other methods or combinations thereof that would be known to one of skill in the art. (See, e.g., Remington's Pharmaceutical Sciences (1990), which is incorporated herein by reference.) In certain embodiments of the present invention, intraperitoneal injection is contemplated.

[0144] The pharmaceutical compositions of the present invention can include various antioxidants to retard the oxidation of one or more components. Additionally, the prevention of the action of microorganisms can be brought about by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0145] The drug may be incorporated into the composition in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, for example, those formed with free amino groups of the protein composition, or with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxides, or organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0146] In certain embodiments of the invention where the composition is in liquid form, the carrier can be a solvent or dispersion medium, including but not limited to water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes), and combinations thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size by dispersing in a carrier such as a liquid polyol or lipid, by the use of surfactants such as hydroxypropylcellulose, or a combination of these methods. In many cases, it is desirable to include isotonicity agents, such as sugars, sodium chloride, or combinations thereof.

[0147] The compounds or compositions of the present invention can be used directly or in admixture with suitable adjuvants and / or carriers. Suitable adjuvants include aluminum salt adjuvants such as aluminum phosphate and aluminum hydroxide, calcium phosphate nanoparticles (BioSante Pharmaceuticals, Inc.), ZADAXIN™, iron oxide nanoparticles, lipid nanoparticles, nucleotides ppGpp and pppGpp, inactivated Bordetella pertussis or components thereof, P40 components from Corynebacterium, cholera toxin and whole or parts of mycobacteria, ISCOMs (DeVries et al., 1988; Morein et al., 199&, Lovgren: al., 1991). Pam3Cys, LPS, ds and ss RNA are also useful as adjuvants. Those skilled in the art are familiar with carriers suitable for pharmaceutical use or for human use. The compositions of the present invention can be administered to different classes of recipients in a variety of ways.

[0148] The compounds and compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid or gas form, such as tablets, capsules, powders, granules, ointments, solutions, deposits, inhalants and injections, which are the usual methods for oral, parenteral or surgical administration. The present invention also encompasses pharmaceutical compositions formulated for local administration, such as implants.

[0149] The compounds and compositions of the present invention can be dissolved, suspended, dispersed or otherwise mixed in the selected vehicle at an effective concentration such that the treated condition is alleviated or ameliorated. Pharmaceutical carriers or vehicles suitable for administration of PI3 kinase antagonists and / or inhibitors include such carriers known to those skilled in the art to be suitable for the particular mode of administration.

[0150] In any event, the compositions and compounds may contain various antioxidants to retard the oxidation of one or more components. Additionally, the prevention of the action of microorganisms may be brought about by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0151] The drug may be incorporated into the composition in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, for example, those formed with free amino groups of the protein composition, and those formed with inorganic acids such as hydrochloric acid and phosphoric acid, and organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, or organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

[0152] In embodiments of the invention where the composition is in liquid form, the carrier can be a solvent or dispersion medium, including but not limited to water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes), and combinations thereof. Proper fluidity can be maintained, for example, by the use of a coating agent such as lecithin, by maintaining the required particle size by dispersion in a carrier such as a liquid polyol or lipid, by the use of surfactants such as hydroxypropylcellulose, polyethylene glycol (PEG), or combinations thereof. In many cases, it is desirable to include isotonicity agents, such as sugars, sodium chloride, or combinations thereof.

[0153] The compounds and compositions of the present invention can be used directly or in admixture with suitable adjuvants and / or carriers. Suitable adjuvants include aluminum salt adjuvants such as aluminum phosphate and aluminum hydroxide, calcium phosphate nanoparticles (BioSante Pharmaceuticals, Inc.), ZADAXIN™, nucleotides ppGpp and pppGpp, inactivated Bordetella pertussis or components thereof, P40 components from Corynebacterium, cholera toxin and whole or parts of mycobacteria, ISCOMs (DeVries et al., 1988; Morein et al., 199&, Lovgren: al., 1991). Also useful as adjuvants include polyinosinic acid:polycytidylic acid (poly I) Pam3Cys, LPS, double-stranded and single-stranded RNA. Those skilled in the art are familiar with carriers suitable for pharmaceutical use or for human use. The compositions of the present invention can be administered to various types of recipients in a variety of ways.

[0154] The formulations of the present invention are administered in pharma- ceutically acceptable solutions, which may routinely contain pharma- ceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

[0155] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Exemplary pharma- ceutically acceptable carriers, particularly for peptides, are described in U.S. Patent No. 5,211,657. Such preparations may routinely include salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. When used as pharmaceuticals, the salts should be pharma- ceutical acceptable, but salts that are not pharma- ceutical acceptable may be conveniently used to prepare pharma- ceutical acceptable salts thereof and are not excluded from the scope of the present invention. Such pharmacologically and pharma- ceutical acceptable salts include, but are not limited to, those prepared from acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. Pharmaceutically acceptable salts may also be prepared as alkali metal or alkaline earth salts, such as sodium, potassium, or calcium salts.

[0156] The compounds and compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, deposits, inhalants, injections, etc., which are conventional methods for oral, parenteral, or surgical administration. The present invention also encompasses pharmaceutical compositions formulated for local administration, such as implants.

[0157] Compounds and compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active ingredient. Other compositions include suspensions in aqueous liquids or non-aqueous liquids, such as a syrup, elixir, or emulsion.

[0158] Other drugs when used in combination with the inhibitors of the present invention can be administered systemically.

[0159] For oral administration, the compositions and compounds can be easily formulated by combining the active ingredients with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the compounds of the present invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by the subject to be treated. Pharmaceutical preparations for oral use can be prepared using solid excipients, and the resulting mixture can be optionally milled, and after adding suitable auxiliary agents, the mixture can be processed into granules to obtain tablet or dragee cores. Suitable excipients are, in particular, sugars including lactose, sucrose, mannitol, or sorbitol, e.g., corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or fillers such as cellulose preparations such as polyvinylpyrrolidone (PVP). Disintegrants can be added, if desired, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, e.g., sodium alginate. Optionally, the oral formulations may be formulated in saline or buffers to neutralize acid conditions in the body, or may be administered without a carrier.

[0160] The sugar-coated core is provided with a suitable coating. For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and concentrated sugar solutions that may optionally contain suitable organic solvents or solvent mixtures can be used. Dyes or pigments can be added to the tablets or druggy coatings for identification or to characterize different combinations of doses of active compounds.

[0161] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin and soft sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. Push-fit capsules can contain the active ingredients mixed with fillers such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compounds can be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers can be added. Microspheres prepared for oral administration can also be used. Such microspheres are well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

[0162] For buccal administration, the compositions can take the form of tablets or lozenges formulated in conventional manner.

[0163] For administration by inhalation, the compounds used according to the present invention can be conveniently administered in the form of aerosol spray using suitable propellants, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases, from pressurized packs or nebulizers. For pressurized aerosols, dosage units can be determined by providing a valve to dispense a metered amount. Capsules and cartridges, such as gelatin capsules and cartridges, for use in inhalers or insufflators can be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch. The techniques for preparing aerosol administration systems are well known to those skilled in the art. In general, such systems should utilize ingredients that do not significantly impair the biological properties of the active agent (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp1694-1712; incorporated by reference). One of ordinary skill in the art can readily determine the various parameters and conditions for producing an aerosol without resorting to undue experimentation.

[0164] The compounds and compositions of the present invention can be formulated for parenteral administration by injection when systemic administration is desired, for example, by bolus injection or continuous infusion. Preparations for injection can be provided in unit dosage form, for example, in ampoules or multi-dose containers, with added preservatives. The compositions can take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents, such as suspending, stabilizing and / or dispersing agents.

[0165] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose). Preservatives and other additives may also be included, such as antibacterial agents, antioxidants, chelating agents, inert gases, and the like. Other forms of administration, such as intravenous administration, may allow for lower dosages. If the subject does not respond adequately to the initial dosage, higher dosages (or substantially higher dosages by another, more localized route of administration) may be employed, as long as the patient tolerates them. Multiple daily doses are contemplated to achieve adequate systemic levels of the compound.

[0166] The pharmaceutical composition of the present invention can be administered in combination with other therapeutic agents, and such administration can be performed simultaneously or sequentially before or after the other therapeutic agents. When other therapeutic agents are administered simultaneously, they can be administered in the same formulation or in separate formulations, but at the same time, at different times, or in combinations thereof. The administration of other therapeutic agents and inhibitors or antagonists can also be separated in time. That is, the therapeutic agents are administered at different times, either before or after the administration of inhibitors or antagonists. The time gap between the administration of these compounds can be a few minutes or it can be longer.

[0167] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose). Preservatives and other additives may also be included, such as antibacterial agents, antioxidants, chelating agents, inert gases, and the like. Other forms of administration, such as intravenous administration, may allow for lower dosages. If the subject does not respond adequately to the initial dosage, higher dosages (or substantially higher dosages via another, more localized route of administration) may be employed, as long as the patient tolerates them. Multiple daily doses are contemplated to achieve adequate systemic levels of the compound.

[0168] Compositions suitable for oral administration can be presented as discrete units such as capsules, tablets, lozenges, each unit containing a predetermined amount of the active ingredient. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir, or emulsion.

[0169] For oral administration, the compounds and compositions of the present invention can be easily formulated by combining the active ingredient with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the compounds of the present invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by the subject to be treated. Pharmaceutical preparations for oral use can be prepared using solid excipients, and the resulting mixture can be milled as required, and the mixture can be granulated to obtain a tablet or dragee core, after adding suitable auxiliary agents. Suitable excipients are, but are not limited to, sugars, including lactose, sucrose, mannitol, or sorbitol, fillers such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or cellulose preparations such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Optionally, the oral formulations can be formulated in saline or buffers to neutralize acid conditions in the body, or can be administered without a carrier.

[0170] The sugar-coated core is provided with a suitable coating. For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and concentrated sugar solutions that may optionally contain suitable organic solvents or solvent mixtures can be used. Dyes or pigments can be added to the tablets or druggy coatings for identification or to characterize different combinations of doses of active compounds.

[0171] Orally usable preparations include push-fit capsules made of gelatin and soft sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. Push-fit capsules can contain the active ingredients mixed with fillers such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compounds can be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers can be added. Microspheres prepared for oral administration can also be used. Such microspheres are well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

[0172] For buccal administration, the compositions can take the form of tablets or lozenges formulated in conventional manner.

[0173] For administration by inhalation, the compounds and compositions for use according to the invention can be conveniently administered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to dispense a metered amount. Capsules and cartridges, for example gelatin capsules and cartridges for use in an inhaler or insufflator, can be formulated to contain a powder mix of the compound and a suitable powder base, for example lactose or starch.

[0174] The techniques for preparing aerosol administration systems are well known to those skilled in the art. In general, such systems can utilize ingredients that do not significantly impair the biological properties of the active agent (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp1694-1712, incorporated by reference). Those skilled in the art can easily determine the various parameters and conditions for producing aerosols without resorting to undue experimentation.

[0175] The compounds and compositions of the present invention can be formulated for parenteral administration by injection when systemic administration is desired, for example, by bolus injection or continuous infusion. Preparations for injection can be provided in unit dosage form, for example, in ampoules or multi-dose containers, with added preservatives. The compositions can take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents, such as suspending, stabilizing and / or dispersing agents.

[0176] C. Manufacturing, packaging, storage of pharmaceutical compositions, kit products The present invention also includes articles of manufacture, which refers to any one or collection of component parts. In some embodiments of the present invention, the articles of manufacture include kits. These articles of manufacture include pharmaceutical or diagnostic grade compounds of the present invention in one or more containers. These articles of manufacture can include instructions or labels that promote or describe the use of the compounds of the present invention.

[0177] As used herein, "promoting" includes all methods of doing business related to the treatment of disease, related to the compositions of the invention, including educational methods, hospital and other clinical instruction, pharmaceutical industry activities including drug sales, and any advertising or other promotional activities, including all forms of written, oral and electronic communication.

[0178] "Instructions" can include a component of a promotional campaign and typically include instructions on or associated with the packaging of the compositions of the invention. Instructions also include oral or electronic instructions provided in any manner.

[0179] Thus, the agents described herein, in some embodiments of the invention, can be assembled into pharmaceutical, diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. The kits can include one or more containers housing the components of the invention and instructions for use. Specifically, such kits can include one or more agents described herein, along with instructions describing the intended therapeutic use of those agents and appropriate methods of administration. In certain embodiments of the invention, the agents in the kits can be provided in pharmaceutical formulations and dosages appropriate for the particular use and method of administration of the agents.

[0180] The kits can be designed to facilitate the use of the methods described herein by the physician and can take many forms. Each composition of the kit can be provided in liquid (e.g., solution) or solid (e.g., dry powder) form, as applicable. In certain cases, some of the compositions can be configurable or processable (e.g., into an active form), for example, by the addition of appropriate solvents or other species (e.g., water or cell culture medium), which may or may not be provided with the kit. As used herein, "instructions" can define a component of instructions and / or promotional activities, and typically includes written instructions on or associated with the packaging of the invention. Instructions can also include oral or electronic instructions provided in any manner that clearly identifies the instructions as being related to the kit, such as, for example, audiovisual (e.g., videotape, DVD, etc.), internet, and / or web-based communications. Written instructions can be in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biologics, and the instructions may also reflect agency approval of the manufacture, use, or sale for administration to humans.

[0181] The kit may include one or more containers containing any one or more of the components described herein. By way of example, in one embodiment, the kit may include instructions for mixing one or more of the components included in the kit and / or for separating or mixing samples and applying to a subject. The kit may include a container containing the agents described herein. These agents may be prepared sterilely, packaged in syringes, and shipped refrigerated. Alternatively, the kit may be contained in a vial or other container for storage. A second container may contain other agents that are prepared sterile. Alternatively, the kit may include premixed active ingredients and shipped in syringes, vials, tubes, or other containers.

[0182] The kits can be in the form of blister pouches, shrink-wrapped pouches, vacuum sealable pouches, sealable thermoformed trays, or similar pouches or trays, with the accessories loosely packed within the pouches or in one or more tubes, containers, boxes, or bags. The kits can be used without packaging the individual accessories within the containers, by sterilizing after the accessories are added. The kits can be sterilized using any suitable sterilization technique, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits can also include other components, such as, for example, containers, cell culture media, salts, buffers, reagents, syringes, needles, cloths such as gauze for applying or removing disinfectants, disposable gloves, supports for the medication prior to administration, etc., depending on the particular application.

[0183] The compositions of the kit can be provided in any suitable form, for example, as a liquid solution or a dry powder. If the composition provided is a dry powder, the powder can be reconstituted by adding a suitable solvent, which may also be provided. In embodiments where the composition is used in liquid form, the liquid form may be concentrated or ready to use. The solvent will vary depending on the compound and the method of use or administration. Solvents suitable for pharmaceutical compositions are well known and available in the literature. The solvent will vary depending on the compound and the method of use or administration. The kits, in one set of embodiments, can include carrier means compartmentalized to receive in close confinement one or more container means, such as vials, tubes, and the like, each containing one of the distinct elements used in the subject methods. For example, one of the containers can hold a positive control for the assay. Additionally, the kits can include containers for other components, such as buffers useful in the assay.

[0184] The present invention also includes pharmaceutical products that are packaged and labeled as finished products. The articles of manufacture contain the appropriate unit dosage forms in suitable containers, such as glass vials or other hermetically sealed containers. In dosage forms suitable for parenteral administration, the active ingredient is sterile and suitable for administration as a particulate-free solution. In other words, the present invention includes both parenteral solutions and lyophilized powders, each of which is sterile, the latter suitable for reconstitution prior to injection. Alternatively, the unit dosage form can be a solid formulation suitable for oral, transdermal, topical or mucosal administration.

[0185] In a preferred embodiment of the invention, the unit dosage form is suitable for intravenous, intramuscular or subcutaneous administration. The invention therefore encompasses solutions, preferably sterile, suitable for each administration route.

[0186] In another preferred embodiment of the present invention, the compositions and compositions of the present invention are stored in a container together with a biocompatible detergent (including but not limited to lecithin, taurocholic acid, cholesterol, etc.) or other proteins (including but not limited to gamma globulin, serum albumin, etc.). More preferably, the compositions of the present invention can be stored together with human serum albumin for human use and bovine serum albumin for animal use.

[0187] As with other pharmaceutical products, the packaging and containers are designed to protect the stability of the product during storage and transportation. Additionally, the products of the present invention include instructions or other informational material advising the physician, technician, or patient how to properly prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instruction means that indicate or suggest a dosing regimen, including, but not limited to, the actual dosage, monitoring procedures (such as methods for monitoring mean absolute lymphocyte count, tumor cell count, tumor size, etc.), and other monitoring information.

[0188] More specifically, the present invention provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, nebulizer, insufflator, intravenous (iv) bag, envelope, etc., and a unit dosage form of at least one pharmaceutical agent contained within the packaging material. The present invention also provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, nebulizer, insufflator, intravenous (iv) bag, envelope, etc., and at least one unit dosage form of each pharmaceutical agent contained within the packaging material. The present invention further provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, nebulizer, insufflator, intravenous (iv) bag, envelope, etc., and at least one unit dosage form of each pharmaceutical agent contained within the packaging material. The present invention further provides an article of manufacture comprising a needle or syringe, preferably packaged in a sterile form, for injecting the formulation, and / or a packaged alcohol pad.

[0189] The present invention also includes articles of manufacture, which refers to any one or collection of component parts. In some embodiments, the articles of manufacture are kits. These articles of manufacture include pharmaceutical or diagnostic grade compounds of the present invention in one or more containers. These articles of manufacture can include instructions or labels that promote or describe the use of the compounds of the present invention.

[0190] As used herein, "promoting" includes all methods of doing business related to the treatment of disease, related to the compositions of the invention, including educational methods, hospital and other clinical instruction, pharmaceutical industry activities including drug sales, and any advertising or other promotional activities, including all forms of written, oral and electronic communication.

[0191] "Instructions" can define a component of a promotional campaign and typically include written instructions on or associated with the packaging of a composition of the invention. Instructions also include oral or electronic instructions provided in any manner.

[0192] Thus, the agents described herein can be assembled into pharmaceutical or diagnostic or research kits in some aspects of the invention to facilitate their use in therapeutic, diagnostic or research applications. The kits can include one or more containers housing the components of the invention and instructions for use. Specifically, such kits can include one or more agents described herein, along with instructions describing the intended therapeutic use of those agents and appropriate methods of administration. In certain embodiments of the invention, the agents in the kits can be provided in pharmaceutical formulations and dosages appropriate for the particular use and method of administration of the agents.

[0193] The kit can be designed to facilitate the use of the methods described herein by the physician and can take many forms. Each composition of the kit can be provided in liquid (e.g., solution) or solid (e.g., dry powder) form, if applicable. In certain cases, some of the compositions can be configurable or processable (e.g., into an active form) by, for example, adding suitable solvents or other species (e.g., water or cell culture medium), which may or may not be provided with the kit.

[0194] As used herein, "instructions" can define a component of instructions and / or promotional activities, and typically include written instructions on or associated with the packaging of the present invention. Instructions can also include oral or electronic instructions provided in any manner that clearly identifies the instructions as being associated with the kit, such as, for example, audiovisual (e.g., videotape, DVD, etc.), internet, and / or web-based communication. Written instructions can be in a format prescribed by a government agency regulating the manufacture, use, or sale of drugs or biologics, and the instructions may also reflect agency approval of manufacture, use, or sale for administration to humans.

[0195] The kit can include any one or more of the components described herein in one or more containers. For example, the kit can include instructions for mixing one or more components of the kit and / or instructions for separating, mixing, and administering samples to a subject. The kit can include a container containing the agents described herein. These agents can be sterilely prepared, packaged in syringes, and shipped refrigerated. Alternatively, they can be contained in vials or other containers for storage. A second container can contain other agents that are sterilely prepared. Alternatively, the kit can include active ingredients premixed and shipped in syringes, vials, tubes, or other containers.

[0196] The kits can be in the form of blister pouches, shrink-wrapped pouches, vacuum sealable pouches, sealable thermoformed trays, or similar pouches or trays, with the accessories loosely packed within the pouches or in one or more tubes, containers, boxes, or bags. The kits can be used without packaging the individual accessories within the containers, by sterilizing after the accessories are added. The kits can be sterilized using any suitable sterilization technique, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits can also include other components, such as, for example, containers, cell culture media, salts, buffers, reagents, syringes, needles, cloths such as gauze for applying or removing disinfectants, disposable gloves, supports for the medication prior to administration, etc., depending on the particular application.

[0197] The compositions of the kit can be provided in any suitable form, for example, as a liquid solution or a dry powder. If the composition provided is a dry powder, the powder can be reconstituted by adding a suitable solvent, which may also be provided. In embodiments where the composition is used in liquid form, the liquid form may be concentrated or ready to use. The solvent will vary depending on the compound and the use or method of administration. Solvents suitable for pharmaceutical compositions are well known and available in the literature. The solvent will vary depending on the compound and the use or method of administration.

[0198] In one embodiment of the invention, the kit may include compartmentalized holding means for containing in close confinement one or more container means, such as vials, tubes, etc., each container means containing one of the individual elements used in the method. For example, one of the containers may contain a positive control for the assay. Additionally, the kit may include containers for other components, such as buffers useful in the assay.

[0199] The present invention also includes pharmaceutical products that are packaged and labeled as finished products. The articles of manufacture contain the appropriate unit dosage forms in suitable containers, such as glass vials or other hermetically sealed containers. In dosage forms suitable for parenteral administration, the active ingredient is sterile and suitable for administration as a particulate-free solution. In other words, the present invention includes both parenteral solutions and lyophilized powders, each of which is sterile, the latter suitable for reconstitution prior to injection. Alternatively, the unit dosage form can be a solid formulation suitable for oral, transdermal, topical or mucosal administration.

[0200] In a preferred embodiment of the invention, the unit dosage form is suitable for intravenous, intramuscular or subcutaneous administration. The invention therefore encompasses solutions, preferably sterile, suitable for each administration route.

[0201] In another preferred embodiment of the present invention, the compositions of the present invention are stored in a container with biocompatible detergents, including but not limited to lecithin, taurocholic acid, and cholesterol, or other proteins, including but not limited to gamma globulin and serum albumin. More preferably, the compositions of the present invention are stored with human serum albumin for human use, and with bovine serum albumin for animal use.

[0202] As with other pharmaceutical products, the packaging and containers are designed to protect the stability of the product during storage and transportation. Additionally, the products of the present invention include instructions or other informational material advising the physician, technician, or patient how to properly prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instruction means that indicate or suggest a dosing regimen, including, but not limited to, the actual dosage, monitoring procedures (such as methods for monitoring mean absolute lymphocyte count, tumor cell count, tumor size, etc.), and other monitoring information.

[0203] More specifically, the present invention provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, nebulizer, insufflator, intravenous (iv) bag, envelope, etc., and at least one unit dosage form of a pharmaceutical agent contained within the packaging material. The present invention also provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, nebulizer, insufflator, intravenous (iv) bag, envelope, etc., and at least one unit dosage form of each pharmaceutical agent contained within the packaging material. The present invention further provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, nebulizer, insufflator, intravenous (iv) bag, envelope, etc., and at least one unit dosage form of each pharmaceutical agent contained within the packaging material. The present invention further provides an article of manufacture comprising a needle or syringe, preferably packaged in sterile form, for injecting the formulation, and / or a packaged alcohol pad.

[0204] VII PI3K Inhibitors in Combination with Checkpoint Inhibitors to Treat Cancer - Methods for Treating Glioblastoma, Breast Cancer, and Triple-Negative Breast Cancer The present invention also includes methods of treating glioblastoma, breast cancer, and / or triple-negative breast cancer.

[0205] A seventh aspect of the present invention includes a method of treating a subject having cancer, including, but not limited to, a) providing a subject in need of treatment for at least one cancer, wherein the cancer is glioblastoma, breast cancer, triple negative breast cancer, or a combination thereof, b) providing at least one pharmaceutical composition, comprising: 1) a pharmacologic effective amount of at least one PI3K inhibitor in a pharmacologic acceptable carrier, and 2) a pharmacologic effective amount of at least one checkpoint inhibitor in a pharmacologic acceptable carrier, c) administering to the subject a pharmacologic effective amount of the at least one pharmaceutical composition, wherein the subject is treated for at least one cancer.

[0206] In one aspect of the invention, the method further comprises, but is not limited to, administering at least one treatment to the subject.

[0207] In another aspect of the invention, the treatment includes, but is not limited to, at least one chemotherapy, at least one immunotherapy, at least one radiation therapy, at least one cryotherapy, at least one hyperthermia, at least one laser therapy, at least one surgery, at least one nanoparticle delivery system, or a combination thereof.

[0208] In a further aspect of the invention, at least one PI3K inhibitor and a checkpoint inhibitor are provided together or separately. In one embodiment of the present invention, the at least one PI3K inhibitor includes, but is not limited to: [ka] TIFF2025511969000012.tif188153 Or a combination thereof.

[0209] In another embodiment of the present invention, the at least one checkpoint inhibitor includes, but is not limited to, a) at least one anti-CTLA-4 inhibitor, b) at least one PD-1 inhibitor, c) at least one anti-PD-L1 inhibitor, or d) a combination thereof.

[0210] In further embodiments of the present invention, anti-CTLA-4 inhibitors include but are not limited to ipilimumab.

[0211] In further aspects of the invention, PD-1 inhibitors include, but are not limited to, pembrolizumab, nivolumab, semipilimab (trade name LIBTAYO®), and dostarlimab (trade name JEMPERLI®), or combinations thereof.

[0212] In one aspect of the invention, anti-PD-L1 inhibitors include, but are not limited to, atezolizumab (trade name TECENTRIQ®), durvalumab (trade name IMFINZI®), and avelumab (trade name BAVENCIO®), or combinations thereof. EXAMPLES

[0213] Example 1: Glioblastoma In this embodiment, reference is made to FIGS.

[0214] The goal of these experiments was to determine how and whether PI3 kinase inhibitors affect glioblastoma (GBM) cells in culture. GL261 mouse glioblastoma cells were cultured at approximately 5 × 10 5 The cells were cultured in tissue culture plates at a concentration of 10 ...

[0215] The second phase of the study was to see if the compound would change the way GBM cells in culture "appear" to the immune system; in other words, whether treatment with GCT would increase the chances that T cells would become "activated" and release the "brake" on the anti-tumor response. In this experiment, cells were cultured at 5 × 10 5 The GBM cells were cultured at 1000x1000 cells / ml. The cells were treated with 1 μg / ml of GCT.Glio.1 (also known as GCT07) for 48 hours. The cells were then harvested, counted, and stained with fluorochrome-conjugated labeled antibodies against mouse MHC class II and mouse PD-L1. The cells were then harvested, washed, and analyzed by flow cytometry. The data show that treatment with GCT increases expression of PD-L1, the target of PD-1:PD-L1-targeted immune checkpoint inhibitors, on the cell surface of GBM, potentially providing a target for "releasing the brakes" by treating patients with "checkpoint inhibitors." The following experiments demonstrate that treatment with either compound results in an increase in MHC class II (Figure 2).

[0216] Figure 1: GL261 mouse GBM cells were cultured in the presence of the indicated concentrations of GCT.GLIO.1 for 24, 48 or 72 hours, after which cells were harvested and counted.

[0217] Figure 2: GL261 cells were cultured with or without GCT.GLIO.1 (also known as GCT007) for 72 hours, after which cells were harvested and stained with anti-PD-L1 as indicated. The histograms show increased cell surface expression of PD-L1 corresponding to increasing doses of GCT.GLIO.1, indicating that increased PD-L1 is a target for immune checkpoint inhibitor therapy.

[0218] Figure 3: GL261 cells were cultured for 7 days with or without GCT.GLIO.1 as indicated, harvested, and stained as indicated to look for changes in cell surface expression of PD-L1 and MHC class II, or expression of LC3, a marker of autophagy. Increased MHC class II and PD-L1 increase the likelihood that the agent will be recognized by CD4 T cells, suggesting that increased PD-L1 may be a target for immune checkpoint inhibitor therapy.

[0219] Results: These results show that while both compounds increase levels of MHC class II, compound GCT.GLIO.1 (NPT520-337) causes an increase in all three molecules, while compound GCT.GLIO.2 (NPT520-322) causes growth arrest and increased expression of MHC class II.

[0220] Example 2: Glioblastoma For this example, refer to FIGS. 4A and 4B to 6.

[0221] Effect of GCT.Glio.1 on GL261 radiosensitivity. In these experiments, we addressed the question of how or whether treatment with GCT.Glio.1 affects subsequent treatment with radiation (Figure 4A and Figure 4B). GL261 cells were cultured for 48 hours with and without 2 Gy radiation (denoted R) in the presence or absence of GCT.GLIO.1 (NPT520-337) at a concentration of 1 μg / ml. Cells were then harvested and viability of the harvested cells was assessed using trypan blue viability dye. Viability data show that GCT.Glio.1 sensitizes cells to 2 Gy, as indicated by decreased viability (increased cell death) following GCT and radiation (Figure 4A and Figure 4B).

[0222] In addition to inducing growth arrest, GCT.GLIO.1 increased cell surface PD-L1 and MHC class II (denoted as mean fluorescence intensity (MFI)), potentially making these cells better targets for immunotherapy (Figure 5).

[0223] Similar results were observed when human U251 glioma cells were treated with increasing doses of GCT.GLIO.1. We found that GCT.GLIO.1 induced growth arrest and cell death in a dose-dependent manner in these cells (Fig. 6).

[0224] Several isoform-specific PI3K inhibitors are in early clinical trials. A major problem has been drug delivery across the blood-brain barrier, but Genentech's GDC0084 is an orally available small molecule PI3K inhibitor that can cross the blood-brain barrier.

[0225] The compound, named GCT.GLIO.X, was also designed to be brain-penetrating. What's novel about GCT.GLIO.1 and GCT.GLIO.2 is that they combine the advantage of being able to cross the blood-brain barrier while also changing how GBM "sees" to the immune system.

[0226] Figure 4A and Figure 4B: Effect of GCT.GLIO.1 on GL261 radiosensitivity and cell cycle arrest. In the next experiments, we sought to identify the optimal radiation dose to achieve cell cycle arrest after treatment with GCT.Glio.1. In these studies, 5 × 10 5GL261 cells cultured at 1000 cells / ml were treated with 1 μg / ml GCT.Glio.1 for 24 h and then irradiated with increasing doses of 2, 4, and 8 Gy. Cells were then harvested and counted. The cell cycle stage of harvested cells was determined by mild detergent lysis with saponin, staining with propidium iodide, followed by mild fixation with paraformaldehyde. Stained cells were analyzed by flow cytometry set up to measure the frequency of cells arrested at each stage of the cell cycle. Retreatment with GCT.Glio.1 significantly amplified the response to radiation and significantly increased cells arrested at the G2 cell cycle checkpoint.

[0227] FIG. 5A and FIG. 5B. Human GBM cell line U251 was incubated at 5×10 5 Cells were cultured at a concentration of 100 cells / ml in complete RPMI medium containing 5% heat-inactivated fetal bovine serum, L-glutamine, HEPES buffer, and an antibiotic cocktail of penicillin and streptomycin, and treated with 1 μg / ml GCT.Glio.1, then harvested, counted, and stained with fluorochrome-conjugated antibodies against PD-L1 or MHC class II to measure changes in cell surface PD-L1 (left panel) and MHC class II (right panel).

[0228] Figure 6A and 6B. Human U251 cell line was cultured at 5 × 10 5 Cells were cultured at a concentration of 1000 cells / ml in complete RPMI medium containing 5% heat-inactivated fetal bovine serum, L-glutamine, HEPES buffer, and an antibiotic cocktail of penicillin and streptomycin. Cells were harvested and counted after treatment with 1 μg / ml GCT.Glio.1, and then assessed for changes in cell number and cell death.

[0229] Example 3: Breast Cancer In this example, reference is made to FIGS.

[0230] Breast cancer is well known to be a serious public health threat, yet despite significant efforts, it remains an area where research and development of effective treatments continues. In particular, drug-resistant breast cancer variants remain a major challenge. Despite decades of therapeutic trials, improved diagnostics, new treatments, and hundreds, if not thousands, of breast cancer clinical trials, breast cancer remains the most deadly cancer for women. The innovations and promise of immunotherapy for many tumors have not been successful in breast cancer. This is because breast cancer tumors are immunologically "silent" or "cold."

[0231] In this application, GCT.BC.1 (also known as GCT007, GCT.GLIO.1, NPT520-337) is found to be a novel small molecule PI3Kinas inhibitor, which can convert immunologically "cold" tumors to those that express many of the target molecules for immunotherapy, thus turning the tumor from "cold" to "hot" with respect to immunotherapy. Radiation penetrates deep, even to bone, can cover small to large areas, and low doses are relatively non-toxic. Thus, radiation-activated prodrugs would also reduce the amount of radiation required to control tumors, thereby significantly reducing damage to normal tissues. If the prodrugs were sequestered and washed out of normal tissues, the drug could be administered with greatly reduced risk to normal peripheral tissues.

[0232] These examples relate to in vitro data from human and mouse cell lines in breast cancer. These experiments were designed to investigate the effect of GCT.BC.1 on breast cancer. These data show that GCT.BC.1 is as effective at killing and preventing cell proliferation as other PI3K inhibitors, and can be effectively combined with other approved chemotherapy agents.

[0233] Figure 7: Cell viability of human MCF7 breast cancer cells. Human MCF7 cells were treated with 1 uM aperisib (Alp), 1 uM GCT.BC.1 (also known as GCT007, GCT.GLIO.1, NPT520-337), or DMSO control for 48 hours. Viability was determined by cleaved caspase-7 and measured using the Caspase Glo assay (Promega) according to the manufacturer's instructions.

[0234] Figure 8: Cell viability of TC11 breast cancer cells in mice. TC11 cells were treated for 48 hours with 100 nM paclitaxel (Pac), 100 nM paclitaxel + 1 uM alpelisib (Alp), 0.50 uM GCT.BC.1 (GCT), 0.50 uM GCT.Glio.1 + 100 nM paclitaxel, 0.50 uM GCT.BC.1 + 7.5 nM fulvestrant, or DMSO control as indicated. Viability was determined by cleaved caspase-7 and measured using the Caspase Glo assay (Promega) according to the manufacturer's instructions.

[0235] Figure 9: Proliferation of 66CL4 breast cancer cells in mice. 66CL4 breast cancer cells in mice were treated with the indicated doses of GCT.BC.01. Proliferation was measured using an Agilent Biotek Biospa system and Biospa software.

[0236] Example 4: Glioblastoma In this embodiment, reference is made to FIGS.

[0237] These examples relate to in vivo animal data. These experiments were designed to validate the efficacy of GCT.Glio.1 (also known as GCT007, GCT.GLIO.1, NPT520-337) in animal models of GBM and to determine the effect of combining GCT.Glio.1 with immune checkpoint inhibitors using luciferase-labeled tumors and bioluminescence to measure tumor growth in vivo. We observed that the combination of GCT.Glio.1 with anti-PD-1 significantly improved overall survival (Figure 10) and delayed tumor growth (Figure 11) compared to anti-PD-1 or GCT.Glio.1 alone.

[0238] Figure 10: Luciferase-labeled GL261 tumor cells were implanted into the caudate nucleus of C57BL6 mice on day 0. Treatment began on day 9. Treatment included no treatment, oral GCT alone, anti-PD-1 + DMSO vehicle, or oral GCT followed by checkpoint inhibitor anti-PD1 24 hours later as indicated. Tumor growth was monitored by MRI. Kaplan-Meier survival curves. Black lines indicate survival time of animals implanted with GL261, red lines indicate survival time of animals treated with GCT.Glio.1, and blue lines indicate animals treated with GCT.Glio.1 followed by 24 hours of anti-PD1 and the cycle repeated. As of day 28, 3 animals in the combination group are alive.

[0239] Figure 11: Luciferase-labeled GL261 tumor cells were implanted into the caudate nucleus of C57B16 mice on day 0. Treatment began on day 9. Treatment included no treatment, oral GCT alone, anti-PD-1 + DMSO vehicle, or oral GCT followed by checkpoint inhibitor anti-PD-1 24 hours later as indicated. GL261 tumors were measured using bioluminescence and observed daily. In the top panel, bioluminescence quantification shows that tumor growth was significantly delayed when treated with GCT.Glio.1 followed by the checkpoint inhibitor anti-PD-1.

[0240] Example 5: Parenteral Formulations In this example, reference is made to a formulation that can be used in the examples.

[0241] Figure 12: Structures of various compounds mentioned in the Examples. (R)-NPT520-232 refers to GCT.2 (GCT.Glio.2), (-)-NPT520-337 refers to GCT.1 (GCT.Glio.1), (-)-NPT520-338 refers to GCT.Glio.3 and GCT.BR.1.

[0242] Precautions for in vitro and in vivo formulations of NPT520-337 enantiomer 2 1. N81520-337: 10 mM stock in DMSO for in vitro use Weigh out compound. Use compound molecular weight to calculate amount of DMSO to add to make 10 mM stock in DMSO. Add this amount and mix by vortexing. Store 10 mM stock at -20°C or -80°C and thaw when ready to use. Compound stocks in DMSO are stable through repeated freeze-thaw cycles. Compounds have been confirmed to be soluble up to 10 uM in aqueous buffers / vehicles.

[0243] 2. BPT520-337: 10 mg / kg and vehicle (oral administration) in vivo studies Composition of excipients: 7,5X NMP, 7.5%-solutol HS-15, 30% PEG-400, 55*(0.95NaCl) saline.

[0244] 3. Preparation of GCT.1 Add the required amount of NMP to the vial containing the compound (using a pipette) and heat the solution to 65-70 °C until the drug is completely dissolved in the NMP, then vortex for 30 s.

[0245] The required amount of PEG-400 was added (using a pipette) and the solution was heated for 2 min. Vortex. Solution should be clear with no precipitate, cloudiness, or turbidity. Add half the required amount of saline (by pipette) and vortex for 30 seconds. Do not heat.

[0246] Add the required amount of Solutol-Hs and vortex for 30 seconds. Rinse the tip twice with solution, making sure the Solutol is completely submerged in solution. Vortex. Add the remaining saline and vortex for 30 seconds.

[0247] 4. NPYS20-337(82) Vehicle Preparation: a. Prepare the vehicle by adding NMP, PEG, Solutol Hs, and saline, adding each component one at a time and vortexing 30 seconds after each addition. b. The vehicle is clear. Notes: 1. Solutol-Hs is an opaque paste. Place the paste in a scintillation vial and heat to 50-60°C until it becomes liquid. At 2.10 mg / mL, the suspension was cloudy. MW=391.44 IM-391.44g / L 10mM.3.9144mg / mL 21.39 3,914H1m9 21.3mg

[0248] Example 6: Triple-negative breast cancer In this embodiment, reference is made to FIGS.

[0249] This example demonstrates that endocrine receptor positive cells develop resistance to endocrine therapy via activation of semaphorin 7A and PI3K.

[0250] Cell culture protocol: Cells were plated at approximately 1 million cells / plate in 10 cm plates and cultured overnight to allow attachment. The cell lines and media used were McCoy's 5a modified medium for the HER2+ breast cancer cell line SKBR3 cells and DMEM for MDA-MB-231, MDA-MB-468, MCF-7, and HS578t. Cells were then treated with PI3K inhibitors: 0uM (control), 1uM, or 10uM. Treated cells were cultured for approximately 72 hours. After culture, cells were imaged, harvested with 0.25% trypsin / EDTA, and counted with trypan blue viability staining using a Nexcelom Cellometer Mini. The MDA-MB-231 cell line is an epithelial human breast cancer cell line established from the pleural effusion of a patient with metastatic breast adenocarcinoma.

[0251] The aim of the following study was to determine whether GCT007 (also called NPT520-337, NPT520-337 / ENT2, or GCT.Br.1), a potent PI3K inhibitor, could reverse semaphorin 7A-dependent resistance to endocrine therapy in breast cancer. SEMA7A is a membrane-bound protein that can inhibit tumor cell death via integrin-mediated activation of PI3K / Akt pro-survival signals. The Traci Lyons lab has shown that semaphorin 7a (SEMA7A) is a potential biomarker for endocrine therapy resistance, increased recurrence, and shorter overall survival in ER+ BC patients. Researchers have found that SEMA7A promotes tumor growth, angiogenesis and lymphangiogenesis, epithelial-mesenchymal transition (EMT), metastasis, and endocrine therapy resistance in preclinical models. Specifically, we show that SEMA7A+ER+MCF7 tumors are resistant to the endocrine therapy, fulvestrant, in part via reduced expression of ER in vivo, leading to lung metastasis. This creates a need to identify novel drug targets for SEMA7A+ER+BC. Thus, the following data support the use of GCT007 as a novel therapeutic agent for endocrine-resistant breast cancer. See, e.g., Figures 13 and 14.

[0252] The purpose of the following study was to test the hypothesis that treatment with GCT007 (also called NPT520-337 / ENT2, or GCT.Br.1) increases cell surface expression of important cell surface receptors, including human epidermal growth factor 2 (HER2). HER2 is a key receptor that provides a necessary drug target for treatment with the breast cancer drug Herceptin™. Thus, treatment with GCT007 may be able to sensitize HER2-negative and triple-negative breast cancer cells to Herceptin™, providing a new breast cancer treatment strategy that combines GCT007 and Herceptin™. See, for example, Figures 15, 16, 17, and 18.

[0253] Figures 13A, 13B, and 13C: SEMA7A overexpressing (OE) cells are poorly responsive to endocrine therapy but become sensitive upon addition of PI3K inhibition. MCF7EV and SEMA7A OE cells were treated with A) fulvestrant (7.5 nM), B) alpelisib (1 μM), or C) the combination for 48 hours.

[0254] 14A, 14B, 14C, 14D, and 14E: Cells expressing SEMA7A are sensitive to both alpelisib and a novel PI3K inhibitor (GCT.BR.1). FIG. 14A) MCF7 cells treated with vehicle, 1 μM alpelisib, and 1 μM GCT.BR.1. Scale bar = 10 microns. FIG. 14B) Quantitative analysis of A. FIG. 14C) MCF7 S7 OE spheroids in suspension were treated with vehicle (DMSO), 1 μM alpelisib, or 0.5 μM GCT and stained with calcein green viability stain 48 hours later. FIG. 14D) Tumor volume in NCG mice bearing MCF7 SEMA7A OE treated every 5 days with fulvestrant starting on day 30 (arrows). FIG. 14E) Size-matched graph.

[0255] Figure 15: SK-BR-3 is a human breast cancer cell line that overexpresses the Her2 (Neu / ErbB-2) gene product. These cells display epithelial morphology in tissue culture and can form poorly differentiated tumors in immunodeficient mice. MDA-MB-231 is a highly aggressive, invasive, and poorly differentiated triple-negative breast cancer (TNBC) cell line that lacks expression of estrogen receptor (ER) and progesterone receptor (PR) as well as amplification of HER2 (human epidermal growth factor receptor 2).

[0256] Figure 16: SKBR3 cells treated with GCT.BR.1 at the indicated doses. Histogram showing changes in HER2 expression analyzed by flow cytometry. Treatment with GCT007 dose-dependently increased HER2 expression in triple-negative SKRB3 breast cancer cells.

[0257] Figure 17: Triple-negative breast cancer cell line MDA MB 231 cells treated with the indicated doses of GCT.BR.1 (GCT007). Histogram showing changes in HER2 expression by flow cytometry. Treatment with GCT007 increased HER2 expression in breast cancer 231 cells in a dose-dependent manner.

[0258] FIG. 18: Quantification of median fluorescence intensity of HER-2 expression in 231 cells.

[0259] The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and can be practiced or carried out in various ways. Moreover, the phraseology and terminology used herein are for the purpose of description and should not be considered as limiting. The use of "including," "comprising," "having," "containing," "involving," and variations thereof herein means to include the items listed thereafter and equivalents thereof as well as additional items. The terms "comprising," "consisting essentially of," and "consisting of," particularly in the claims, refer to the legal meaning of these terms as set forth in, for example, Section 2111.03 of the USTPO Manual of Patent Examining Procedure (MPEP). Briefly, the transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim and closes the claim to the inclusion of materials other than those recited, except for impurities ordinarily associated therewith. Additionally, the transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0260] All publications, including patent documents and scientific articles, mentioned in this application and in the references and appendices are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference.

[0261] All headings and titles are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless specifically stated.

Claims

1. A pharmaceutical composition for use in a method of treating a subject with cancer, The PI3K inhibitor is administered to the subject in a pharmaceutically acceptable carrier in a pharmaceutically effective amount of at least one PI3K inhibitor, wherein the PI3K inhibitor is administered to the subject in a pharmaceutically acceptable carrier in a pharmaceutically effective amount of at least one checkpoint inhibitor. Optionally, further comprising administering at least one treatment to the subject, A pharmaceutical composition in which the treatment comprises chemotherapy, immunotherapy, radiotherapy, laser therapy, surgery, or a nanoparticle delivery system, or a combination thereof.

2. The at least one PI3K inhibitor and the checkpoint inhibitor are provided together or separately. Optionally, at least one of the PI3K inhibitors, 【Chemistry 1】 【change】 or a combination thereof, Optionally, at least one of the PI3K inhibitors, 【Chemistry 2】 The pharmaceutical composition according to claim 1, or a combination thereof.

3. The at least one checkpoint inhibitor is a) At least one anti-CTLA-4 inhibitor, b) At least one PD-1 inhibitor, c) At least one anti-PD-L1 inhibitor, or d) The pharmaceutical composition according to claim 1, comprising a combination thereof.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the cancer is glioblastoma, breast cancer, or triple-negative breast cancer.

5. The at least one PI3K inhibitor is 【Chemistry 2】 A pharmaceutical composition according to any one of claims 1 to 3, or a combination thereof.

6. The at least one PI3K inhibitor is 【Chemistry 2-1】 A pharmaceutical composition according to any one of claims 1 to 3, comprising or a salt thereof.

7. The pharmaceutical composition according to claim 3, wherein the anti-CTLA-4 inhibitor comprises ipilimumab.

8. The pharmaceutical composition according to claim 3, wherein the PD-1 inhibitor comprises pembrolizumab, nivolumab, semiprimab (trade name LIBTAYO®), and dostallimab (trade name JEMPERLI®), or a combination thereof.

9. The pharmaceutical composition according to claim 3, wherein the anti-PD-L1 inhibitor comprises atezolizumab (trade name TECENTRIQ®), durvalumab (trade name IMFINZI®), and avelumab (trade name BAVENCIO®), or a combination thereof.

10. a) A pharmaceutically effective amount of at least one PI3K inhibitor in a pharmaceutically acceptable carrier, and b) A pharmaceutical composition comprising a pharmaceutically effective amount of at least one checkpoint inhibitor in a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10, wherein the at least one PI3K inhibitor and the checkpoint inhibitor are provided together or separately.

12. The at least one PI3K inhibitor is 【Transformation 3】 The pharmaceutical composition according to claim 10, or comprising a salt thereof.

13. The at least one checkpoint inhibitor is a) At least one anti-CTLA-4 inhibitor, b) At least one PD-1 inhibitor, c) At least one anti-PD-L1 inhibitor, or d) Combinations of those Includes, Optionally, the anti-CTLA-4 inhibitor includes ipilimumab. Optionally, the PD-1 inhibitor may include pembrolizumab, nivolumab, semiprimab (trade name LIBTAYO®), and dostallimab (trade name JEMPERLI®), or a combination thereof. The pharmaceutical composition according to claim 10, wherein the anti-PD-L1 inhibitor optionally comprises atezolizumab (trade name TECONTRIQ®), durvalumab (trade name IMFINZI®), and avelumab (trade name BAVENCIO®), or a combination thereof.

14. A pharmaceutical composition as defined in claim 4, for use in a method of treating a subject having cancer, Optionally, further comprising administering at least one treatment to the subject, Optionally, a pharmaceutical composition in which the treatment comprises at least one chemotherapy, at least one immunotherapy, at least one radiotherapy, at least one cryotherapy, at least one hyperthermia, at least one laser therapy, at least one surgery, at least one nanoparticle administration system, or a combination thereof.

15. A method for screening at least one test compound, at least one procedure, or a combination thereof, for the ability to make at least one cancer cell more sensitive to one or more therapies for reducing the number of cancer cells in a cell population, a. To provide one or more cancer cells that express one or more markers, b. To provide one or more of the following: at least one test compound, at least one procedure, or a combination thereof. c. Contacting one or more cancer cells with one or more test compounds, d. Measuring the change in the expression of one or more markers by one or more cancer cells in the presence or absence of one or more test compounds, A method for reasonably predicting that changes in the expression of one or more markers in one or more cancer cells in the presence of one or more test compounds, compared to the absence of one or more test compounds, indicate that one or more cancer cells are more sensitive to one or more treatments for reducing the number of cancer cells.

16. The test compound, procedure, or combination thereof comprises at least one chemical compound, at least one chemotherapy, at least one immunotherapy, at least one radiotherapy, at least one cryotherapy, at least one hyperthermia, at least one laser therapy, at least one surgery, at least one nanoparticle administration system, or a combination thereof. Optionally, the nanoparticle administration system includes at least one scintillator. Optionally, the nanoparticle administration system includes at least one cleavable linker, The method according to claim 15, wherein optionally the cleavable linker is located between the drug and the nanoparticles.

17. A pharmaceutically acceptable carrier contains a pharmaceutically effective amount of the compound according to claim 15, Optionally, include at least one additional cancer treatment. Optionally, a pharmaceutical composition for use in a method of treating a subject having cancer, wherein the treatment includes chemotherapy, immunotherapy, radiotherapy, laser therapy, surgery, nanoparticle delivery systems, or a combination thereof.

Images