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

Abstract

The present invention has recognized that a method for treating cancer has been required for a long time. A first aspect of the invention generally relates to methods of screening for cancer treatments. A second aspect of the invention relates generally to compounds for the treatment of cancer. A third aspect of the invention relates generally to pharmaceutical compositions for the treatment of cancer. A fourth aspect of the invention generally relates to methods of treating cancer. A fifth aspect of the invention relates generally to PI3K inhibitors and checkpoint inhibitors for the treatment of cancer. A sixth aspect of the invention generally relates to methods of treating cancer using PI3K inhibitors with checkpoint inhibitors or other immunotherapies. The seventh aspect of the invention relates generally to methods of treating glioblastoma, breast cancer, and / or triple negative breast cancer using PI3K inhibitors with checkpoint inhibitors or other immunotherapies.

Description

[0001] Priority declaration

[0002] This application:

[0003] Claims the benefit of now-expired U.S. Provisional Application No. 63 / 329,096, filed on April 8, 2022; and

[0004] Claiming the benefit of U.S. Provisional Application No. 63 / 368,499, filed on July 15, 2022, currently pending;

[0005] Each of these applications is incorporated herein by reference in its entirety. Technical Field

[0006] The present invention relates generally to the field of cancer treatment. Background Art

[0007] Glioblastoma multiforme (GBM) is a serious and aggressive central nervous system cancer with no reported cure. Unlike breast and lung cancer, its survival has not improved much in the past 40 years. GBM can progress from an existing astrocytoma, or develop de novo. About 90% of GBMs occur in patients with an average age of 64 years as primary tumors, but they can occur at any age. Current treatment involves resection, if possible, combined with chemotherapy, radiation, steroids, and / or viral therapy. Despite treatment, the median survival is 9-22 months.

[0008] One of the most promising new strategies for treating many cancers is to develop therapies that target the body’s own immune system, or components of it, to fight the disease. Promising approaches within immunotherapy include drugs that target “immune checkpoint” molecules. For example, cancer-free survival rates for patients with advanced melanoma who are treated with immune checkpoint inhibitor (ICI) drugs range from 30%-40%. While this is a positive outcome for some melanoma patients, the question remains why a large proportion of melanoma patients (approximately 60%-70%) do not respond well to other blockbuster ICI drugs. Similarly, the question remains why ICI drugs are somewhat effective in treating some forms of cancer, but not as effective as those who receive temozolomide (TMZ) or Avastin. TM There was no improvement in outcomes for GBM patients treated with ICI drugs compared with GBM patients treated with (currently used GBM treatments).

[0009] The phosphatidylinositol 3-kinase (PI3K) / AKT / mTOR pathway is an important intracellular signaling pathway that is involved in a variety of biological processes, including proliferation, apoptosis, angiogenesis, and glucose metabolism. The pathway is initiated by the binding of extracellular growth factors to receptor tyrosine kinases (RTKs), upon which PI3K is activated and subsequently activates serine / threonine protein kinase B (PKB or AKT), leading to the phosphorylation of a series of downstream substrates that mediate cellular responses. Overactivation of the (PI3K) / Akt pathway is common in cancers, including glioblastoma and breast cancer, and the pathway plays a central role in regulating tumor cell survival, growth, angiogenesis, and metabolism. Many PI3K inhibitors, including pan-PI3K inhibitors, isoform-selective inhibitors, and dual PI3K / mTOR inhibitors, have shown promising preclinical results and have entered clinical trials for a range of malignancies. Currently, several PI3K inhibitors are in Phase I / II clinical trials for breast cancer.

[0010] The core problem that limits the use of PI3K inhibitors for the treatment of breast cancer, glioblastoma multiforme and diffuse intrinsic pontine glioma (DIPG) is the potential toxicity of these drugs throughout the body. A potential therapeutic solution is to produce only local high drug concentrations at the tumor tissue site, thereby protecting normal tissues from toxic effects. Many cancer researchers have proposed the concept of prodrugs and focused on potential activators (such as tumor-specific enzymes, light or heat), but this brings multiple problems. For example, enzymes are usually unevenly distributed, difficult to obtain, and are usually not completely tumor-specific. Heat and light are difficult to accurately apply to deep tissue structures, and blood flow may disperse and dissipate energy deposition.

[0011] Recent studies of tumor cell expression of major histocompatibility complex class II (MHCII) molecules have identified, at least in part, why ICI drugs have failed to effectively treat some melanoma patients and may also explain why ICI drugs have been ineffective to date for treating GBM. Specifically, recent studies have shown that patients whose tumors respond to ICI therapy are those that express cell surface MHC class II molecules. It is well known that MHCII expression at the cell surface provides the first (and perhaps essential) signal for T cell activation, a key cellular event in the antitumor immune response, and under appropriate conditions, can lead to cell death of the MHCII-expressing cells. These MHCII-related studies have important implications for improving the potential efficacy of ICI drugs in treating various cancers and generally support the hypothesis that MHC II expression on GBM cells, combined with blockade of immune checkpoints, may provide a missing component that may be necessary to increase the frequency with which patients respond to ICI drugs via effective antitumor immunity. In the paper titled “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 MHCII or were able to induce MHC II expression. They found that HLA-DR expression correlated with a positive response to PD-1-targeted immune checkpoint therapy. They also found that HLA-DR expression was positively correlated with the percentage of CD4+ T cell infiltration. While the authors hypothesized that CD4+ helper cells were involved in this process, they did not identify the involvement of subsets of CD4+ cells, including poor T regs that suppress anti-tumor T cell responses.

[0012] MHC II-encoded molecules are usually present on professional antigen presenting cells (APCs), including macrophages, dendritic cells, and B cells, but many cells can be induced to express these molecules and have the potential to act as APCs. In humans, MHC class II molecules are called human leukocyte antigen gene complexes (HLA Dr). MHC II molecules present antigens (Ag) in their grooves to T cell receptors (TCRs) expressed on T lymphocytes. T cell activation also requires a second co-signaling molecule, which is provided by the interaction between presenting cells and T cells. Under the action of some co-stimulatory signals, T cells become activated. Under the action of other signals, the expanded CD4+T cells are regulatory and appear to inhibit conventional T cell activation, which is most likely achieved through the interaction between PD1 and PDL-1. Summary of the invention

[0013] The present invention recognizes that there has been a long felt need for methods of treating cancer.

[0014] A first aspect of the invention generally relates to methods of screening for cancer treatments.

[0015] A second aspect of the present invention generally relates to compounds for use in treating cancer.

[0016] A third aspect of the present invention generally relates to pharmaceutical compositions for treating cancer.

[0017] A fourth aspect of the invention generally relates to methods of treating cancer.

[0018] A fifth aspect of the invention generally relates to pharmaceutical compositions comprising a PI3K inhibitor and a checkpoint inhibitor or other immunotherapy.

[0019] A sixth aspect of the invention generally relates to methods of treating cancer using PI3K inhibitors with checkpoint inhibitors or other immunotherapies.

[0020] A seventh aspect of the invention generally relates to methods of treating glioblastoma, breast cancer and / or triple-negative breast cancer using a PI3K inhibitor with a checkpoint inhibitor or other immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 G1261 mouse GBM cells were cultured in the presence of the indicated concentrations of GCT.GLIO.1 for 24, 48, or 72 hours. The cells were then harvested and counted.

[0022] Figure 2 GL261 cells were cultured with or without GCT.GLIO.1 for 72 hours, harvested, and stained with anti-PD-L1 as indicated. The histograms demonstrate that increases in cell surface PD-L1 expression correspond to increasing doses of GCT.GLIO.1 and suggest that increased PD-L1 could provide a target for immune checkpoint inhibitor therapy.

[0023] Figure 3 GL261 cells were cultured with and without GCT.GLIO.1 for 7 days as indicated, harvested, and stained as indicated for changes in cell surface expression of PD-L1 and MHC class II; or for expression of the autophagy marker LC3. Increases in MHC class II and PD-L1 indicate that the drug will increase the likelihood of CD4 T cell recognition and that increased PD-L1 will provide a target for immune checkpoint inhibitor therapy.

[0024] Figure 4A and Figure 4BEffect of GCT.Glio.1 on Radiosensitivity and Cell Cycle Arrest of GL261. In the next experiment, we sought to identify the optimal radiation dose following GCT.Glio.1 treatment. In these studies, we treated with GCT.Glio.1 for 24 hours and then increased radiation doses, including 2, 4, and 8 Gy. The results showed that re-treatment with GCT.Glio.1 significantly enhanced the response to radiation and resulted in a significant increase in cells arrested at the G2 cell cycle checkpoint, see Figures 5 to 6.

[0025] Figure 5A and Figure 5B The human GBM cell line U251 was 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.

[0026] Fig. 6A and Figure 6B Human U251 cell line was treated with GCT.Glio.1, harvested, counted, and evaluated for changes in cell number and dead cells. The results showed that GCT.Glio.1 induced growth arrest, which increased with increasing compound dose (left panel) and increased dead cell numbers (right panel).

[0027] Figure 7 .Cell viability of human MCF7 breast cancer cells. Human MCF7 cells were treated with 1 uM apellis (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) according to the manufacturer's instructions.

[0028] Figure 8 Cell viability of mouse TC11 breast cancer cells. TC11 cells were treated with 100 nM paclitaxel (Pac), 100 nM paclitaxel + 1 uM apellis (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 for 48 hours. Viability was determined by cleaved caspase-7 and measured using the Caspase Glo assay (Promega) according to the manufacturer's instructions.

[0029] Fig. 9.Proliferation of 66CL4 mouse breast cancer cells. 66CL4 mouse breast cancer cells were treated with the indicated doses of GCT.BC.01. Proliferation was determined using the Agilent Biotek Biospa system and Biospa software.

[0030] Fig.10 .On day 0, luciferase-labeled GL261 tumor cells were implanted into the caudate nucleus of C57Bl6 mice. Treatment started on day 9. Treatments included no treatment, oral administration of GCT alone, anti-PD-1 plus DMSO vehicle, or oral administration of GCT and 24 hours later the indicated checkpoint inhibitor anti-PD1. Tumor growth was confirmed by MRI. Kaplan Meier survival curves. The black line represents the animal survival of animals implanted with GL261; the red line represents the survival of animals treated with GCT.1; the blue line represents animals treated with GCT.1 and 24 hours later with anti-PD1 (and so on). As of day 28, 3 animals in the combination group were still alive.

[0031] Fig.11 .Luciferase-labeled GL261 tumor cells were implanted into the caudate nucleus of C57Bl6 mice on day 0. Treatments began on day 9. Treatments included no treatment, oral administration of GCT alone, anti-PD-1 plus DMSO vehicle, or oral administration of GCT and 24 hours later the indicated checkpoint inhibitor anti-PD1. GL261 tumors were measured using bioluminescence and monitored daily. In the upper panel, bioluminescence quantification demonstrates that tumor growth was significantly delayed when animals were treated with GCT.1 followed by the checkpoint inhibitor anti-PD-1.

[0032] Fig. 12A , Fig. 12B and Fig. 12C . The structures of the 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.

[0033] Fig.13A , Fig. 13B and Fig. 13C SEMA7A overexpressing (OE) cells responded poorly to endocrine therapy but became sensitive after the addition of Pl3K inhibition. MCF7EV and SEMA7A OE cells were treated with Fig.13A ) Fulvestrant (7.5nM), Fig. 13B ) Apellis (1uM) or Fig. 13C ) combination for 48 hours.

[0034] Fig.14A , Fig. 14B , Fig. 14C , Fig.14D and Fig.14E . Cells expressing SEMA7A are sensitive to both apellisib and a novel PI3K inhibitor (GCT.BR.1). Fig.14A ) MCF7 cells treated with vehicle, 1 uM Apellisin, and 1 uM GCT.BR.1. Scale bar = 10 microns. Fig. 14B )Quantification of A. Fig. 14C ) MCF7 S7OE spheroids in suspension were treated with vehicle (DMSO), 1 uM apellisin or 0.5 uM GCT and stained with Calcein green viability dye after 48 h. Fig.14D ) Tumor volume in NCG mice bearing MCF7 SEMA7 A OE that were treated with fulvestrant every 5 days starting from day 30 (arrows). Fig.14E ) of matching sizes.

[0035] Fig.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 are able to form poorly differentiated tumors in immunocompromised mice. MDA-MB-231 is a highly aggressive, invasive, and poorly differentiated triple-negative breast cancer (TNBC) cell line as it lacks estrogen receptor (ER) and progesterone receptor (PR) expression, as well as HER2 (human epidermal growth factor receptor 2) amplification.

[0036] Fig.16 SKBR3 cells were treated with the indicated doses of GCT.BR.1. The histogram depicts changes in HER2 expression analyzed by flow cytometry. Treatment with GCT007 resulted in a dose-dependent increase in HER2 expression on triple-negative SKRB3 breast cancer cells.

[0037] Fig.17 .231 cells were treated with the indicated doses of GCT.BR.1 (GCT007). The histogram depicts changes in HER2 expression analyzed by flow cytometry. Treatment with GCT007 resulted in a dose-dependent increase in HER2 expression on breast cancer 231 cells

[0038] Fig.18 Quantification of median fluorescence intensity of HER-2 expression on .231 cells. DETAILED DESCRIPTION

[0039] definition

[0040] Unless otherwise defined, the meaning of all technical terms and scientific terms used herein is the same as that generally understood by those of ordinary skill in the art to which the present invention belongs. In general, the nomenclature used herein and the laboratory procedures in cell culture, chemistry, microbiology, molecular biology, cell science, flow cytometry and cell culture described below are well-known and commonly used in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references, such as conventionally known in the art. Where a term is provided in singular form, the inventors also contemplate the plural form of the term. The nomenclature used herein and the laboratory procedures described below are well-known and commonly used in the art. As used throughout the disclosure, unless otherwise indicated, the following terms are understood to have the following meanings:

[0041] "Direct" refers to the direct cause and effect of a process, which requires no intermediate steps.

[0042] "Indirect" refers to indirect causal relationships that require intermediate steps.

[0043] "Subject" refers to a human or vertebrate mammal, including but not limited to dogs, cats, horses, goats, and primates, e.g., monkeys. Thus, the present invention can also be used to treat cancer in non-human subjects. Preferably, the subject is a human.

[0044] When used with respect to a condition, "treat," "treated," or "treating" refers to both prophylactic treatment, which increases a subject's resistance to developing a disease, or in other words, reduces the likelihood that a subject will develop a disease, as well as treatment after a subject has developed a disease in order to combat the disease, prevent the disease from getting worse, or slow the progression of the disease compared to what would occur in the absence of therapy.

[0045] Other technical terms used herein have their ordinary meanings used in the art as exemplified by various technical dictionaries.

[0046] introduce

[0047] The present invention recognizes that there has been a long felt need for methods of treating cancer.

[0048] As a non-limiting introduction to the invention, the invention includes several general and useful aspects, including:

[0049] 1) Methods for screening cancer treatments;

[0050] 2) Compounds for treating cancer;

[0051] 3) Pharmaceutical compositions for treating cancer;

[0052] 4) Methods for treating cancer;

[0053] 5) Pharmaceutical compositions comprising PI3K inhibitors and checkpoint inhibitors or other immunotherapies;

[0054] 6) Methods of treating cancer using PI3K inhibitors in combination with checkpoint inhibitors or other immunotherapies; and

[0055] 7) Methods of using PI3K inhibitors with checkpoint inhibitors or other immunotherapies to treat glioblastoma, breast cancer, and / or triple-negative breast cancer.

[0056] These aspects of the invention, as well as other aspects described herein, can be achieved by using the methods, articles, and compositions of matter described herein.To fully appreciate the scope of the invention, it will be further appreciated that various aspects of the invention can be combined to form desired embodiments of the invention.

[0057] I Methods for screening cancer treatments

[0058] The invention includes methods of screening for cancer therapeutics.

[0059] 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 the 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:

[0060] a. Providing one or more cancer cells expressing one or more markers;

[0061] b. providing one or more of said at least one test compound, at least one procedure, or a combination thereof;

[0062] c. contacting the one or more cancer cells with the one or more test compounds;

[0063] d. measuring changes 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;

[0064] wherein a change in expression of the one or more markers in the one or more cancer cells in the presence of the one or more test compounds relative to the absence of the one or more test compounds reasonably predicts that the one or more cancer cells are more susceptible to the one or more treatments for reducing the number of cancer cells.

[0065] 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 aspect of the invention, the activity of an inhibitor or antagonist is determined by using various experimental animal models, including but not limited to cancer animal models, such as immunocompetent wild-type animals implanted with genetically compatible tumors; SCID mouse models or nude mice with human tumor transplants.

[0066] Various in vitro and in vivo assays to test the activity of inhibitors or antagonists are used during the purification of the inhibitors or antagonists.The regimens 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.

[0067] Compounds for use in therapy can be tested in appropriate and acceptable or accepted animal model systems, including but not limited to rats, mice, chickens, cows, monkeys, rabbits, etc., prior to testing in humans.

[0068] Toxicity and efficacy of the prophylactic and / or therapeutic regimens of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index and can be expressed as the ratio LD 50 / ED 50 Preferred are prophylactic and / or therapeutic agents that exhibit large therapeutic indices. While prophylactic and / or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue to minimize potential damage to uninfected cells and thereby reduce side effects.

[0069] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for the prophylactic and / or therapeutic agents for use in humans. The dosage of such agents preferably lies within the range that includes the ED 50 The dose may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC determined in cell culture. 50 (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.

[0070] In certain aspects of the invention, the pharmaceutical composition may include, for example, at least about 0.1% of the active compound. In other aspects of the invention, the active compound may account for between about 2% to about 75% of the unit weight, or for example between about 25% to about 60%, and any range derivable therein.

[0071] The compounds and compositions described herein can be used alone or conjugated to other molecules (such as detection agents or cytotoxic agents) in the detection and treatment methods of the invention, as described in more detail herein.

[0072] Typically, one of the components may include a detectable label, or be coupled or conjugated to a detectable label for specific binding assays known in the art. A detectable label is a portion whose presence can be determined directly or indirectly. In some cases, detection of a label involves the energy emission of the label. The label can be directly detected by its ability to emit and / or absorb photons or other atomic particles of a specific wavelength (e.g., radioactivity, luminescence, optical or electron density, etc.). The label can be indirectly detected by its ability to bind, recruit, and in some cases cut another portion, which itself can emit or absorb light of a specific wavelength (e.g., epitope tags, such as FLAG epitopes, enzyme tags, such as horseradish peroxidase, etc.). An example of indirect detection is the use of a first enzyme label that cuts a substrate into a visible product. The label can have chemical, peptide or nucleic acid molecule properties, but is not limited thereto. Other detectable labels include: radioisotopes, such as P 32 or H 3 ); luminescent markers, such as fluorescent dyes, optical or electron density markers, etc.; or epitope tags, such as FLAG epitope or HA epitope, biotin, avidin; and enzyme tags, such as horseradish peroxidase, β-galactosidase, etc. The label can be bound to the peptide during or after peptide synthesis. There are many different labels and labeling methods known to those of ordinary 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 ordinary skill in the art will be aware of other suitable labels for the peptides described herein, or will be able to determine these labels using routine experiments. In addition, the coupling or conjugation of these labels to the peptides of the present invention can be performed using standard techniques commonly used by those of ordinary skill in the art.

[0073] Another class of detectable labels includes, but is not limited to, diagnostic and imaging labels (often referred to as in vivo detectable labels), such as magnetic resonance imaging (MRI): Gd(DOTA); for nuclear medicine: 201Tl, γ-radionuclide 99mTc; for positron emission tomography (PET): positron emitting isotopes, (18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride, copper-64, gadolinium and radioactive isotopes of Pb(II), such as 203Pb; 111In.

[0074] The conjugation or modification described herein employs conventional 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 monobifunctional linkers and heterobifunctional linkers) is well documented in the literature and will not be described in detail here.

[0075] As used herein, "conjugation" means two entities that are stably bound to each other by any physicochemical means. Importantly, the nature of the connection is such that it does not significantly impair the effectiveness of either entity. Taking these parameters into account, any covalent or non-covalent connection known to those of ordinary skill in the art may be used. In some aspects of the invention, covalent attachment is preferred. Non-covalent conjugation includes hydrophobic interactions, ionic interactions, high-affinity interactions (such as biotin-avidin and biotin-streptavidin complexes) and other affinity interactions. Such connection modes and methods are well known to those of ordinary skill in the art.

[0076] Depending on the nature of the label and other assay components, various methods can be used to detect the label. For example, the label can be detected when it is bound to a solid substrate or after separation from the solid substrate. The label can be directly detected by optical or electron density, radioactive emission, non-radiative energy transfer, etc., or indirectly detected with antibody conjugates, streptavidin-biotin conjugates, etc. Methods for detecting labels are known in the art.

[0077] A. Test Compounds, Procedures, or Combinations thereof

[0078] One aspect of the invention includes wherein the test compound, procedure, or a combination thereof comprises at least one chemical compound, at least one chemotherapy, at least one immunotherapy, at least one radiation therapy, at least one cryogenic treatment, at least one hyperthermia, at least one laser, at least one surgery, at least one nanoparticle delivery system, or a combination thereof.

[0079] Another aspect of the present invention includes wherein the nanoparticle delivery system comprises at least one scintillator.

[0080] Another aspect of the invention includes wherein the nanoparticle delivery system comprises at least one cleavable linker.

[0081] Another aspect of the invention includes wherein the cleavable linker is located between the drug and the nanoparticle.

[0082] B. Cancer cells

[0083] One aspect of the invention includes wherein the cancer cells are derived from endoderm, mesoderm, ectoderm, or a combination thereof.

[0084] Another aspect of the invention includes wherein the cancer cell comprises an epithelial cancer cell, a sarcoma cell, a leukemia cell, a lymphoma cell, a myeloma cell, or a combination thereof.

[0085] Another aspect of the invention includes wherein the cancer cell comprises a skin cell, a melanoma cell, a non-melanoma cell, a brain cell, a prostate cell, a breast cell, a colorectal cell, a kidney cell, a bladder cell, a non-Hodgkin lymphoma cell, a thyroid cell, an endometrial cell, a head cell, a neck cell, a brain cell, a glioblastoma cell, or a combination thereof.

[0086] C. More susceptible to treatment

[0087] Another aspect of the invention includes wherein the greater susceptibility to treatment includes the cancer cells being more susceptible to cell death, having a reduced growth rate, having an increased doubling time, being more susceptible to DNA damage, having a poorer ability to repair DNA damage, being more likely to be recognized by immune cells, or a combination thereof.

[0088] D. Treatment to reduce the number of cancer cells

[0089] One aspect of the invention includes wherein the treatment for reducing the number of cancer cells comprises at least one chemotherapy, at least one immunotherapy, at least one radiation therapy, at least one cryogenic treatment, at least one hyperthermia treatment, at least one laser treatment, at least one surgery, at least one nanoparticle delivery system, or a combination thereof.

[0090] E. Cell population

[0091] Another aspect of the invention includes wherein the cell population comprises a pure culture, a mixed culture, a biopsy, in vivo, in situ, or a combination thereof.

[0092] F. Markers

[0093] Another aspect of the invention includes wherein the one or more markers include one or more of the following:

[0094] i.PD-L1;

[0095] ii. MHC class II;

[0096] iii.CD80;

[0097] iv.CD86;

[0098] v.CD40;

[0099] vi.CD95;

[0100] vii. MHC Class I

[0101] viii.Lag-3

[0102] ix.Vista

[0103] x.LC3;

[0104] 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, CD166, and combinations thereof; and

[0105] xii. Their combinations.

[0106] G. Measuring changes in expression

[0107] Another aspect of the invention includes wherein said measuring changes in expression comprises immunological methods, nucleic acid methods, proteomic methods, flow cytometric methods, genomic methods, or a combination thereof pure culture, mixed culture, biopsy, or a combination thereof.

[0108] H. Changes in expression

[0109] One aspect of the invention includes wherein the change in expression comprises an increase, a decrease, or a combination thereof from a pure culture, a mixed culture, a biopsy, or a combination thereof.

[0110] I. Reasonable prediction

[0111] Another aspect of the invention includes wherein the reasonable prediction includes a statistically significant difference at a p-value of less than 0.10, 0.05, or acceptable to one skilled in the art.

[0112] J. Further testing

[0113] Another aspect of the invention includes further comprising testing the cancer cells with the one or more test compounds and one or more treatments for reducing the number of cancer cells.

[0114] II Compounds used to treat cancer

[0115] The present invention also includes compounds for use in the treatment of cancer.

[0116] A second aspect of the present invention comprises a compound identified by the method of aspect I of the present invention.

[0117] III Pharmaceutical composition for treating cancer

[0118] The present invention includes pharmaceutical compositions for treating cancer.

[0119] The third aspect of the present invention includes a pharmaceutical composition comprising: a pharmaceutically effective amount of a compound of the present invention in a pharmaceutically acceptable carrier.

[0120] One aspect of the present invention includes the pharmaceutical compositions of the present invention further comprising at least one additional cancer treatment.

[0121] Another aspect of the invention includes the pharmaceutical compositions of the invention, wherein the treatment comprises at least one chemotherapy, at least one immunotherapy, at least one radiotherapy, at least one hypothermia, at least one hyperthermia, at least one laser, at least one surgery, at least one nanoparticle delivery system, or a combination thereof.

[0122] Another aspect of the present invention includes a pharmaceutical composition comprising:

[0123] At least one pan-PI3K inhibitor;

[0124] Pitiliximab (GDC-0941);

[0125] Copanex (BAY 80–6946);

[0126] Taselis (GDC-0032);

[0127] Sonolis (PX-866);

[0128] Genentech GDC 0084;

[0129]

[0130] or a combination thereof.

[0131] IV Methods of treating cancer

[0132] The invention also includes methods of treating cancer.

[0133] A fourth aspect of the invention includes a method of treating a subject having cancer, the method comprising:

[0134] a. providing a subject in need of treatment for at least one cancer;

[0135] b. providing at least one compound or pharmaceutical composition of the present invention or a combination thereof;

[0136] c. administering to the subject a pharmaceutically effective amount of at least one compound;

[0137] wherein the subject is being treated for the at least one cancer.

[0138] One aspect of the invention further comprises administering at least one therapy to said subject.

[0139] Another aspect of the invention includes wherein the treatment comprises at least one chemotherapy, at least one immunotherapy, at least one radiation therapy, at least one cryogenic treatment, at least one hyperthermia treatment, at least one laser treatment, at least one surgery, at least one nanoparticle delivery system, or a combination thereof.

[0140] V PI3K inhibitors and checkpoint inhibitors or other immunotherapies for the treatment of cancer - drug combinations

[0141] The present invention includes PI3K inhibitors with checkpoint inhibitors or other immunotherapies for treating cancer. The present invention may include other components described herein, such as but not limited to CAR-T cells and antibodies.

[0142] A fifth aspect of the present invention includes a pharmaceutical composition comprising:

[0143] a) a pharmaceutically effective amount of at least one PI3K inhibitor in a pharmaceutically acceptable carrier; and

[0144] b) a pharmaceutically effective amount of at least one checkpoint inhibitor or other immunotherapy, including CAR T cells or antibodies.

[0145] One aspect of the invention includes wherein the at least one PI3K inhibitor and the checkpoint inhibitor are provided together or separately.

[0146] A. PI3K inhibitors and agonists

[0147] In some aspects of the invention, PI3K inhibitors may also be administered alone or in combination with other components of the invention.

[0148] Any PI3K inhibitor can be used in the present invention. Many PI3K inhibitors are disclosed herein, and additional PI3K inhibitors are disclosed in the following documents: U.S. Published Patent Application No. 2021 / 0163462A1 to Stocking et al., published on June 3, 2021; U.S. Patent No. 11,492,348 to Stocking et al., published on November 8, 2022; WO 2009 / 066087 to Hoffman-La Roche et al., published on May 28, 2009; WO 2007 / 084786 to Novartis, published on July 26, 2007; U.S. Patent No. 8,921,361 to Cmiljanovic et al., published on December 30, 2014; Each of these documents is incorporated herein by reference in its entirety.

[0149] Preferred PI3K inhibitors are disclosed in U.S. Patent No. 11,492,348, issued on November 8, 2022 to Stocking et al., such as, but not limited to, the following:

[0150] In one aspect, the present disclosure provides a compound of formula (I):

[0151]

[0152] in

[0153] R 1 For –(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; wherein

[0154] m, n, o and p are each independently 0, 1 or 2;

[0155] R a , R b , R c , R d , R e , R f , R g and R h are each independently H, halogen or C 1-4 alkyl,

[0156] or R a and R b together with the carbon to which it is attached, form a cycloalkyl ring,

[0157] or R a and R b Together they form =CH2 or =O;

[0158] Exists in R 1 Each aryl, heteroaryl, heterocycloalkyl or cycloalkyl in is unsubstituted or substituted by one or two R x Substituent substitution;

[0159] Each R x The substituents are independently halogen, C 1-4 Alkyl, cycloalkyl, -C 1-2 -Halogenated alkyl, -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 halide), -CO2C 1-4 alkyl, -CO2 aryl, -C(O)CH2-aryl (optionally substituted with methyl or halogen), -CH2-aryl (optionally substituted with methyl or halogen) or monocyclic heterocycloalkyl (optionally substituted with methyl, -C(O)C 1-4 Alkyl or –CO2C 1-4 Alkyl substitution);

[0160] Where R i , R j , R k and R l Each independently is H, C 1-4 Alkyl, -C 1-4 Alkyl-OH or -C 1-4 Alkyl-OC 1-4 alkyl,

[0161] Where R q and R r Each independently is H, C 1-4 Alkyl, -C 1-4 Alkyl-OH, -C 1-4 Alkyl-OC 1-4 Alkyl, -C(O)C 1-4Alkyl, -CO2C 1-4 Alkyl or -SO2C 1-4 alkyl;

[0162] 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 it is attached, they form a cycloalkyl ring;

[0163] X is O, S, NH, N(CO2C 1-4 alkyl), N(SO2C 1-4 alkyl), N(SO2 cycloalkyl) or CH2;

[0164] Y1, Y2 and Y3 are each independently CH or N; wherein when L is not -S(O)2-, Y2 and Y3 are each CH;

[0165] G2 is N or CR 2 ;

[0166] G3 is N or CR 3 ;

[0167] G4 is N, NR 4b or CR 4a ;

[0168] G5 is N or CR 5 ;and

[0169] G6 is N or CR 6 ;

[0170] Where 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 ;

[0171] or R 4b With R 6 and the atoms to which they are attached together form a heteroaryl ring or a heterocyclic ring; wherein R 4b and R 6 The heteroaryl ring includes no more than one N and is optionally substituted with alkyl, and includes R 4b and R6 The heterocyclic ring is optionally substituted by an oxo group,

[0172] R u H or C 1-4 alkyl;

[0173] R v H, C 1-4 Alkyl, monocyclic cycloalkyl, -C(O)C 1-4 Alkyl or -C(O)NR w R y ;

[0174] Among them, there are v Each alkyl group in the alkyl group is unsubstituted or replaced by –OH, -NH2, -NH(C 1-4 alkyl) or –N(C 1-4 Alkyl)2 substituted,

[0175] R w and R y Each independently is H or C 1-4 alkyl;

[0176] in is unsubstituted phenyl; and

[0177] R 7 and R 8 are independently hydrogen or C 1-4 alkyl,

[0178] or R 7 and R 8 Together they form -CH2CH2-;

[0179] or a pharmaceutically acceptable salt thereof.

[0180] In certain embodiments, the compound of Formula (I) is a compound selected from those species described or exemplified in the detailed description herein.

[0181] In some embodiments of Formula (I) or any variation thereof, R 1 For –(CR a R b ) m -aryl. In some embodiments, R 1 (CR c R d ) n -heteroaryl. In some embodiments, R 1 (CR e R f ) o -heterocycloalkyl or (CR g R h )p -cycloalkyl.

[0182] In some embodiments of Formula (I) or any variation thereof, L is -S(O)2-. In some embodiments, L is -C(O)-, -CH2-, -CF2-, C(CH3)2, -C(=CH2)-, 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.

[0183] In other aspects there are provided compounds of formula (II):

[0184]

[0185] in

[0186] X is O, S, NH, N(CO2C 1-4 alkyl), N(SO2C 1-4 alkyl), N(SO2 cycloalkyl) or CH2;

[0187] Y1, Y2 and Y3 are each independently CH or N;

[0188] G2 is N or CR 2 ;

[0189] G3 is N or CR 3 ;

[0190] G4 is N, NR 4b or CR 4a ;

[0191] G5 is N or CR 5 ;and

[0192] G6 is N or CR 6 ;

[0193] Where 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 ;

[0194] or R 4b With R 6and the atoms to which they are attached together form a heteroaryl ring or a heterocyclic ring; wherein R 4b and R 6 The heteroaryl ring includes no more than one N and is optionally substituted with alkyl, and includes R 4b and R 6 The heterocyclic ring is optionally substituted by an oxo group,

[0195] R u H or C 1-4 alkyl;

[0196] R v H, C 1-4 Alkyl, monocyclic cycloalkyl, -C(O)C 1-4 Alkyl or -C(O)NR w R y ;

[0197] Among them, there are v Each alkyl group in the alkyl group is unsubstituted or replaced by –OH, -NH2, -NH(C 1-4 alkyl) or –N(C 1-4 alkyl)2, and

[0198] R w and R y Each independently is H or C 1-4 alkyl;

[0199] in is an unsubstituted phenyl group;

[0200] R 7 and R 8 are independently hydrogen or C 1-4 alkyl,

[0201] or R 7 and R 8 Together they form -CH2CH2-;

[0202] R 9a and R 9b are each independently hydrogen or halogen;

[0203] R 10 , R 11 , R 12 , R 13 and R 14 are independently hydrogen, halogen, -OH, -CN, -alkyl, -Oalkyl, -haloalkyl, heterocycloalkyl, -O-haloalkyl, -SO2C 1-4 Alkyl or –NR aa R bb ;

[0204] Raa For hydrogen, C 1-4 Alkyl or -C 1-4 Alkyl-OH;

[0205] R bb is hydrogen or C 1-4 alkyl;

[0206] or R 9a With R 10 and the inserted atoms together form a heteroaryl ring or a heterocyclic ring;

[0207] or R 11 With R 12 and the atoms to which they are attached together form a heteroaryl ring or a heterocyclic ring;

[0208] or a pharmaceutically acceptable salt thereof.

[0209] In other aspects there are provided compounds of formula (III):

[0210]

[0211] in

[0212] R 1 For –(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, wherein when L is SO2, present in R 1 The heteroaryl and heterocycloalkyl in are each a monocyclic ring;

[0213] m is 0 or 2;

[0214] n, o and p are each independently 0, 1 or 2;

[0215] R a , R b , R c , R d , R e , R f , R g and R h are each independently H, halogen or C 1-4 alkyl,

[0216] or R a and Rb together with the carbon to which it is attached, form a cycloalkyl ring,

[0217] or R a and R b Together they form =CH2 or =O;

[0218] Exists in R 1 Each aryl, heteroaryl, heterocycloalkyl or cycloalkyl in is unsubstituted or substituted by one or two R x Substituent substitution;

[0219] Each R x The substituents are independently halogen, C 1-4 Alkyl, cycloalkyl, -C 1-2 -Halogenated alkyl, -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 halide), -CO2C 1-4 alkyl, -CO2 aryl, -C(O)CH2-aryl (optionally substituted with methyl or halogen), -CH2-aryl (optionally substituted with methyl or halogen) or monocyclic heterocycloalkyl (optionally substituted with methyl, -C(O)C 1-4 Alkyl or –CO2C 1-4 Alkyl substitution);

[0220] Where R i , R j , R k and R l Each independently is H, C 1-4 Alkyl, -C 1-4 Alkyl-OH or -C 1-4 Alkyl-OC 1-4 alkyl,

[0221] Where R q and R r Each independently is 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 -SO2C1-4 alkyl;

[0222] or R 1 for

[0223] in

[0224] R 10 , R 11 , R 12 , R 13 and R 14 are independently hydrogen, halogen, -OH, -CN, -alkyl, -Oalkyl, -haloalkyl, heterocycloalkyl, -O-haloalkyl, -SO2C 1-4 Alkyl or –NR aa R bb ;

[0225] R aa For hydrogen, C 1-4 Alkyl or -C 1-4 Alkyl-OH;

[0226] R bb is hydrogen or C 1-4 alkyl;

[0227] or R 10 With R 11 and the atoms to which they are attached together form a heteroaryl ring or a heterocyclic ring;

[0228] or R 11 With R 12 and the atoms to which they are attached together form a heteroaryl ring or a heterocyclic ring;

[0229] 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 it is attached, they form a cycloalkyl ring;

[0230] X is O, S, NH, N(CO2C 1-4 alkyl), N(SO2C 1-4 alkyl), N(SO2cyclo-alkyl), or CH2;

[0231] Y1, Y2 and Y3 are each independently CH or N; wherein when L is not -S(O)2-, Y2 and Y3 are each CH;

[0232] G2 is N or CR2 ;

[0233] G3 is N or CR 3 ;

[0234] G4 is N, NR 4b or CR 4a ;

[0235] G5 is N or CR 5 ;and

[0236] G6 is N or CR 6 ;

[0237] Where 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 ;

[0238] or R 4b With R 6 and the atoms to which they are attached together form a heteroaryl ring or a heterocyclic ring; wherein R 4b and R 6 The heteroaryl ring includes no more than one N and is optionally substituted with alkyl, and includes R 4b and R 6 The heterocyclic ring is optionally substituted by an oxo group,

[0239] R u H or C 1-4 alkyl;

[0240] R v H, C 1-4 Alkyl, monocyclic cycloalkyl, -C(O)C 1-4 Alkyl or -C(O)NR w R y ;

[0241] Among them, there are v Each alkyl group in the alkyl group is unsubstituted or replaced by –OH, -NH2, -NH(C 1-4 alkyl) or –N(C 1-4 Alkyl)2 substituted,

[0242] R w and R y are independently H or C 1-4 alkyl;

[0243] in is unsubstituted phenyl; and

[0244] R 7 and R 8 are independently hydrogen or C 1-4 alkyl,

[0245] or R 7 and R 8 Together they form -CH2CH2-;

[0246] or a pharmaceutically acceptable salt thereof.

[0247] 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.

[0248] In some embodiments of any of the compounds of Formula (I), (II) or (III), X is O. In some embodiments, X is NH, N(CO2C 1-4 alkyl), N(SO2C 1-4 alkyl) or N(SO2 cyclo-alkyl).

[0249] In some embodiments of compounds of any of Formula (I), (II) or (III), G2 and G4 are each N, and G6 is CR 6 In some embodiments of compounds of any of Formula (I), (II) or (III), G3 is CR 3 And G5 is CR 5 In some embodiments of compounds of any of Formula (I), (II), or (III), one of G2 and G4 is N.

[0250] In some embodiments of compounds of any of Formula (I), (II) or (III), R 6 For –NR u R v In some embodiments of compounds of any of Formula (I), (II) or (III), R 4b With R 6 Together with the atoms to which they are attached, they form a heteroaryl ring or a heterocyclic ring.

[0251] On the other hand, the present disclosure provides a pharmaceutical composition comprising at least one compound of formula (I), (II) or (III) or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to the embodiment may further comprise a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition is provided, comprising (a) at least one compound of formula (I), (II) or (III) or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable excipient.

[0252] PI3K inhibitors can be administered in combination with other anticancer agents (such as checkpoint inhibitors, CAR T cells or antibodies). PI3K inhibitors can be administered by any appropriate formulation, dosage, route of administration and regimen. Although the PI3K inhibitors of the present invention are preferably administered topically, they can be administered by any appropriate route of administration. Any other agent can be administered by any appropriate route of administration, including systemic or local.

[0253] PI3K antagonists and PI3K inhibitors can also be used alone or in combination with other components of the present invention. Any PI3K agonist can be used in the present invention. Many PI3K agonists are disclosed herein, and additional PI3K agonists are disclosed in PCT patent application WO 2019 / 199865 and U.S. Patent No. 11,492,348 published in the United States, each of which is incorporated herein by reference in its entirety.

[0254] B. Checkpoint inhibitors

[0255] In some aspects of the invention, checkpoint inhibitors may also be administered alone or in combination with other components of the invention.

[0256] Any checkpoint inhibitor can be used in the present invention. Many checkpoint inhibitors are disclosed herein, and additional checkpoint agonists are disclosed in, for example, PMID: 27054314 and https: / / doi.org / 10.3389 / fimmu.2020.01508, each of which is incorporated herein by reference in its entirety.

[0257] 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 that play important roles in all stages of T cell and other cell function. CTLA-4, cytotoxic T lymphocyte-associated protein 4 (CD152), is involved in controlling T cell proliferation.

[0258] The PD-1 receptor is expressed on the surface of activated T cells (and B cells) and, under normal circumstances, binds to its ligands (PD-L1 and PD-L2) expressed on the surface of antigen presenting cells (such as dendritic cells or macrophages). This interaction signals the T cell to inhibit it. Cancer cells exploit this system by driving high levels of PD-L1 expression on their surface. This allows cancer cells to gain control of the PD-1 pathway and shut down PD-1-expressing T cells that can enter the tumor microenvironment, thereby suppressing the anti-cancer immune response. Pembrolizumab (formerly known as MK-3475 and lambrolizumab, marketed as ) is a human antibody used in cancer immunotherapy and targets the PD-1 receptor.

[0259] Some aspects of the present invention further relate to administering a checkpoint inhibitor to a subject. In some aspects of the present invention, the checkpoint inhibitor may be an anti-CTLA4 antibody or an antigen-binding fragment thereof selected from anti-PD1 antibodies or antigen-binding fragments thereof that specifically bind to CTLA4, anti-PD1 antibodies or antigen-binding fragments thereof that specifically bind to PD1, anti-PD-L1 antibodies or antigen-binding fragments thereof that specifically bind to PD-L1, antibodies selected from anti-VISTA antibodies or antigen-binding fragments thereof that specifically bind to VISTA, or a combination thereof. In other aspects of the present invention, the checkpoint inhibitor may be an anti-PD-L1 antibody selected from atezolizumab, avelumab, durvalumab, or a combination thereof. In other aspects of the present invention, the checkpoint inhibitor is an anti-CTLA-4 antibody, which may be selected from tremelimumab or ipilimumab, or a combination thereof. In yet other embodiments of the present invention, the checkpoint inhibitor may be an anti-PD1 antibody, which may be selected from nivolumab or pembrolizumab, or a combination thereof.

[0260] In some aspects of the invention, the checkpoint inhibitor 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, CHK 1, 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, CHK 1, CHK2, A2aR, and B-7 family ligands, or combinations thereof. In some aspects of the invention, the anti-PD-1 antibody may be BMS-936558 (nivolumab). In other aspects of the invention, the anti-CTLA-4 antibody may be ipilimumab (trade name: In another aspect of the invention, the checkpoint inhibitor may be J43 (anti-PD1 antibody), RMP1-14 (anti-PD1 antibody), atezolizumab ( In yet other aspects of the present invention, the checkpoint inhibitor may be pembrolizumab.

[0261] Pembrolizumab is a potent humanized immunoglobulin G4 monoclonal antibody with high binding specificity for the PD-1 receptor, thereby inhibiting its interaction with PD-L1 and programmed cell death 1 ligand 2. Based on preclinical in vitro data, pembrolizumab has high affinity and potent receptor blocking activity for PD-1. Pembrolizumab has an acceptable preclinical safety profile and is currently in clinical development as an IV immunotherapy for advanced malignancies. (pembrolizumab) is approved for the treatment of patients with multiple indications. Pembrolizumab is approved for several cancer types and is being studied in several stages of clinical development for additional cancers.

[0262] 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, cemiplimab (trade name: ) and dotalizumab (trade name: ) or a combination thereof. Preferred anti-PD-L1 inhibitors include but are not limited to atezolizumab (trade name: ), durvalumab (trade name ) and avelumab (trade name: ) or a combination thereof.

[0263] In some aspects of the invention, the invention comprises administering at least one immune checkpoint inhibitor, as described herein or otherwise known. In some aspects of the invention, a combination of immune checkpoint inhibitors is administered.

[0264] C.CAR-T cells

[0265] In some aspects of the invention, cell therapy (such as CAR-T cells) may also be administered alone or in combination with other components of the invention.

[0266] Any suitable CAR-T cell can be used in the present invention.Many CAR-T cells are disclosed herein, and additional CAR-T cells are known in the literature.

[0267] Preferably, PI3K inhibitors can be administered with such CAR-T cells. As used herein, CAR-T cells refer to T cells into which chimeric antigen receptors have been introduced to redirect receptor specificity to selected antigens. Such receptors include an extracellular domain combined with a cytoplasmic signaling domain that recognizes antigens independent of MHC restrictions. Many different proteins can be introduced into T cells as extracellular domains of chimeric antigen receptors. Examples include, but are not limited to, nanobodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, human antibodies, antibody fragments, or combinations thereof.

[0268] CAR-T cells can be cells (e.g., T cells) engineered to express CAR, wherein CAR-T cells preferably exhibit anti-tumor properties. CAR can be engineered to include an extracellular domain peptide fused to an intracellular signaling domain of a T cell antigen receptor complex ζ chain (e.g., CD3ζ). When expressed in T cells, CAR can redirect antigen recognition according to antigen binding specificity. Antigen binding peptides are preferably fused with intracellular domains from one or more of the costimulatory molecules and ζ chains. In some aspects of the invention, antigen binding peptides are fused with one or more intracellular domains selected from the group consisting of: CD137 (4-1BB) signaling domain, CD28 signaling domain, CD3ζ signaling domain, and combinations thereof.

[0269] In some aspects of the invention, the invention relates to administering antigen-specific CAR-T cells to a subject with cancer in the presence or absence of a PI3 kinase inhibitor. For example, the CAR-T cells may have a modified PI3 kinase receptor, an effective amount of which may be used to treat cancer.

[0270] D. Antibodies against cancer cells

[0271] Another form of anti-cancer therapy involves the administration of antibodies specific for cell surface antigens, for example, of cancer cells.

[0272] In some aspects of the invention, antibodies directed against cancer cells may also be administered alone or in combination with other components of the invention.

[0273] Any suitable antibody against cancer cells may be used in the present invention.Many antibodies against cancer cells are disclosed herein, and additional antibodies against cancer cells are known in the art.

[0274] In one aspect of the invention, the antibody can be Ributaxin, Herceptin, Rituximab, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, 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, CMA676, Monopharm-C, 4B5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1D10 Ab, SMART ABL364Ab, 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 can be obtained from commercial sources or can be synthesized de novo.

[0275] Other cancer therapies and their dosages, routes of administration, and recommended uses are known in the art and have been described in such literature.

[0276] VI PI3K inhibitors with checkpoint inhibitors or other immunotherapies, including CAR-T cells for cancer treatment or antibody

[0277] The invention also includes methods of treating cancer.

[0278] A sixth aspect of the 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 invention or a combination thereof; and c) administering to the subject a pharmaceutically effective amount of at least one compound; wherein the subject is treated for the at least one cancer.

[0279] In some aspects, the present invention provides methods for treating cancer (including improving one or more symptoms) in subjects refractory to existing monotherapy, comprising administering to the subject a therapeutically effective amount of a composition comprising an inhibitor or antagonist and a therapeutically effective amount of one or more therapeutic agents other than the inhibitor or antagonist. The present invention also provides methods for treating cancer by administering a combination of a composition comprising an inhibitor or antagonist and any other anti-cancer treatment (e.g., checkpoint inhibitors, CAR-T cells, radiotherapy, chemotherapy, or surgery) to patients who have been proven to be refractory to other treatments or who may benefit from adjuvant therapy. The present invention also provides methods for treating patients who have cancer and are immunosuppressed due to having previously received one or more other cancer therapies. The present invention also provides alternative methods for treating cancer in cases where chemotherapy, radiotherapy, hormone therapy, and / or biological therapy / immunotherapy have been proven or can be proven to be too toxic (e.g., causing unacceptable or intolerable side effects to the subject being treated).

[0280] A. Subjects

[0281] The subject can be any suitable subject, including but not limited to humans and non-human primates. Companion animals (such as but not limited to cats and dogs) are also subjects. Any test animal from mice to humans is also considered a subject of the present invention.

[0282] B. Type of cancer to be treated

[0283] Cancers that can be treated by the present invention include, but are not limited to, neoplasms, malignancies, metastases, or any disease or condition characterized by uncontrolled cell growth such that it is considered cancerous. Cancer can be primary or metastatic cancer. Such cancers further include, but are not limited to: brain cancer, including glioblastoma and medulloblastoma; bile duct cancer; bladder cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; blood tumors, including acute lymphoblastic leukemia and myeloid leukemia; multiple myeloma; AIDS-related leukemia and adult T-cell leukemia lymphoma; intraepithelial neoplasms, including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphoma, including Hodgkin's disease (Hodgkin's disease) and lymphocytic lymphoma; neuroblastoma; oral cancer, including squamous cell carcinoma; ovarian cancer, including those caused by epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcoma, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi's sarcoma, basal cell carcinoma and squamous cell carcinoma; testicular cancer, including reproductive tumors such as seminoma, non-seminoma, teratoma, choriocarcinoma; stromal tumors and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma; and renal cancer, including adenocarcinoma and Wilms' tumor. Common cancers include breast cancer, prostate cancer, lung cancer, colorectal cancer and brain cancer. In some embodiments, the cancer is not gastric cancer or gastrointestinal cancer.

[0284] Preferred cancers to be treated include, but are not limited to, glioblastoma, breast cancer, triple-negative breast cancer, and combinations thereof.

[0285] Glioblastoma is the most common type of primary brain cancer. Astrocytes cells, and they grow very fast. Brain tumor cells can also inhibit Immune system As a result, patients with glioblastoma or a variant called liposarcoma have a poor long-term prognosis.

[0286] Breast cancer is a disease in which breast cells grow out of control and may form tumors. There are several types of breast cancer. If the cancer cells spread to other parts of the body, the disease is called metastatic breast cancer. About 12% of women in the United States, or 1 in 8 women, will develop breast cancer in their lifetime. It is the second most common cancer among women in the United States, after skin cancer. Breast cancer mainly occurs in women who are 50 years old or older, but men can also develop the disease. About 245,000 women and 2,200 men are diagnosed with breast cancer in the United States each year. While many risk factors that increase a woman's chance of developing breast cancer have been identified, it is not clear 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.

[0287] Triple-negative breast cancer is a type of breast cancer that does not have any of the receptors commonly found in breast cancer. ， The second is a female hormone, and the third is a protein called human epidermal growth factor (HER2).

[0288] The compounds and compositions of the present invention can also be administered to prevent progression to a tumor or malignant state. Such preventive use refers to conditions known or suspected to have previously developed into neoplasia or cancer, specifically in cases where non-tumor cell growth consisting of hyperplasia, metaplasia, or most specifically dysplasia has occurred. Hyperplasia is a controlled form of cell proliferation that involves an increase in the number of cells in a tissue or organ without significant changes in structure or function. Endometrial hyperplasia usually precedes endometrial cancer. Metaplasia is a controlled form of cell growth in which one type of adult or fully differentiated cell replaces another type of adult cell. Metaplasia can occur in epithelial cells or connective tissue cells. Typical metaplasia involves somewhat disordered metaplastic epithelium. Dysplasia is usually a precursor to cancer and is found primarily in epithelial cells; it is the most disordered form of non-tumor cell growth, involving the loss of uniformity and cell structure orientation of individual cells. Dysplastic cells usually have abnormally large, darkly stained nuclei and exhibit polymorphism. Dysplasia characteristically occurs where chronic irritation or inflammation exists and is frequently found in the cervix, respiratory tract, oral cavity, and gallbladder.

[0289] Alternatively or in addition to the presence of abnormal cell growth characterized by hyperplasia, metaplasia or dysplasia, the presence of one or more characteristics of a transformed phenotype or a malignant phenotype exhibited in vivo or in vitro by a cell sample from a patient may indicate the need for prophylactic / therapeutic administration of the compositions of the invention. Such characteristics of the transformed phenotype include morphological changes, looser underlying connections, loss of contact inhibition, loss of anchorage dependence, release of proteases, increased sugar transport, decreased serum requirements, expression of fetal antigens.

[0290] In other aspects of the invention, a patient who exhibits one or more of the following malignancy susceptibility factors is treated by administering an effective amount of the composition of the invention: a chromosomal translocation associated with malignancy (e.g., Philadelphia chromosome t(9:22) for chronic myeloid leukemia, t(14;18) for follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (a possible precursor to colon cancer), a benign monoclonal gammopathy (a possible precursor to multiple myeloma), a first degree genetic disease associated with cancer or a pro-cancer disease that exhibits a Mendelian inheritance pattern. family history (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostoses, multiple endocrine neoplasia, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, von Recklinghausen's neurofibromatosis, retinoblastoma, carotid body tumors, cutaneous melanoma, intraocular melanoma, xeroderma pigmentosum, ataxia-telangiectasia, Seddick-Donghal syndrome, albinism, Fanconi's aplastic anemia, and Bloom's syndrome; see Robbins and Angell, 1976, Basic Pathology, 2nd ed., WB Saunders Co., Philadelphia, pp. 112-113), etc.), and exposure to carcinogens (e.g., smoking and inhalation or contact with certain chemicals).

[0291] In one set of aspects of the invention, the invention includes a method of treating a subject susceptible to cancer or showing symptoms of cancer. The cancer can be primary, metastatic, recurrent or multi-drug resistant. In some cases, the cancer is drug resistant or multi-drug resistant. As used herein, "drug resistant cancer" is a cancer that is resistant to conventional, well-known cancer therapies. Examples of conventional cancer therapies include treatment of cancer with methotrexate, trimetrexate, adriamycin, taxotere, doxorubicin, 5-fluorouracil, vincristine, vinblastine, pamidronate disodium, anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab, megestrol, tamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate, and the like. A "multidrug-resistant cancer" is a cancer that is resistant to more than one type or class of cancer agents, for example, the cancer is resistant to a first drug with a first mechanism of action and a second drug with a second mechanism of action.

[0292] C. Dosage of the Pharmaceutical Composition

[0293] The active agents of the present invention may be administered to a subject in an amount effective for treating a disorder such as cancer.

[0294] For example, a "pharmaceutically effective amount" is an amount necessary or sufficient to achieve a desired biological effect. An "effective amount for autoimmune disease" may be an amount sufficient to prevent or inhibit T cell proliferation compared to levels in the absence of peptide treatment. H According to some aspects of the invention, an effective amount is an amount of a compound of the invention alone or in combination with another drug, which, when combined or co-administered or administered alone, results in a therapeutic response to the disease in the prevention or treatment of the disease. The biological effect can be an improvement and / or absolute elimination of symptoms caused by the disease. In another embodiment, the biological effect is complete elimination of the disease, as evidenced, for example, by the absence of disease symptoms.

[0295] The pharmaceutically effective amount of the compounds of the present invention in treating the diseases described herein can vary according to the specific compound used, the delivery mode of the compound, and whether it is used alone or in combination. The effective amount of any particular application can also vary according to factors such as the disease being treated, the specific compound being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a specific molecule of the present invention without unnecessary experiments. In conjunction with the teachings provided herein, by selecting among various active compounds and weighting factors (such as efficacy, relative bioavailability, patient weight, severity of adverse side effects, and preferred modes of administration), an effective preventive or therapeutic treatment regimen can be planned that does not cause substantial undesirable toxicity, but is still fully effective for treating a specific subject.

[0296] For example, a pharmaceutically effective dose should preferably produce a serum concentration of a PI3 kinase antagonist and / or inhibitor of about 0.1 ng / ml to about 50-100 μg / ml. The pharmaceutical composition should generally provide a dosage of about 0.001 mg to about 2000 mg of PI3 kinase antagonist and / or inhibitor per kg of body weight per day (such as about 0.01 mg to about 200 mg of PI3 kinase antagonist and / or inhibitor per kg of body weight per day, or about 0.1 mg to about 20 mg of PI3 kinase antagonist and / or inhibitor per kg of body weight per day, or about 1 mg to about 10 mg of PI3 kinase antagonist and / or inhibitor per kg of body weight per day, or about 1 mg to about 5 mg of PI3 kinase antagonist and / or inhibitor per kg of body weight per day). A pharmaceutical dosage unit form is prepared to provide about 1 mg to about 1000 mg (such as about 10 to about 500 mg) of the activating agent or a combination of the activating agents per dosage unit form.

[0297] The therapeutically effective dose preferably produces a serum concentration of the PI3 kinase antagonist and / or inhibitor of about 0.1 ng / ml to about 50-100 μg / ml. The pharmaceutical composition should generally provide a dosage of about 0.001 mg to about 2000 mg of PI3 kinase antagonist and / or inhibitor per kg of body weight per day (such as about 0.01 mg to about 200 mg of PI3 kinase antagonist and / or inhibitor per kg of body weight per day, or about 0.1 mg to about 20 mg of PI3 kinase antagonist and / or inhibitor per kg of body weight per day, or about 1 mg to about 10 mg of PI3 kinase antagonist and / or inhibitor per kg of body weight per day, or about 1 mg to about 5 mg of PI3 kinase antagonist and / or inhibitor per kg of body weight per day). The pharmaceutical dosage unit form is prepared to provide about 1 mg to about 1000 mg (such as about 10 to about 500 mg) of the activating agent or a combination of the activating agents per dosage unit form.

[0298] The dosage range of the compounds described herein is generally about 0.1 μg to 10,000 mg, more generally about 1 μg / day to 8000 mg, and most generally about 10 μg to 100 μg. In terms of subject body weight, a typical dosage range is about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of ranges that can be derived from the numbers listed herein, based on the above numbers, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc. can be administered. The absolute amount will depend on a variety of factors, including concurrent treatment, number of doses, and individual patient parameters, including age, physical condition, body size, and weight. These are factors well known to those of ordinary skill in the art and can be addressed only by routine experimentation. It is generally preferred to use a maximum dose, i.e., the highest safe dose based on sound medical judgment. Multiple doses of the molecules of the invention are also contemplated.

[0299] When used in combination with the therapies of the present invention, the dosage of known therapies may be reduced in some cases to avoid side effects.

[0300] In some aspects of the present invention, the present invention provides a method for treating cancer, the method comprising administering a therapeutically effective amount of a composition comprising an inhibitor or antagonist to a subject in need of such treatment. The composition of the present invention can be used, for example, as a first, second, third or fourth line cancer treatment. In some aspects of the present invention, the present invention provides a method for treating a subject's cancer (including improving its symptoms) that is refractory to one or more conventional therapies for such cancers, the methods comprising administering a therapeutically effective amount of a composition comprising an inhibitor or antagonist to the subject. When at least some significant portions of cancer cells are not killed in response to treatment or their cell division is not prevented, it can be determined that cancer is refractory to therapy. This determination can be performed in vivo or in vitro by any method known in the art for determining the effectiveness of treatment of cancer cells, in which case the art-recognized "refractory" meaning is used. In a specific aspect of the present invention, cancer is refractory when the number of cancer cells is not significantly reduced or increased.

[0301] D. Routes of Administration of Pharmaceutical Compositions

[0302] According to the method of the present invention, the compounds and compositions can be administered in the form of a pharmaceutical composition. Generally speaking, the pharmaceutical composition comprises a compound of the present invention and a pharmaceutically acceptable carrier.

[0303] "Local administration" refers to administration of an agent to a target tissue, muscle or subcutaneous location or its vicinity by a non-systemic route (e.g., by subcutaneous, intramuscular, subdermal, intradermal, subcutaneous, intraperitoneal, intraorgan (e.g., injection into cancerous tissue) or transdermal route) or any other route. Thus, local administration does not include systemic (e.g., to the blood circulation system) routes of administration, such as intravenous or oral administration. Peripheral administration means administration to the periphery (e.g., to a location on or within the patient's face, limbs, trunk, or head), as opposed to visceral or intestinal (e.g., to visceral) administration. In some embodiments, local administration is administration to the tumor site, for example, by intratumoral injection.

[0304] "Systemically administering" or "systemic administration" refers to administering an agent (e.g., by intravenous, inhalation, oral administration, etc.) to or near a central tissue (such as the blood, lungs, or intestines) that is remote from the local tissue in need of treatment. 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.

[0305] The compounds of the present invention can be administered directly to tissues. Direct tissue administration can be achieved by direct injection or other acceptable means and methods (such as but not limited to electroporation or direct contact with tissues or organs). These compounds can be administered all at once, or alternatively, they can be administered multiple times. If multiple administrations are administered, the compounds can be administered by different routes. For example, the first (or first few) administrations can be directly into the affected tissue, while subsequent administrations can be systemic.

[0306] When used in combination with the inhibitors of the invention, the other agents may be delivered systemically.

[0307] Injections are designed for local and systemic administration. Typically, a therapeutically effective dose is formulated to contain a PI3 kinase antagonist and / or inhibitor at a concentration of at least about 0.1% w / w up to about 90% w / w or higher (such as more than 1% w / w) for the treated tissue. The PI3 kinase antagonist and / or inhibitor can be administered in a single dose, or can be divided into multiple smaller doses for administration at intervals. It should be understood that the exact dose and duration of treatment vary with the treated tissue and can be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data. It should be noted that concentrations and dosage values ​​may also vary with the age of the individual being treated. It should be further understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering the formulation or supervising the administration of the formulation, and the concentration ranges described herein are exemplary only and are not intended to limit the scope or practice of the claimed formulations.

[0308] Injections are designed for local and systemic administration. Typically, a therapeutically effective dose is formulated to contain a PI3 kinase antagonist and / or inhibitor at a concentration of at least about 0.1% w / w up to about 90% w / w or higher (such as more than 1% w / w) for the treated tissue. The PI3 kinase antagonist and / or inhibitor can be administered in a single dose, or can be divided into multiple smaller doses for administration at intervals. It should be understood that the exact dose and duration of treatment vary with the treated tissue and can be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data. It should be noted that concentrations and dosage values ​​may also vary with the age of the individual being treated. It should be further understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering the formulation or supervising the administration of the formulation, and the concentration ranges described herein are exemplary only and are not intended to limit the scope or practice of the claimed formulations.

[0309] A. Regimens of Pharmaceutical Compositions

[0310] The pharmaceutical compositions of the present invention (such as, but not limited to, PI3 kinase antagonists and / or inhibitors) can be administered in a single dose, or can be divided into multiple smaller doses for administration at intervals. It should be understood that the precise dosage and duration of treatment varies with the disease state being treated, and can be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data. It should be noted that concentrations and dosage values ​​may also vary with the severity of the condition to be alleviated. It should be further understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering the composition or supervising the administration of the composition, and the concentration ranges described herein are exemplary only and are not intended to limit the scope or practice of the present invention.

[0311] The pharmaceutical composition of the present invention can be administered in a single dose, or can be divided into multiple multiple doses for administration at intervals. It should be understood that the precise dosage and duration of treatment vary with the disease state being treated, and can be determined empirically using known test protocols or by extrapolating from in vivo or in vitro test data. It should be noted that concentration and dosage values ​​may also vary with the severity of the condition to be alleviated. It should be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to individual needs and the professional judgment of the person administering the composition or supervising the administration of the composition, and the concentration ranges described herein are only exemplary and are not intended to limit the scope or practice of the claimed method.

[0312] The compounds and compositions of the present invention can be directly administered to tissues. Direct tissue administration can be achieved by direct injection. The compounds and compositions can be administered once, or alternatively, they can be administered multiple times. If multiple administrations are given, the compounds and compositions can be administered by different routes. For example, the first (or first few) administrations can be directly into the affected tissue, while subsequent administrations can be systemic.

[0313] B. Preparation of pharmaceutical compositions

[0314] According to the method of the present invention, the pharmaceutical composition of the present invention can be administered to a subject in the form of a pharmaceutical composition. Generally speaking, the pharmaceutical composition comprises a compound of the present invention and a pharmaceutically acceptable carrier.

[0315] The formulations of the present invention are administered in the form of pharmaceutically acceptable solutions, which may generally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants and optionally other therapeutic ingredients.

[0316] The pharmaceutical composition of the present invention comprises one or more medicaments of effective amount dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other adverse reactions when applied to animals (such as, for example, humans) when appropriate. In addition, for animal (including humans and non-human subjects) application, it should be understood that the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards. The compounds and compositions of the present invention are generally suitable for application to humans and non-humans. Preferably, the compound or composition is nontoxic and pure enough so that no further operation of the compound or composition is required before being applied to humans.

[0317] The pharmaceutical composition of the present invention comprises one or more medicaments of effective amount dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other adverse reactions when applied to animals (such as, for example, people) when appropriate. In addition, for animal (e.g., humans and non-human) use, it should be understood that the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards. The compound is generally suitable for application to humans and non-humans. The term requires that the compound or composition is nontoxic and pure enough so that no further operation of the compound or composition is required before being applied to humans.

[0318] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, materials like these, and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences (1990), which is incorporated by reference). Except in the case where any conventional carrier is incompatible with the active ingredient, its use in therapeutic compositions or pharmaceutical compositions or is contemplated.

[0319] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, materials like these, and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences (1990), which is incorporated by reference). Except in the case where any conventional carrier is incompatible with the active ingredient, its use in therapeutic compositions or pharmaceutical compositions or is contemplated.

[0320] Pharmaceutically acceptable carriers include, but are not limited to, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Exemplary pharmaceutically acceptable carriers for peptides are specifically described in U.S. Patent No. 5,211,657. Such preparations may generally contain salts, buffers, preservatives, compatible carriers, and optional other therapeutic agents. When used in medicine, salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may be conveniently used to prepare pharmaceutically acceptable salts thereof, and are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. In addition, pharmaceutically acceptable salts may be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0321] In general, by way of example and not limitation, the compositions of the invention are preferably formulated for single-dose administration, such as administration from a single container or pill, rather than multiple doses, although multiple such doses are included within the scope of the invention. To formulate the compositions of the invention, the weight fraction of the PI3 kinase inhibitor and / or agonist or other composition of the invention is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the condition being treated is alleviated or improved. Pharmaceutical carriers or vehicles suitable for administering compositions (such as PI3 kinase inhibitors and / or agonists) may include any such carriers known to those skilled in the art to be suitable for a particular mode of administration.

[0322] A pharmaceutical formulation may include different types of carriers depending on whether the formulation is to be administered in solid, liquid or aerosol form and whether the formulation needs to be sterile for routes of administration such as injection. The present invention can be administered intravenously, intradermally, intraarterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravascularly, intramucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, inhaled (e.g., aerosol inhalation), injected, infused, continuously infused, directly locally perfused into target cells, administered via a catheter, administered by lavage, administered in a cream, administered in a lipid composition (e.g., liposomes), or by other methods as would be known to one of ordinary skill in the art or any combination of the above methods (see, e.g., Remington's Pharmaceutical Sciences (1990), which is incorporated herein by reference). In a specific embodiment of the present invention, intraperitoneal injection is contemplated.

[0323] Pharmaceutical compositions of the present invention may include various antioxidants to delay the oxidation of one or more components. In addition, the effects of microorganisms may be prevented 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 a combination thereof.

[0324] The medicament can be formulated as a composition in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (e.g., salts formed with free amino groups of protein components), or they are formed with inorganic acids (such as, for example, hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid or mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or organic bases, such as isopropylamine, trimethylamine, histidine or procaine.

[0325] In certain aspects of the invention, when 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. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin; maintaining the desired particle size by dispersing in a carrier such as, for example, a liquid polyol or lipid; using a surfactant such as, for example, hydroxypropylcellulose; or such methods and combinations thereof. In many cases, it is preferred to include an isotonic agent such as, for example, a sugar, sodium chloride, or a combination thereof.

[0326] The compounds or compositions of the present invention may be used directly or may be mixed with suitable adjuvants and / or carriers. Suitable adjuvants include aluminum salt adjuvants, such as aluminum phosphate or aluminum hydroxide, calcium phosphate nanoparticles (BioSante Pharmaceuticals, Inc.), ZADAXIN TM , iron oxide nanoparticles, lipid nanoparticles, nucleotides ppGpp and pppGpp, inactivated Bordetella pertussis or its components, P40 components derived from Corenybacterium, cholera toxin and all or part of mycobacteria and ISCOM (DeVries et al., 1988; Morein et al., 199&, Lovgren et al., 1991). Pam3Cys, LPS, ds and ss RNA can also be used as adjuvants. Those skilled in the art are familiar with carriers suitable for pharmaceutical use or suitable for use in humans.

[0327] The compositions of the present invention can be administered to different categories of recipients in a variety of ways.

[0328] Compounds and compositions of the present invention can be formulated into solid, semisolid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, storage agents, inhalants and injections, and for oral, parenteral or surgical use of conventional means. The present invention also includes a pharmaceutical composition formulated for topical use (such as by implant).

[0329] The compounds and compositions of the present invention can be dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration so that the condition being treated is alleviated or improved. Pharmaceutical carriers or vehicles suitable for administering PI3 kinase antagonists and / or inhibitors include any such carriers known to those skilled in the art to be suitable for a particular mode of administration.

[0330] In any case, the compositions and compounds may contain various antioxidants to retard oxidation of one or more components. In addition, the action of microorganisms may be prevented 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.

[0331] The medicament can be formulated as a composition in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (e.g., salts formed with free amino groups of protein components), or they are formed with inorganic acids (such as, for example, hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid or mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or organic bases, such as isopropylamine, trimethylamine, histidine or procaine.

[0332] In embodiments of the present invention, when 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. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin; maintaining the desired particle size by dispersing in a carrier such as, for example, a liquid polyol or lipid; using a surfactant such as, for example, hydroxypropylcellulose; polyethylene glycol (PEG), or such methods and combinations thereof. In many cases, it is preferred to include an isotonic agent such as, for example, sugar, sodium chloride, or a combination thereof.

[0333] The compounds and compositions of the present invention can be used directly or can be mixed with suitable adjuvants and / or carriers. Suitable adjuvants include aluminum salt adjuvants, such as aluminum phosphate or aluminum hydroxide, calcium phosphate nanoparticles (BioSante Pharmaceuticals, Inc.), ZADAXIN TM , nucleotides ppGpp and pppGpp, inactivated Bordetella pertussis or its components, P40 components derived from Corynebacterium, cholera toxin and all or part of mycobacteria and ISCOM (DeVries et al., 1988; Morein et al., 199&, Lovgren et al., 1991). Also useful as adjuvants are polyinosinic acid: polycytidylic acid (polyI:C) Pam3Cys, LPS, ds and ss RNA. Those skilled in the art are familiar with carriers suitable for pharmaceutical use or for use in humans.

[0334] The compositions of the present invention can be administered to different categories of recipients in a variety of ways.

[0335] The formulations of the present invention are administered in the form of pharmaceutically acceptable solutions, which may generally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants and optionally other therapeutic ingredients.

[0336] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Exemplary pharmaceutically acceptable carriers for peptides are specifically described in U.S. Patent No. 5,211,657. Such preparations may generally contain salts, buffers, preservatives, compatible carriers, and optional other therapeutic agents. When used in medicine, salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may be conveniently used to prepare pharmaceutically acceptable salts thereof, and are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. In addition, pharmaceutically acceptable salts may be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0337] Compounds and compositions of the present invention can be formulated into solid, semisolid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, storage agents, inhalants and injections, and for oral, parenteral or surgical administration of conventional means. The present invention also includes pharmaceutical compositions formulated for topical administration (such as by implants).

[0338] Compounds and compositions suitable for oral administration can be presented in the form of discrete units, such as capsules, tablets, lozenges, each of which contains a predetermined amount of active agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids (such as syrups, elixirs or emulsions).

[0339] When used in combination with the inhibitors of the invention, the other agents may be delivered systemically.

[0340] For oral administration, compositions and compounds can be easily formulated by combining active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the present invention to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral intake of subjects to be treated. Oral pharmaceutical preparations can be obtained as solid excipients, optionally grinding the resulting mixture after adding suitable adjuvants (if necessary), and processing the granular mixture to obtain tablets or dragee cores. Suitable excipients are specifically fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methyl-cellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). Disintegrants such as cross-linked polyvinylpyrrolidone, agar or alginic acid or its salt (such as sodium alginate) can be added as needed. Optionally, the oral formulation may also be formulated in saline or buffer for neutralizing internal acidic conditions, or may be administered without any carrier.

[0341] Suitable coatings are provided for dragee cores. For this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talcum powder, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to tablets or dragee coatings for identification or characterization of different combinations of active compound dosages.

[0342] Orally usable pharmaceutical preparations include push-fit capsules made of gelatin, and soft sealed capsules made of gelatin and plasticizers (such as glycerol or sorbitol). Such push-fit capsules can contain active ingredients, which are mixed with fillers such as lactose, adhesives such as starch, and / or lubricants such as talc or magnesium stearate and optional stabilizers. In soft capsules, the active compound can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin or liquid polyethylene glycol. In addition, stabilizers can be added. Microspheres formulated for oral administration can also be used. Such microspheres have been clearly defined in the art. All formulations for oral administration should be suitable for such administration.

[0343] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0344] For inhalation administration, the compounds used according to the present invention can be conveniently delivered in the form of an aerosol spray from a pressurized package or nebulizer using a suitable propellant (e.g., 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 deliver a metered amount. Capsules and cartridges (e.g., gelatin) for inhalers or insufflators can be formulated into a powder mixture containing the compound and a suitable powder matrix (such as lactose or starch). The technology for preparing aerosol delivery systems is well known to those skilled in the art. In general, such systems should utilize components that will not significantly impair the biological properties of the active agent (see, for example, Sciarra and Cutie, "Aerosols," Remington's Pharmaceutical Sciences, 18th edition, 1990, pages 1694-1712; which are incorporated by reference). Those skilled in the art can easily determine the various parameters and conditions for generating an aerosol without resorting to excessive experimentation.

[0345] When systemic delivery of the compounds and compositions of the present invention is desired, they can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection can be present in unit dosage form, for example, in ampoules or in multidose containers, and preservatives are added. The compounds and compositions can be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulation agents, such as suspending agents, stabilizers, and / or dispersants.

[0346] Preparations for parenteral administration include aseptic 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 liquid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents and inert gases, etc. Other forms of administration, such as intravenous administration, will produce lower dosages. In the case of insufficient response of the subject at the initial dose applied, a higher dosage (or an effective higher dosage by a different, more local third approach) can be used within the range allowed by patient tolerance. It is envisioned that multiple doses per day are used to reach the appropriate systemic level of the compound.

[0347] Pharmaceutical composition of the present invention can be used in combination with other therapeutic agents, and such use can be simultaneous or sequential, before or after other therapeutic agents. When other therapeutic agents are used simultaneously, they can be used in identical or separate formulations, but use at the same time or use at different times, or its combination. The use of other therapeutic agents and inhibitors or antagonists can also be separated in time, which means that therapeutic agents are used at different times, before or after using inhibitors or antagonists. The time interval between the use of these compounds can be a few minutes or can be longer.

[0348] Preparations for parenteral administration include, but are not limited to, aseptic 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 liquid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents and inert gases, etc. Other forms of administration, such as intravenous administration, will produce lower doses. In the case of insufficient response of the subject at the initial dose applied, a higher dose (or an effective higher dose by a different, more local third approach) can be used within the range allowed by patient tolerance. It is envisioned that multiple doses per day are used to reach the appropriate systemic level of the compound.

[0349] Compositions suitable for oral administration may be presented in the form of discrete units such as capsules, tablets, lozenges, each containing a predetermined amount of active agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids (such as syrups, elixirs or emulsions).

[0350] For oral administration, the compositions and compounds of the present invention can be easily formulated by combining the active compound with a pharmaceutically acceptable carrier well known in the art. Such carriers allow the compounds of the present invention to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral intake by subjects to be treated. Oral pharmaceutical preparations can be obtained as solid excipients, optionally grinding the resulting mixture after adding suitable adjuvants (if necessary), and processing the granular mixture to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methyl-cellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). Disintegrants such as cross-linked polyvinylpyrrolidone, agar or alginic acid or its salt (such as sodium alginate) can be added as needed. Optionally, the oral formulation may also be formulated in saline or buffer for neutralizing internal acidic conditions, or may be administered without any carrier.

[0351] Suitable coatings are provided for dragee cores. For this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talcum powder, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to tablets or dragee coatings for identification or characterization of different combinations of active compound dosages.

[0352] Orally usable pharmaceutical preparations include push-fit capsules made of gelatin, and soft sealed capsules made of gelatin and plasticizers (such as glycerol or sorbitol). Such push-fit capsules can contain active ingredients, which are mixed with fillers such as lactose, adhesives such as starch, and / or lubricants such as talc or magnesium stearate and optional stabilizers. In soft capsules, the active compound can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin or liquid polyethylene glycol. In addition, stabilizers can be added. Microspheres formulated for oral administration can also be used. Such microspheres have been clearly defined in the art. All formulations for oral administration should be suitable for such administration.

[0353] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0354] For administration by inhalation, the compounds and compositions of the invention used according to the invention may be conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer, using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0355] The technology for preparing aerosol delivery systems is well known to those skilled in the art. In general, such systems can utilize components that will not significantly impair the biological properties of the active agent (see, for example, Sciarra and Cutie, "Aerosols," Remington's Pharmaceutical Sciences, 18th edition, 1990, pages 1694-1712; which are incorporated by reference). Those skilled in the art can easily determine the various parameters and conditions for generating aerosols without resorting to excessive experimentation.

[0356] When systemic delivery of the compounds and compositions of the invention is desired, they can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection can be in unit dosage form, for example, in ampoules or in multidose containers, with the addition of preservatives. Compositions can be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulation agents, such as suspending agents, stabilizers, and / or dispersants.

[0357] C. Preparation, packaging, storage and kits of pharmaceutical compositions

[0358] The present invention also includes articles of manufacture, which refers to any one component or collection of components. In some aspects of the present invention, articles of manufacture include kits. Articles of manufacture include pharmaceutical or diagnostic grade compounds of the present invention in one or more containers. Articles of manufacture may include instructions or labels promoting or describing the use of the compounds of the present invention.

[0359] As used herein, "promotion" includes all methods of business related to the compositions of the present invention for treating disease, including educational methods, hospital and other clinical guidance, pharmaceutical industry activities (including drug sales), and any advertising or other promotional activities (including any form of written, oral and electronic communications).

[0360] "Instructions" may include promotional components and generally refer to written instructions on the packaging of the composition of the invention or written instructions associated with the composition of the invention. Instructions may also include any oral or electronic instructions provided in any manner.

[0361] Therefore, in some embodiments of the present invention, the medicaments described herein can be assembled into a drug or diagnostic or research kit to facilitate their use in treatment, diagnosis or research applications. The kit may include one or more containers containing the components of the present invention and instructions for use. Specifically, such kits may include one or more medicaments described herein, as well as instructions describing the intended therapeutic application of these medicaments and the correct administration. In certain embodiments of the present invention, the medicaments in the kit may be in a pharmaceutical formulation and dosage suitable for a particular application and the method of administration of the medicament.

[0362] The kit can be designed to facilitate the use of the methods described herein by doctors, and can take a variety of forms. Where applicable, each composition in the kit can be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In some cases, some compositions can be composed or processable (e.g., processed into active forms), for example, by adding suitable solvents or other substances (e.g., water or cell culture media), which may or may not be provided with the kit. As used herein, "instructions" can define the components of guidance and / or promotion, and generally relate to written instructions on the packaging of the present invention or written instructions related to the present invention. Instructions can also include any oral or electronic instructions provided in any manner, for example, audio-visual (e.g., videotapes, DVDs, etc.), the Internet and / or network-based communications, etc., so that the user will clearly recognize that the instructions will be associated with the kit. The written instructions can be in the form specified by a government agency that regulates the production, use or sale of drugs or biological products, and the instructions can also reflect that the production, use or sales agency is approved for human administration.

[0363] The kit may contain any one or more of the components described herein in one or more containers. For example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or separating and mixing samples and applying to a subject. The kit may include a container for holding medicaments described herein. These medicaments may be aseptically prepared, packaged in syringes and transported refrigerated. Alternatively, the kit may be contained in a vial or used to store other containers. The second container may have other medicaments prepared aseptically. Alternatively, the kit may include an active agent premixed and transported in a syringe, vial, tube or other container.

[0364] The kit can have a variety of forms, such as blister pouches, shrink-wrap bags, vacuum-sealed bags, sealable thermoformed trays, or similar pouches or tray forms, wherein the accessories are loosely packed in a pouch, one or more tubes, containers, boxes, or bags. The kit can be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise disassembled. The kit can be sterilized using any appropriate sterilization technique (such as radiation sterilization, heat sterilization, or other sterilization methods known in the art). Depending on the specific application, the kit may also include other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, fabrics (such as gauze) for applying or removing disinfectants, disposable gloves, supports for the drug before application, etc.

[0365] The composition of the kit can be provided in any suitable form, for example, as a liquid solution or as a dry powder. When the composition provided is a dry powder, the powder can be reconstituted by adding a suitable solvent, which can also be provided. In embodiments using a liquid form of the composition, the liquid form can be concentrated or ready-to-use. The solvent can depend on the compound and the mode of use or administration. Suitable solvents for pharmaceutical compositions are well known and available from the literature. The solvent can depend on the compound and the mode of use or administration.

[0366] In one set of embodiments, the kit may include a carrier device that is separated to receive in close confinement one or more container devices, such as vials, tubes, etc., each of which includes one of the individual elements to be used in the method. For example, one of the containers may include a positive control for the assay. Additionally, the kit may include containers for other components, for example, a buffer that may be used in the assay.

[0367] The present invention also encompasses packaged and labeled finished pharmaceutical products. The product is included in a suitable unit dosage form in a suitable vessel or container (such as a glass bottle or other sealed container). In the case of a dosage form suitable for parenteral administration, the active ingredient is sterile and suitable for use as a particle-free solution. In other words, the present invention encompasses both parenteral solutions and lyophilized powders, each of which is sterile, and the latter is suitable for reconstitution before injection. Alternatively, the unit dosage form can be a solid suitable for oral, transdermal, topical or mucosal delivery.

[0368] In a preferred embodiment of the invention, the unit dosage form is suitable for intravenous, intramuscular or subcutaneous delivery. Therefore, the present invention encompasses solutions suitable for each delivery route, preferably sterile solutions.

[0369] In another preferred embodiment of the present invention, the composition and composition of the present invention are stored in a container with a biocompatible detergent (including but not limited to lecithin, taurocholic acid and cholesterol); or with other proteins (including but not limited to gamma globulin and serum albumin). More preferably, the composition of the present invention can be stored with human serum albumin for human use, and can be stored with bovine serum albumin for veterinary use.

[0370] As with any pharmaceutical product, packaging materials and containers are designed to protect the stability of the product during storage and transportation. Further, the product of the present invention includes instructions for use or other informational materials that advise the physician, technician, or patient how to properly prevent or treat the disease or condition in question. In other words, the article of manufacture includes instructions that indicate or suggest a dosing regimen, including but not limited to actual dosage, monitoring procedures (such as methods for monitoring mean absolute lymphocyte counts, tumor cell counts, and tumor size), and other monitoring information.

[0371] More specifically, the present invention provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, sprayer, insufflator, intravenous (iv) bag, envelope, etc.; and at least one unit dosage form of a medicament included in the packaging material. The present invention also provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, sprayer, insufflator, intravenous (iv) bag, envelope, etc.; and at least one unit dosage form of each medicament included in the packaging material. The present invention further provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, sprayer, insufflator, intravenous (iv) bag, envelope, etc.; and at least one unit dosage form of each medicament included in the packaging material. The present invention further provides an article of manufacture comprising a needle or syringe for injection of a formulation, preferably packaged in a sterile form, and / or a packaged alcohol pad.

[0372] The present invention also includes articles, which refer to any one component or collection of components. In some embodiments, the article is a kit. The article includes a pharmaceutical grade or diagnostic grade compound of the present invention in one or more containers. The article may include instructions or labels that promote or describe the use of the compound of the present invention.

[0373] As used herein, "promotion" includes all methods of business related to the compositions of the present invention for treating disease, including educational methods, hospital and other clinical guidance, pharmaceutical industry activities (including drug sales), and any advertising or other promotional activities (including any form of written, oral and electronic communications).

[0374] "Instructions" may define an integral part of the promotion and generally refer to the written instructions on the packaging of the composition of the invention or the written instructions associated with the composition of the invention. Instructions may also include any oral or electronic instructions provided in any manner.

[0375] Therefore, in some aspects of the invention, the medicaments described herein can be assembled into a drug or diagnostic or research kit to facilitate their use in treatment, diagnosis or research applications. The kit can include one or more containers containing components of the present invention and instructions for use. Specifically, such kits can include one or more medicaments described herein, as well as instructions for describing the expected therapeutic application of these medicaments and the correct application. In certain aspects of the invention, the medicaments in the kit can be in a pharmaceutical formulation and dosage suitable for a particular application and the method of administration of the medicament.

[0376] Kits can be designed to facilitate the use of the methods described herein by doctors, and can take a variety of forms. Where applicable, each composition in the kit can be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In some cases, some compositions can be composed or processable (e.g., processed into active forms), for example, by adding a suitable solvent or other substance (e.g., water or cell culture medium), which may or may not be provided with the kit.

[0377] As used herein, "instructions" may define a component of guidance and / or promotion, and generally refers to written instructions on the packaging of the invention or written instructions associated with the invention. Instructions may also include any oral or electronic instructions provided in any manner, such as audio-visual (e.g., videotape, DVD, etc.), Internet and / or network-based communications, etc., so that the user will clearly recognize that the instructions will be associated with the kit. The written instructions may be in a form prescribed by a governmental agency that regulates the production, use, or sale of a drug or biological product, and the instructions may also reflect approval by the production, use, or sales agency for human administration.

[0378] The kit may include any one or more of the components described herein in one or more containers. For example, the kit may include instructions for mixing one or more components of the kit and / or separating and mixing samples and applying to a subject. The kit may include a container for holding medicaments described herein. These medicaments may be aseptically prepared, packaged in syringes and transported refrigerated. Alternatively, it may be contained in a vial or used to store other containers. A second container may have other medicaments prepared aseptically. Alternatively, the kit may include an active agent premixed and transported in a syringe, vial, tube or other container.

[0379] The kit can have a variety of forms, such as blister pouches, shrink-wrap bags, vacuum-sealed bags, sealable thermoformed trays, or similar pouches or tray forms, wherein the accessories are loosely packed in a pouch, one or more tubes, containers, boxes, or bags. The kit can be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise disassembled. The kit can be sterilized using any appropriate sterilization technique (such as radiation sterilization, heat sterilization, or other sterilization methods known in the art). Depending on the specific application, the kit may also include other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, fabrics (such as gauze) for applying or removing disinfectants, disposable gloves, supports for the drug before application, etc.

[0380] The composition of the kit can be provided in any suitable form, for example, as a liquid solution or as a dry powder. When the composition provided is a dry powder, the powder can be reconstituted by adding a suitable solvent, which can also be provided. In embodiments where a liquid form of the composition is used, the liquid form can be concentrated or ready-to-use. The solvent will depend on the compound and the mode of use or administration. Suitable solvents for pharmaceutical compositions are well known and available from the literature. The solvent will depend on the compound and the mode of use or administration.

[0381] In one set of aspects of the invention, the kit may include a carrier device that is separated to receive in close confinement one or more container devices, such as vials, tubes, etc., each of which includes one of the individual elements to be used in the method. For example, one of the containers may include a positive control for the assay. In addition, the kit may include containers for other components, for example, a buffer that may be used for the assay.

[0382] The present invention also encompasses packaged and labeled finished pharmaceutical products. The product is included in a suitable unit dosage form in a suitable vessel or container (such as a glass bottle or other sealed container). In the case of a dosage form suitable for parenteral administration, the active ingredient is sterile and suitable for use as a particle-free solution. In other words, the present invention encompasses both parenteral solutions and lyophilized powders, each of which is sterile, and the latter is suitable for reconstitution before injection. Alternatively, the unit dosage form can be a solid suitable for oral, transdermal, topical or mucosal delivery.

[0383] In a preferred aspect of the invention, the unit dosage form is suitable for intravenous, intramuscular or subcutaneous delivery. Thus, the invention encompasses solutions suitable for each delivery route, preferably sterile solutions.

[0384] In another preferred embodiment of the present invention, the composition of the present invention is stored in a container with a biocompatible detergent (including but not limited to lecithin, taurocholic acid and cholesterol); or with other proteins (including but not limited to gamma globulin and serum albumin). More preferably, the composition of the present invention is stored with human serum albumin for human use, and can be stored with bovine serum albumin for veterinary use.

[0385] As with any pharmaceutical product, packaging materials and containers are designed to protect the stability of the product during storage and transportation. Further, the product of the present invention includes instructions for use or other informational materials that advise the physician, technician, or patient how to properly prevent or treat the disease or condition in question. In other words, the article of manufacture includes instructions that indicate or suggest a dosing regimen, including but not limited to actual dosage, monitoring procedures (such as methods for monitoring mean absolute lymphocyte counts, tumor cell counts, and tumor size), and other monitoring information.

[0386] More specifically, the present invention provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, sprayer, insufflator, intravenous (iv) bag, envelope, etc.; and at least one unit dosage form of a medicament included in the packaging material. The present invention also provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, sprayer, insufflator, intravenous (iv) bag, envelope, etc.; and at least one unit dosage form of each medicament included in the packaging material. The present invention further provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, sprayer, insufflator, intravenous (iv) bag, envelope, etc.; and at least one unit dosage form of each medicament included in the packaging material. The present invention further provides an article of manufacture comprising a needle or syringe for injection of a formulation, preferably packaged in a sterile form, and / or a packaged alcohol pad.

[0387] VII PI3K inhibitors used together with checkpoint inhibitors to treat cancer - glioblastoma, breast cancer and Sanyin

[0388] Breast cancer treatment methods

[0389] The present invention also includes methods of treating glioblastoma, breast cancer and / or triple-negative breast cancer.

[0390] A seventh aspect of the present invention includes a method for treating a subject having cancer, the method 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 pharmaceutically effective amount of at least one PI3K inhibitor in a pharmaceutically acceptable carrier; and 2) a pharmaceutically effective amount of at least one checkpoint inhibitor in a pharmaceutically acceptable carrier; c) administering a pharmaceutically effective amount of at least one pharmaceutical composition to the subject; wherein the subject is being treated for at least one cancer.

[0391] In one aspect of the invention, the method further includes, but is not limited to, administering to the subject at least one therapy.

[0392] 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 radiotherapy, at least one cryogenic treatment, at least one hyperthermia treatment, at least one laser treatment, at least one surgery, at least one nanoparticle delivery system, or a combination thereof.

[0393] In another aspect of the invention, the at least one PI3K inhibitor and a checkpoint inhibitor are provided together or separately.

[0394] In one aspect of the invention, the at least one PI3K inhibitor includes but is not limited to:

[0395] Pitiliximab (GDC-0941);

[0396] Copanex (BAY 80–6946);

[0397] Taselis (GDC-0032);

[0398] Sonolis (PX-866);

[0399] Genentech GDC 0084;

[0400]

[0401]

[0402] or a combination thereof.

[0403] In another aspect of the 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.

[0404] In another aspect of the present invention, anti-CTLA-4 inhibitors include but are not limited to: ipilimumab.

[0405] In another aspect of the present invention, PD-1 inhibitors include but are not limited to pembrolizumab, nivolumab, cemiplimab (trade name ) and dotalizumab (trade name: ) or a combination thereof.

[0406] In one aspect of the present invention, anti-PD-L1 inhibitors include but are not limited to atezolizumab (trade name: ), durvalumab (trade name Avelumab (trade name: ) or a combination thereof.

[0407] Examples

[0408] Example 1: Glioblastoma

[0409] This example refers to Figures 1 to 3 .

[0410] The purpose of these experiments was to investigate how and whether PI3 kinase inhibitors affect glioblastoma (GBM) cells grown in culture. GL261 mouse glioblastoma cells were cultured at approximately 5 x 10 5 Cells were cultured at a concentration of 10 cells / ml in tissue culture plates in complete RPMI medium containing 5% heat-inactivated fetal bovine serum, L-glutamine, HEPES buffer, and an antibiotic cocktail of penicillin and streptomycin. The cells were treated with several doses of GCT.Glio.1 ranging from 10 nanomolar to 10 micromolar final concentrations for 48 hours. The cells were then harvested and counted. The first experiment showed that treatment with the compound resulted in a much lower number of cells after treatment than in the untreated group, as shown by cell counts. The greatest reduction in cell number occurred when the Glio.1 (also known as GCT0007) compound was at a final concentration of 10 micromolar, as shown in Figure 2. Figure 1 Quantification of cell viability showed that the remaining cells were alive with no evidence of cell death, suggesting that the compounds reduced the number of responding cells, most likely as a result of growth arrest in the rapidly dividing GL261 cells. Ongoing studies have confirmed that these compounds cause cell cycle arrest at the G2 / M cell cycle checkpoint.

[0411] The second step in the study was to determine whether the compound changed the way the immune system “sees” GBM cells in culture. In other words, would treatment with a dose of GCT increase the likelihood that T cells would “kick into gear” and release the “brakes” on an anti-tumor response. In this experiment, cells were inoculated at approximately 5 x 10 5 GCT.Glio.1 (also known as GCT07) was treated with 1 microgram / ml GCT.Glio.1 (also known as GCT07) for 48 hours. The cells were then harvested, counted, and stained with fluorescent dye-conjugated antibodies to mouse MHC class II and mouse PD-L1. The cells were then harvested, washed, and analyzed using flow cytometry. The data suggest that GCT treatment increases PD-L1 expression on the surface of GBM cells (the target of PD-1:PD-L1 targeted immune checkpoint inhibitors), which may provide a target for "releasing the brakes" by treating patients with "checkpoint inhibitors." In the following experiments, we demonstrate that treatment with either compound increases MHC class II, Figure 2 .

[0412] 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. The cells were then harvested and counted.

[0413] Figure 2 GL261 cells were cultured with or without GCT.GLIO.1 (also known as GCT007) for 72 hours, harvested, and stained with anti-PD-L1 as indicated. The histogram demonstrates that increases in cell surface PD-L1 expression correspond to increasing doses of GCT.GLIO.1 and suggests that increased PD-L1 could provide a target for immune checkpoint inhibitor therapy.

[0414] Figure 3 GL261 cells were cultured with and without GCT.GLIO.1 for 7 days as indicated, harvested, and stained as indicated to determine changes in cell surface PD-L1 and MHC class II expression; or for expression of the autophagy marker LC3. Increases in MHC class II and PD-L1 indicate that the drug will increase the likelihood of CD4 T cell recognition and that increased PD-L1 will provide a target for immune checkpoint inhibitor therapy.

[0415] Results: These results show that both compounds increased MHC class II levels, but compound GCT.GLIO.1 (NPT520-337) caused an increase in all three molecules, while compound GCT.GLIO.2 (NPT520-322) caused growth arrest and increased MHC class II expression.

[0416] Example 2: Glioblastoma

[0417] This example refers to Figure 4A and Figure 4B To Figure 6.

[0418] Effects of GCT.Glio.1 on the radiosensitivity of GL261. In these experiments, we explored the question of how or whether GCT.Glio.1 treatment might affect subsequent irradiation therapy, Figure 4A and Figure 4B GL261 cells were cultured at 1 microgram / ml for 48 hours with or without GCT indicated by GCT.GLIO.1 (NPT520-337), with or without 2 Gy irradiation treatment (indicated as R). Cells were then harvested and the viability of recovered cells was assessed using trypan blue viability dye. Viability data indicated that GCT.Glio.1 sensitized cells to 2 Gy, as indicated by decreased viability (increased cell death) after GCT and irradiation, Figure 4A and Figure 4B .

[0419] In addition to inducing growth arrest, GCT.GLIO.1 also increased cell surface PD-L1 and MHC class II (expressed as mean fluorescence intensity (MFI)), potentially making these cells better targets for immunotherapy (Figure 5).

[0420] Similar results were found 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 these cells in a dose-dependent manner (Figure 6).

[0421] There are several isoform-specific PI3K inhibitors in early clinical trials. A key issue is drug delivery beyond the blood-brain barrier, however Genentech's GDC 0084 is an oral small molecule PI3K inhibitor that can penetrate the blood-brain barrier.

[0422] The compounds named GCT.GLIO.X were also designed to be brain penetrant. The novelty of GCT.GLIO.1 and GCT.GLIO.2 lies in the combined advantage of being able to cross the blood-brain barrier and alter the "visibility" of GBM to the immune system.

[0423] Figure 4A and Figure 4B Effect of GCT.Glio.1 on Radiosensitivity and Cell Cycle Arrest of GL261. In the next experiment, we sought to identify the optimal radiation dose to achieve cell cycle arrest after GCT.Glio.1 treatment. In these studies, GL261 cells cultured at 5x 105 cells / ml were treated with 1 microgram / ml GCT.Glio.1 for 24 hours and then irradiated with increasing doses, including 2, 4, and 8 Gy. The cells were then harvested and counted. The cell cycle phase of the recovered cells was determined by mild detergent lysis with saponin, followed by staining with propidium iodide and then photofixation in paraformaldehyde. The stained cells were analyzed by flow cytometry to determine the frequency of cells arrested at each stage of the cell cycle. The results showed that re-treatment with GCT.Glio.1 significantly enhanced the response to radiation and resulted in a significant increase in cells arrested at the G2 cell cycle checkpoint.

[0424] Figure 5A and Figure 5B Human GBM cell line U251 was cultured at 5x 10 5 Cells / ml were cultured in complete RPMI medium containing 5% heat-inactivated fetal bovine serum, L-glutamine, HEPES buffer, and an antibiotic cocktail of penicillin and streptomycin, treated with 1 μg / ml GCT.Glio.1, harvested, counted, and stained with fluorescent dye-conjugated antibodies against PD-L1 or MHC class II to determine changes in cell surface PD-L1 (left panel) and MHC class II (right panel).

[0425] Fig. 6A and 6B Human U251 cells were cultured at a density of approximately 5 x 10 5 Cells were cultured at a concentration of 10 cells / ml in tissue culture plates 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 treated with 1 microgram / ml GCT.Glio.1, harvested, counted, and assessed for changes in cell number and dead cells.

[0426] Example 3: Breast cancer

[0427] This example refers to Figure 7 , Figure 8 and Fig. 9 .

[0428] Breast cancer is a well-documented and serious public health threat that, despite significant efforts, remains an active area of ​​research and development of effective treatments, particularly because drug-resistant variants of breast cancer remain a significant problem. Despite decades of therapeutic trials, improved diagnostics, new therapies, and hundreds, if not thousands, of clinical trials for breast cancer, breast cancer remains the deadliest form of cancer in women. Because breast cancer is immunologically silent or "cold," the innovation and excitement surrounding immunotherapies for many tumors has not been as successful in breast cancer.

[0429] In the present application, it has been found that GCT.BC.1 (also known as GCT007, GCT.GLIO.1, NPT520-337) is a novel small molecule PI3 kinase inhibitor, and GCT.BC.1 can transform immunologically "cold" tumors into tumors expressing many immunotherapy target molecules, thereby turning tumors from "cold" to "hot" in terms of immunotherapy. Radiation can penetrate deeply, even through bones, it can cover small or large areas, and low doses are relatively non-toxic. Therefore, radiation-activated prodrugs will also reduce the amount of radiation required to control tumors, thereby significantly reducing normal tissue damage. If the prodrug is isolated and washed away from normal tissues, the harm of the drug to normal peripheral tissues during delivery will be greatly reduced.

[0430] These examples involve in vitro human and mouse cell line data for breast cancer. These experiments were designed to test the effects of GCT.BC.1 on breast cancer. These data suggest that GCT.BC.1 is as effective as other PI3K inhibitors in killing and preventing cell proliferation and can be effectively combined with other approved chemotherapy drugs.

[0431] Figure 7 .Cell viability of human MCF7 breast cancer cells. Human MCF7 cells were treated with 1 uM Apellis (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.

[0432] Figure 8, Cell viability of mouse TC11 breast cancer cells. TC11 cells were treated with 100nM paclitaxel (Pac), 100nM paclitaxel + 1uM apellis (Alp), 0.50uM GCT.BC.1 (GCT), 0.50uM GCT.Glio.1 + 100nM paclitaxel, 0.50uM GCT.BC.1 + 7.5nM fulvestrant or DMSO control as indicated for 48 hours. Viability was determined by cleaved caspase-7 and measured using the Caspase Glo assay (Promega) according to the manufacturer's instructions.

[0433] Fig. 9 , Proliferation of 66CL4 mouse breast cancer cells. 66CL4 mouse breast cancer cells were treated with the indicated doses of GCT.BC.01. Proliferation was determined using the Agilent Biotek Biospa system and Biospa software.

[0434] Example 4: Glioblastoma

[0435] This example refers to Figure 10 to Figure 11 .

[0436] These examples involve in vivo animal data. These experiments were designed to test the efficacy of GCT.Glio.1 (also known as GCT007, GCT.GLIO.1, NPT520-337) in GBM animal models and to determine the impact of combining GCT.Glio.1 with immune checkpoint inhibitors by measuring in vivo tumor growth using luciferase-labeled tumors and bioluminescence. The study found that the combination of GCT.Glio.1 and anti-PD-1 significantly improved overall survival ( 14.4 months) compared to anti-PD-1 or GCT.Glio.1 alone. Fig.10 ) and delayed tumor growth ( Fig.11 ).

[0437] Fig.10 , on day 0, luciferase-labeled GL261 tumor cells were implanted into the caudate nucleus of C57BL6 mice. Treatment started on day 9. Treatments included no treatment, oral administration of GCT alone, anti-PD-1 plus DMSO vehicle, or oral administration of GCT and anti-PD1, the indicated checkpoint inhibitor, 24 hours later. Tumor growth was confirmed by MRI. Kaplan Meier survival curves. The black line represents the animal survival of animals implanted with GL261; the red line represents the survival of animals treated with GCT.Glio.1; the blue line represents animals treated with GCT.Glio.1 and anti-PD1 treatment 24 hours later (and so on). As of day 28, 3 animals in the combination group were still alive.

[0438] Fig.11 , luciferase-labeled GL261 tumor cells were implanted into the caudate nucleus of C57Bl6 mice on day 0. Treatment started on day 9. Treatments included no treatment, oral administration of GCT alone, anti-PD-1 plus DMSO vehicle, or oral administration of GCT and 24 hours later with the indicated checkpoint inhibitor anti-PD1. GL261 tumors were measured using bioluminescence and monitored daily. In the figure above, bioluminescence quantification shows that tumor growth was significantly delayed when animals were treated with GCT.Glio.1 and then with the checkpoint inhibitor anti-PD-1.

[0439] Example 5: General Formulation

[0440] The Examples refer to formulations that can be used in the Examples.

[0441] 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), and (-)-NPT520-338 refers to GCT.Glio.3 and GCT.BR.1.

[0442] In vitro and in vivo formulation description of NPT 520-337 Enantiomer 2

[0443] 1.N81520-337: 10 mM stock solution in DMSO for in vitro use

[0444] Weigh the compound. Using the molecular weight of the compound, calculate the volume of DMSO to add to make a 10 mM stock solution in DMSO. Add this volume and vortex to mix. Store the 10 mM stock solution at -20C or -80C and thaw for use. Stock solutions of the compound in DMSO remain stable after repeated freeze-thaw cycles. Compounds have been shown to be soluble up to 10 uM in aqueous buffers / media.

[0445] 2. BPT520-337: 10 mg / kg and vehicle (PO administration) in vivo study

[0446] Composition of excipients: 7, 5X NMP, 7.5%-solutol HS-15, 30% PEG-400, 55*(0.95NaCl) saline.

[0447] 3. Preparation of GCT.1

[0448] The required volume of NMP was added (by using a pipette) to the vial containing the compound and the solution was heated to 65-70 C until the drug was completely dissolved in the NMP and then vortexed for 30 seconds.

[0449] Add the required volume of PEG-400 (by using a pipette) and heat for 2 minutes, then vortex the solution. The solution should be clear without any precipitation, turbidity or haziness. Add half the required volume of normal saline (via a pipette) and vortex the solution for 30 seconds. Do not heat.

[0450] Add the required volume of Solutol-HS and vortex for 30 seconds. Rinse the tip twice with the drug solution and make sure the Solutol is completely in solution. Vortex. Add the remaining saline and vortex for 30 seconds.

[0451] 4. Preparation of NPYS20-337(82) vector:

[0452] a. Prepare vehicle by adding NMP, PEG, Solutol Hs, and saline one component at a time and vortex for 30 seconds after each addition.

[0453] b. Veins are transparent Notes:

[0454] 1. Solutol-Hs is an opaque paste. Heat the paste to 50-60°C in a scintillation vial until it becomes liquid.

[0455] 2. At 10 mg / mL, the suspension is cloudy / turbid. MW = 391.44

[0456] IM-391.44g / L

[0457] 10mM. 3.9144mg / mL 21.39

[0459] 3,914H1 m9

[0460] 21.3mg

[0461] Example 6: Triple-negative breast cancer

[0462] This example refers to Figures 13 to Fig.18 .

[0463] This example refers to the fact that endocrine receptor-positive cells become resistant to endocrine therapy via activation of semaphorin 7A and PI3K.

[0464] Cell culture protocol: Cells were plated at approximately 1 million cells / plate in 10 cm plates and incubated overnight to allow them to adhere. The cell lines and culture media used included McCoy 5a modified medium for HER2+ breast cancer cell line SKBR3 cells, and for MDA-MB-231, MDA-MB-468, MCF-7, and HS578t, the culture medium was DMEM. The cells were then treated with PI3K inhibitors: 0uM (control), 1uM, or 10uM. The treated cells were incubated for approximately 72 hours. After incubation, the cells were imaged and then harvested with 0.25% trypsin / EDTA and counted using trypan blue viability stain and NexcelomCellometer 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 cancer.

[0465] The purpose of the following study was to determine whether GCT007 (also known as NPT520-337.NPT520-337 / ENT2 or GCT.Br.1), a potent PI3K inhibitor, could reverse semaphorin7A-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 signaling. The Traci Lyons laboratory showed that Semaphorin7a (SEMA7A) is a potential biomarker for endocrine therapy resistance, increased recurrence, and decreased overall survival in ER+ BC patients. They found that SEMA7A promotes tumor growth, angiogenesis and lymphangiogenesis, epithelial-mesenchymal transition (EMT), metastasis, and endocrine therapy resistance in preclinical models. Specifically, they showed that SEMA7A+ER+MCF7 tumors develop lung metastases that are resistant to the endocrine therapy fulvestrant, in part due to ER downregulation in vivo - creating a need to identify novel treatable targets for SEMA7A+ER+BC. Therefore, the following data support the use of GCT007 as a novel therapy for endocrine-resistant breast cancer. See, e.g., Figures 13 and 14.

[0466] The purpose of the following study was to test the hypothesis that treatment with GCT007 (also known as NPT520-337 / ENT2 or GCT.Br.1) would increase cell surface expression of important cell surface receptors including human epidermal growth factor 2 (HER2). HER2 is an important receptor for the breast cancer drug Herceptin. TM Therefore, treatment with GCT007 can render HER2-negative and triple-negative breast cancer cells resistant to Herceptin. TMThus, a method for combining GCT007 with the drug Herceptin is provided. TM Combination of novel therapeutic approaches for the treatment of breast cancer. See e.g. Fig.15 , Fig.16 , Fig.17 ,and Fig.18 .

[0467] Fig.13A , Fig. 13B and Fig. 13C SEMA7A overexpressing (OE) cells respond poorly to endocrine therapy but become sensitive after addition of PI3K inhibition. MCF7EV and SEMA7A OE cells were treated with A) fulvestrant (7.5 nM), B) apellisidone (1 uM), or C) the combination for 48 hours.

[0468] Fig.14A , Fig. 14B , Fig. 14C , Fig.14D and Fig.14E . Cells expressing SEMA7A are sensitive to both apellisib and a novel PI3K inhibitor (GCT.BR.1). Fig.14A ) MCF7 cells treated with vehicle, 1 uM Apellisin, and 1 uM GCT.BR.1. Scale bar = 10 microns. Fig. 14B )Quantification of A. Fig. 14C ) MCF7 S7OE spheroids in suspension were treated with vehicle (DMSO), 1 uM apellisin or 0.5 uM GCT and stained with Calcein green viability dye after 48 h. Fig.14D ) Tumor volume in NCG mice bearing MCF7 SEMA7 A OE that were treated with fulvestrant every 5 days starting from day 30 (arrows). Fig.14E ) of matching sizes.

[0469] Fig.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 are able to form poorly differentiated tumors in immunocompromised mice. MDA-MB-231 is a highly aggressive, invasive, and poorly differentiated triple-negative breast cancer (TNBC) cell line as it lacks estrogen receptor (ER) and progesterone receptor (PR) expression, as well as HER2 (human epidermal growth factor receptor 2) amplification.

[0470] Fig.16SKBR3 cells were treated with the indicated doses of GCT.BR.1. The histogram depicts changes in HER2 expression analyzed by flow cytometry. Treatment with GCT007 resulted in a dose-dependent increase in HER2 expression on triple-negative SKRB3 breast cancer cells.

[0471] Fig.17 .Triple negative breast cancer cell line MDA MB 231 cells treated with labeled doses of GCT.BR.1 (GCT007). Histogram depicts changes in HER2 expression analyzed by flow cytometry. Treatment with GCT007 resulted in a dose-dependent increase in HER2 expression on breast cancer 231 cells

[0472] Fig.18 Quantification of median fluorescence intensity of HER-2 expression on .231 cells.

[0473] The present invention is not limited in its application to the details of the construction and arrangement of the parts set forth in the following description or illustrated in the accompanying drawings. The present invention can have other embodiments and can be practiced or implemented in various ways. Likewise, the words and terms used herein are for descriptive purposes and should not be considered restrictive. The use of "including", "comprising" or "having", "containing", "involving" and variations thereof herein is intended to cover the items listed thereafter and their equivalents as well as additional items. The terms "comprising", "consisting essentially of" and "consisting of", especially in the claims, refer to the legal meaning of these terms, as set forth, for example, in the USTPO Manual of Patent Examining Procedure (MPEP), such as Section 2111.03. In short, the transitional term "comprising," which is synonymous with "comprising," "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 elements, steps, or ingredients not specified in the claim and is meant to close the claim to containing materials other than the recited materials, except for impurities normally associated therewith; and 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.

[0474] All publications, including patent documents and databases, mentioned in this application and in the bibliography and appendix are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference.

[0475] All headings and names throughout the text are for the convenience of the reader and should not be used to limit the meaning of the text following the heading unless otherwise indicated.

Claims

1. A method of treating a subject suffering from cancer, the method comprising: 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, said at least one pharmaceutical composition comprising: 1) a pharmaceutically effective amount of at least one PI3K inhibitor in a pharmaceutically acceptable carrier; and 2) a pharmaceutically effective amount of at least one checkpoint inhibitor in a pharmaceutically acceptable carrier; c) administering to the subject a pharmaceutically effective amount of the at least one pharmaceutical composition; wherein the subject is being treated for the at least one cancer.

2. The method according to claim 1, It further comprises administering at least one therapy to the subject.

3. The method according to claim 1, The treatment comprises at least one chemotherapy, at least one immunotherapy, at least one radiotherapy, at least one cryogenic treatment, at least one hyperthermia treatment, at least one laser treatment, at least one surgery, at least one nanoparticle delivery system, or a combination thereof.

4. The method according to claim 1, wherein the at least one PI3K inhibitor and the checkpoint inhibitor are provided together or separately.

5. The method according to claim 1, wherein the at least one PI3K inhibitor comprises: or a combination thereof.

6. The method according to claim 1, wherein the at least one PI3K inhibitor comprises: or a combination thereof.

7. The method according to claim 1, wherein the at least one checkpoint inhibitor comprises: 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 thereof.

8. The method according to claim 7, The anti-CTLA-4 inhibitors include: Ipilimumab.

9. The method according to claim 7, The PD-1 inhibitors include: Pembrolizumab, nivolumab, and cimiprilimab (trade names: ) and dotalizumab (trade name: ) or a combination thereof.

10. The method according to claim 7, The anti-PD-L1 inhibitors include: Atezolizumab (trade name: ), durvalumab (trade name ) and avelumab (trade name: ) or a combination thereof.

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

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

13. The pharmaceutical composition according to claim 11, wherein the at least one PI3K inhibitor comprises: or a combination thereof.

14. The pharmaceutical composition according to claim 11, wherein the at least one PI3K inhibitor comprises: or a combination thereof.

15. The pharmaceutical composition according to claim 11, wherein the at least one checkpoint inhibitor comprises: 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 thereof.

16. The pharmaceutical composition according to claim 15, The anti-CTLA-4 inhibitors include: Ipilimumab.

17. The pharmaceutical composition according to claim 15, The PD-1 inhibitors include: Pembrolizumab, nivolumab, and cimiprilimab (trade names: ) and dotalizumab (trade name: ) or a combination thereof.

18. The pharmaceutical composition according to claim 15, The anti-PD-L1 inhibitors include: Atezolizumab (trade name: ), durvalumab (trade name ) and avelumab (trade name: ) or a combination thereof.

19. A method of treating a subject suffering from cancer, the method comprising: a) providing a subject in need of treatment for at least one cancer; b) providing at least one pharmaceutical composition according to claim 11; c) administering to the subject a pharmaceutically effective amount of the at least one pharmaceutical composition; wherein the subject is being treated for the at least one cancer.

20. The method according to claim 19, It further comprises administering at least one therapy to the subject.

21. The method according to claim 20, The treatment comprises at least one chemotherapy, at least one immunotherapy, at least one radiotherapy, at least one cryogenic treatment, at least one hyperthermia treatment, at least one laser treatment, at least one surgery, at least one nanoparticle delivery system, or a combination thereof.

22. A method of screening at least one test compound, at least one procedure, or a combination thereof for the 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 said 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. measuring changes 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 a change in expression of the one or more markers in the one or more cancer cells in the presence of the one or more test compounds relative to the absence of the one or more test compounds reasonably predicts that the one or more cancer cells are more susceptible to the one or more treatments for reducing the number of cancer cells.

23. The method according to claim 22, The test compounds, procedures, or combinations thereof include at least one chemical compound, at least one chemotherapy, at least one immunotherapy, at least one radiotherapy, at least one cryogenic treatment, at least one hyperthermia, at least one laser, at least one surgery, at least one nanoparticle delivery system, or combinations thereof.

24. The method according to claim 23, Wherein the nanoparticle delivery system comprises at least one scintillator.

25. The method according to claim 23, Wherein the nanoparticle delivery system comprises at least one cleavable linker.

26. The method according to claim 25, The cleavable linker is located between the drug and the nanoparticle.

27. The method according to claim 22, The cancer cells are derived from endoderm, mesoderm, ectoderm or a combination thereof.

28. The method according to claim 22, The cancer cells include epithelial cancer cells, sarcoma cells, leukemia cells, lymphoma cells, myeloma cells or a combination thereof.

29. The method according to claim 22, The cancer cells include skin cells, melanoma cells, non-melanoma cells, brain cells, prostate cells, breast cells, colorectal cells, kidney cells, bladder cells, non-Hodgkin's lymphoma cells, thyroid cells, endometrial cells, head cells, neck cells, brain cells, glioblastoma cells or a combination thereof.

30. The method according to claim 22, Wherein said being more susceptible to treatment comprises said cancer cells being more susceptible to cell death, having a reduced growth rate, having an increased doubling time, being more susceptible to DNA damage, having a poorer ability to repair DNA damage, being more likely to be recognized by immune cells, or a combination thereof.

31. The method according to claim 22, The treatment for reducing the number of cancer cells comprises at least one chemotherapy, at least one immunotherapy, at least one radiotherapy, at least one hypothermia, at least one hyperthermia, at least one laser, at least one surgery, at least one nanoparticle delivery system, or a combination thereof.

32. The method according to claim 22, The cell populations include pure cultures, mixed cultures, biopsies, in vivo, in situ or a combination thereof.

33. The method according to claim 22, The one or more markers include one or more of the following: i.PD-L1; ii. MHC class II; iii.CD80; iv.CD86; v.CD40; vi.CD95; vii.CD40 viii.MHC Class I ix.Lag-3 x.Vista xi.LC3; xii. 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, CD166, and combinations thereof; and xiii. Combinations thereof.

34. The method according to claim 22, Wherein said measuring the change in expression comprises immunological methods, nucleic acid methods, proteomic methods, flow cytometric methods, genomic methods or a combination thereof pure culture, mixed culture, biopsy or a combination thereof.

35. The method according to claim 22, The change in expression includes an increase, a decrease or a combination thereof from a pure culture, a mixed culture, a biopsy or a combination thereof.

36. The method according to claim 22, Wherein the reasonable prediction includes a statistically significant difference at a p-value of less than 0.10, 0.05 or acceptable to those skilled in the art.

37. The method according to claim 22; It further comprises testing the cancer cells with the one or more test compounds and the one or more treatments for reducing the number of cancer cells.

38. A compound identified by the method of claim 22.

39. A pharmaceutical composition comprising: A pharmaceutically effective amount of a compound according to claim 38 in a pharmaceutically acceptable carrier.

40. The pharmaceutical composition according to claim 39, It further comprises at least one additional cancer treatment.

41. The pharmaceutical composition according to claim 40, The treatment comprises at least one chemotherapy, at least one immunotherapy, at least one radiotherapy, at least one cryogenic treatment, at least one hyperthermia treatment, at least one laser treatment, at least one surgery, at least one nanoparticle delivery system, or a combination thereof.

42. A method of treating a subject suffering from 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 according to claim 38; c. administering to the subject a pharmaceutically effective amount of at least one compound; wherein the subject is being treated for the at least one cancer.

43. The method according to claim 42, It further comprises administering at least one therapy to the subject.

44. The method according to claim 43, The treatment comprises at least one chemotherapy, at least one immunotherapy, at least one radiotherapy, at least one cryogenic treatment, at least one hyperthermia treatment, at least one laser treatment, at least one surgery, at least one nanoparticle delivery system, or a combination thereof.

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