Cancer treatment using bosentan in combination with checkpoint inhibitors

Abstract

The present invention is directed to a combination treatment using bosentan and a checkpoint inhibitor that is effective in treating cancer or inhibiting tumor cell growth in a subject and / or that can elicit, enhance, or prolong an immune response against tumor cells. The effectiveness of cancer immunotherapy depends on the ability of T cells to migrate into tumors and migrate adjacent to malignant cells to recognize and kill them. The present invention provides a means to solve this problem.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefit of priority under U.S. Provisional Application No. 63 / 124,448, filed on 11 December 2020, titled "CANCER TREATMENT USING BOSENTAN IN COMBINATION WITH A CHECKPOINT INHIBITOR," the contents of which are incorporated herein by reference in all respects.

[0002] Field of Invention The present invention discloses a combination therapy using bosentan and a checkpoint inhibitor that is effective in treating cancer in a subject or inhibiting the proliferation of tumor cells, and / or can induce, enhance, or prolong an immune response against tumor cells. [Background technology]

[0003] background The effectiveness of cancer immunotherapy depends on whether T cells can migrate to the tumor, travel to locations adjacent to malignant cells, recognize them, and kill them. One barrier to T cell homing is the tumor's vascular wall, which inhibits T cell attachment and migration through the endothelin B receptor, but antagonizing this receptor has not yet led to clinically approved drugs. One reason may be hypoperfusion in the tumor, which can limit the surface area of ​​perfusion vessels to which anti-tumor T cells can attach. If the collapsed tumor vessels can be decompressed and reperfused by reducing mechanical compression (i.e., solid stress), then antagonizing the endothelin B receptor could increase the effectiveness of cancer immunotherapy.

[0004] Bosentan (Tracleer®; Stayveer®) is a dual endothelin receptor antagonist used to treat pulmonary arterial hypertension (PAH). Bosentan is a competitive antagonist of endothelin-1 at endothelin-A (ET-A) and endothelin-B (ET-B) receptors. Under normal conditions, endothelin-1 binding to the ET-A receptor causes vasoconstriction of pulmonary blood vessels. Conversely, the binding of endothelin-1 to the ET-B receptor is associated with both vasodilation and vasoconstriction of vascular smooth muscle, depending on the ET-B subtype (ET-B1 or ET-B2) and tissue. Bosentan blocks both ET-A and ET-B receptors, but is thought to exert a greater effect on the ET-A receptor, leading to an overall reduction in pulmonary vascular resistance.

[0005] Immune checkpoints, which act as off-switches in T cells of the immune system, are being studied to restore the immune response to targeted agonists, thus indirectly treating cancer by activating the body's immune system.

[0006] International patent applications WO2002086083, WO2004004771, WO2004056875, WO2006121168, WO2008156712, WO2010077634, WO2011066389, WO2014055897, and WO2014100079 report methods for identifying PD-1, PD-L1 inhibitory antibodies and / or such antibodies. Furthermore, U.S. Patent No. 8735553 and U.S. 8 U.S. patents such as No. 168757 report PD-1 or PD-L1 inhibitory antibodies and / or fusion proteins. The disclosures of WO2002086083, WO2004004771, WO2004056875, WO2006121168, WO2008156712, WO2010077634, WO2011066389, WO2014055897, and WO2014100079, as well as U.S. patents 8735553 and 8168757, are incorporated herein by reference in their entirety.

[0007] Furthermore, international patent applications WO2011161699, WO2012168944, WO2013144704, WO2013132317, and WO2016044900 report peptides or peptide mimetic compounds capable of repressing and / or inhibiting the programmed cell death 1 (PD-1) signaling pathway. The disclosures of WO2011161699, WO2012168944, WO2013144704, WO2013132317, and WO2016044900 are incorporated herein by reference in their entirety.

[0008] Furthermore, international patent applications WO2016142852, WO2016142894, WO2016142886, WO2016142835, and WO2016142833 report small molecule compounds capable of repressing and / or inhibiting the programmed cell death 1 (PD-1) signaling pathway, and / or treating the impairment by inhibiting immunosuppressive signals induced by PD-1, PD-L1, or PD-L2. The disclosures of WO2016142852, WO2016142894, WO2016142886, WO2016142835, and WO2016142833 are incorporated herein by reference in their entirety.

[0009] Recently, ipilimumab (Yervoy®), a monoclonal antibody targeting cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and nivolumab (Opdivo®), a monoclonal antibody targeting programmed cell death protein 1 pathway (PD-1) on the surface of T cells, have been approved by the U.S. Food and Drug Administration for the treatment of advanced melanoma, advanced renal cell carcinoma, and non-small cell lung cancer. However, current checkpoint inhibitor therapies are effective in treating cancers in relatively small cancer target populations, partly due to the presence of existing immune-activating and inhibitory receptors. Immune checkpoint blockade (ICB) with checkpoint inhibitors has revolutionized the treatment of many types of solid tumors, but it is now estimated that less than 20% of cancer patients benefit. Increasing the proportion of patients who respond and the duration of that response is an urgent clinical unmet need. Therefore, there is a need to develop methods and combination therapies to induce or enhance the efficacy of checkpoint inhibitors in both unresponsive and responsive target populations.

[0010] Antitumor T cells must circulate within the tumor via blood vessels, bind to the endothelium, traverse the vessel walls, and migrate through cancer-associated fibroblasts (CAFs) and the extracellular matrix (ECM) before encountering cancer cells. However, since up to 80% of tumor blood vessels lack perfusion, the area of ​​the vessel wall through which T cells can migrate is limited.

[0011] Compressed blood vessels impair blood flow and oxygen delivery to tumors, leading to increased hypoxia in tumors and resistance to immunotherapy through multiple mechanisms. Strategies to decompress blood vessels enhance the efficacy of ICBs in ICB-resistant mouse models of metastatic breast cancer. If there is a method to decompress tumor blood vessels while also facilitating T cell adhesion and migration to the tumor parenchyma, the proportion of cancer patients who respond to ICBs will increase. All references cited herein, including patent applications, patent publications, and scientific papers, are incorporated herein by reference in their entirety as if each individual reference is specifically and individually indicated to be incorporated herein by reference.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0013] Abstract A method for treating solid tumors in a subject requiring such treatment is provided herein, comprising administering an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with a checkpoint inhibitor to the subject. A method for inducing, enhancing, or prolonging the effect of a checkpoint inhibitor in a subject requiring such treatment, or enabling the subject to respond to a checkpoint inhibitor, comprising administering an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with a checkpoint inhibitor to the subject, wherein the subject has a solid tumor. A method for enhancing the effect of a checkpoint inhibitor in a subject requiring such treatment is provided herein, comprising administering an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with a checkpoint inhibitor to the subject, wherein the subject has a solid tumor. A method for increasing blood flow to a solid tumor in a subject is also provided herein, comprising administering an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with a checkpoint inhibitor to the subject, wherein increasing blood flow to the solid tumor enhances the effect of the checkpoint inhibitor. In some embodiments, blood flow is measured using ultrasound-based hemodynamics or histological techniques for measuring hypoxia. In some embodiments, blood flow is measured using ultrasound-based hemodynamics. In some embodiments, blood flow is measured using histological techniques for measuring hypoxia. In some embodiments, blood flow is measured using histological techniques for measuring hypoxia in a biopsy from a solid tumor. Also provided herein are methods for improving the delivery or efficacy of a checkpoint inhibitor in a subject, comprising administering an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with the checkpoint inhibitor, wherein the subject has a solid tumor, and thereby improving the delivery or efficacy of the treatment in the subject. In some embodiments, administering bosentan or a pharmaceutically acceptable salt thereof increases the number of antitumor T cells co-localizing with the solid tumor. In some embodiments, administering bosentan or a pharmaceutically acceptable salt thereof reduces the tissue stiffness of the solid tumor.In some embodiments, the tissue stiffness of solid tumors is measured using ultrasound elastography. In some embodiments, administration of bosentan or a pharmaceutically acceptable salt thereof reduces the levels of extracellular matrix proteins in solid tumors. In some embodiments, the extracellular matrix proteins are collagen I or hyaluronan-binding protein (HABP). In some embodiments, administration of bosentan or a pharmaceutically acceptable salt thereof reduces hypoxia in solid tumors. In some embodiments, the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, BMA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof. In some embodiments, the checkpoint inhibitor is a CTLA-4, PD-L1, PD-L2, or PD-1 inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the checkpoint inhibitor is selected from the group consisting of MEDI0680, AMP-224, nivolumab, pembrolizumab, pidilizumab, MEDI4736, atezolizumab, ipilimumab, tremelimumab, and BMS-936559. In some embodiments, the checkpoint inhibitor is a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to the subject once daily. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to the subject twice daily. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of approximately 0.01 mg / kg to approximately 5 mg / kg. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to the subject in doses ranging from approximately 100 mg to approximately 1200 mg. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to the subject in doses ranging from approximately 125 mg to approximately 500 mg.In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of approximately 125 mg. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of approximately 500 mg. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to the subject before administering the checkpoint inhibitor to the subject. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to the subject starting at least one day before administering the checkpoint inhibitor to the subject. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to the subject starting at least two days before administering the checkpoint inhibitor to the subject. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to the subject starting at least three days before administering the checkpoint inhibitor to the subject. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to the subject starting at least five days before administering the checkpoint inhibitor to the subject. In some embodiments, administration of bosentan or a pharmaceutically acceptable salt thereof to the subject is maintained for at least a portion of the period during which the checkpoint inhibitor is administered to the subject. In some embodiments, administration of bosentan or a pharmaceutically acceptable salt thereof to a subject is maintained for the entire duration of administration of the checkpoint inhibitor to the subject. In some embodiments, one or more therapeutic effects in the subject are improved compared to baseline after administration of bosentan or a pharmaceutically acceptable salt thereof and the checkpoint inhibitor. In some embodiments, one or more therapeutic effects are selected from the group consisting of tumor size derived from cancer, objective response rate, duration of response, time to response, progression-free survival, and overall survival. In some embodiments, tumor size derived from cancer is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% compared to tumor size derived from cancer before administration of bosentan or a pharmaceutically acceptable salt thereof and the checkpoint inhibitor.In some embodiments, the objective response rate is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%. In some embodiments, subjects exhibit progression-free survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of bosentan or a pharmaceutically acceptable salt thereof and a checkpoint inhibitor. In some embodiments, subjects demonstrate overall survival of at least approximately 1 month, at least approximately 2 months, at least approximately 3 months, at least approximately 4 months, at least approximately 5 months, at least approximately 6 months, at least approximately 7 months, at least approximately 8 months, at least approximately 9 months, at least approximately 10 months, at least approximately 11 months, at least approximately 12 months, at least approximately 18 months, at least approximately 2 years, at least approximately 3 years, at least approximately 4 years, or at least approximately 5 years after administration of bosentan or a pharmaceutically acceptable salt thereof, and a checkpoint inhibitor. In some embodiments, the duration of response to the antibody-drug conjugate is at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of bosentan or a pharmaceutically acceptable salt thereof and a checkpoint inhibitor.In some embodiments, the solid tumor is selected from the group consisting of breast cancer, lung metastasis of breast cancer, sarcoma, pancreatic cancer, ovarian cancer, liver metastasis, prostate cancer, brain cancer, melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, lung cancer, head and neck squamous cell carcinoma, urothelial carcinoma, esophageal squamous cell carcinoma, gastric cancer, esophageal cancer, cervical cancer, Merkel cell carcinoma, endometrial cancer, mesothelioma, and cutaneous squamous cell carcinoma. In some embodiments, the solid tumor is breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the subject is human.

[0014] A kit is also provided herein that includes an effective amount of bosentan or a pharmaceutically acceptable salt thereof; an effective amount of a checkpoint inhibitor; and instructions for using bosentan or a pharmaceutically acceptable salt thereof and the checkpoint inhibitor according to any of the methods described herein.

[0015] A method for determining an effective dose of a vasodilator in a subject having a solid tumor, comprising: (a) measuring the blood flow and / or stiffness of the solid tumor; (b) administering an effective dose of the vasodilator to the subject; and (c) measuring the blood flow and / or stiffness of the solid tumor after administration of the vasodilator, wherein an increase in blood flow and / or decrease in stiffness after administration of the vasodilator to the subject indicates that the administered dose was effective. A method for treating a solid tumor in a subject requiring such treatment is also provided, comprising: (a) measuring the blood flow and / or stiffness of the solid tumor; (b) administering an effective dose of the vasodilator to the subject; (c) measuring the blood flow and / or stiffness of the solid tumor after administration of the vasodilator; and (d) administering a chemotherapeutic agent if the blood flow to the solid tumor increases and / or the stiffness of the solid tumor decreases after administration of the vasodilator. A method for treating a solid tumor in a subject requiring such treatment is also provided herein, comprising: (a) measuring the blood flow and / or stiffness of the solid tumor; (b) administering an effective amount of a vasodilator to the subject; (c) measuring the blood flow and / or stiffness of the solid tumor after administration of the vasodilator; (d) determining that the subject is responsive to a chemotherapeutic agent based on an increase in blood flow or a decrease in the stiffness of the solid tumor after administration of the vasodilator; and (e) administering a chemotherapeutic agent to a subject that has been determined to be responsive to a chemotherapeutic agent based on an increase in blood flow or a decrease in the stiffness of the solid tumor after administration of the vasodilator. A method for predicting a response to treatment with a chemotherapeutic agent is also provided herein, comprising: (a) measuring the blood flow and / or stiffness of a solid tumor; (b) administering an effective amount of a vasodilator to a subject; and (c) measuring the blood flow and / or stiffness of a solid tumor after administration of the vasodilator, wherein an increase in blood flow or a decrease in the stiffness of a solid tumor after administration of the vasodilator indicates that the subject is likely to respond to treatment with a chemotherapeutic agent.In some embodiments, the effective dose of a vascular decompressant is determined by measuring changes in blood flow and / or stiffness of a solid tumor after administration of the vascular decompressant to the target, and an increase in blood flow and / or decrease in stiffness after administration of the vascular decompressant indicates that the administered dose was an effective dose. In some embodiments, the method includes a step of measuring blood flow to a solid tumor, and the blood flow to the solid tumor increases after administration of the vascular decompressant. In some embodiments, the method includes a step of measuring the stiffness of a solid tumor, and the stiffness of the solid tumor decreases after administration of the vascular decompressant. In some embodiments, the vascular decompressant is administered for at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days prior to the administration of a chemotherapeutic agent. In some embodiments, the vascular decompressant is administered in a dose that increases blood flow to a solid tumor and / or decreases the stiffness of a solid tumor. In some embodiments, the vascular decompressant is bosentan or a pharmaceutically acceptable salt thereof. In some embodiments, blood flow and / or stiffness of the solid tumor are measured using ultrasound. In some embodiments, blood flow in solid tumors is measured using histological techniques to assess hypoxia. In some embodiments, the chemotherapeutic agent is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, BMA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof. In some embodiments, the checkpoint inhibitor is a CTLA-4, PD-L1, PD-L2, or PD-1 inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the checkpoint inhibitor is selected from the group consisting of MEDI0680, AMP-224, nivolumab, pembrolizumab, pidilizumab, MEDI4736, atezolizumab, ipilimumab, tremelimumab, and BMS-936559.In some embodiments, the checkpoint inhibitor is a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody. In some embodiments, the solid tumor is selected from the group consisting of breast cancer, lung metastases of breast cancer, sarcoma, pancreatic cancer, ovarian cancer, liver metastases, prostate cancer, brain cancer, melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, lung cancer, head and neck squamous cell carcinoma, urothelial carcinoma, esophageal squamous cell carcinoma, gastric cancer, esophageal cancer, cervical cancer, Merkel cell carcinoma, endometrial cancer, mesothelioma, and cutaneous squamous cell carcinoma. In some embodiments, the solid tumor is breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the subject is human.

[0016] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention will be further described by the following detailed description. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for treating a solid tumor in a subject requiring such treatment, comprising administering to the subject an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with a checkpoint inhibitor. (Item 2) A method for inducing, enhancing, or prolonging the effect of a checkpoint inhibitor in a subject requiring such effect, or enabling the subject to respond to a checkpoint inhibitor, comprising administering to the subject an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with a checkpoint inhibitor, wherein the subject has a solid tumor. (Item 3) A method for enhancing the effect of a checkpoint inhibitor in a subject having a checkpoint inhibitor, comprising administering to the subject an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with the checkpoint inhibitor, wherein the subject has a solid tumor. (Item 4) A method for increasing blood flow to a solid tumor in a subject, comprising administering to the subject an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with a checkpoint inhibitor, wherein increasing blood flow to the solid tumor enhances the effect of the checkpoint inhibitor. (Item 5) The method according to item 4, wherein blood flow is measured using ultrasound-based blood flow measurement or using histological techniques for measuring hypoxia. (Item 6) A method for improving the delivery or efficacy of a checkpoint inhibitor in a subject, comprising administering an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with the checkpoint inhibitor, wherein the subject has a solid tumor, and thereby the delivery or efficacy of the treatment in the subject is improved. (Item 7) The method according to any one of items 1 to 6, wherein administration of bosentan or a pharmaceutically acceptable salt thereof increases the number of antitumor T cells co-localizing with a solid tumor. (Item 8) Administering bosentan or a pharmaceutically acceptable salt thereof to reduce the tissue hardness of solid tumors, as described in any one of items 1-7. (Item 9) The method according to item 8, wherein the tissue hardness of the solid tumor is measured using ultrasound elastography. (Item 10) The method according to any one of items 1 to 9, wherein administration of bosentan or a pharmaceutically acceptable salt thereof reduces the level of extracellular matrix proteins in the solid tumor. (Item 11) The method according to item 10, wherein the extracellular matrix protein is collagen I or hyaluronan-binding protein (HABP). (Item 12) The method according to any one of items 1 to 11, wherein administration of bosentan or a pharmaceutically acceptable salt thereof reduces the hypoxic state in the solid tumor. (Item 13) The method according to any one of items 1 to 12, wherein the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, BMA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof. (Item 14) The method according to item 13, wherein the checkpoint inhibitor is a CTLA-4, PD-L1, PD-L2, or PD-1 inhibitor. (Item 15) The method according to item 13, wherein the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. (Item 16) The method according to item 13, wherein the checkpoint inhibitor is selected from the group consisting of MEDI0680, AMP-224, nivolumab, pembrolizumab, pidilizumab, MEDI4736, atezolizumab, ipilimumab, tremelimumab, and BMS-936559. (Item 17) The method according to any one of items 13 to 16, wherein the checkpoint inhibitor is a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody. (Item 18) The method according to any one of items 1 to 17, wherein the bosentan or a pharmaceutically acceptable salt thereof is administered to the subject once daily. (Item 19) The method according to any one of items 1 to 17, wherein the bosentan or a pharmaceutically acceptable salt thereof is administered to the subject twice daily. (Item 20) The method according to any one of items 1 to 19, wherein bosentan or a pharmaceutically acceptable salt thereof is administered to the subject in a dose of about 0.01 mg / kg to about 5 mg / kg. (Item 21) The method according to any one of items 1 to 19, wherein bosentan or a pharmaceutically acceptable salt thereof is administered to the subject in a dose of about 100 mg to about 1200 mg. (Item 22) The method according to any one of items 1 to 19, wherein bosentan or a pharmaceutically acceptable salt thereof is administered to the subject in a dose of about 125 mg to about 500 mg. (Item 23) The method according to any one of items 1 to 19, wherein bosentan or a pharmaceutically acceptable salt thereof is administered to the subject in a dose of about 125 mg. (Item 24) The method according to any one of items 1 to 19, wherein bosentan or a pharmaceutically acceptable salt thereof is administered to the subject in a dose of about 500 mg. (Item 25) The method according to any one of items 1 to 24, wherein the bosentan or a pharmaceutically acceptable salt thereof is administered to the subject before administering the checkpoint inhibitor to the subject. (Item 26) The method according to item 25, wherein the bosentan or a pharmaceutically acceptable salt thereof is administered to the subject starting at least one day before administering the checkpoint inhibitor to the subject. (Item 27) The method according to item 25, wherein the bosentan or a pharmaceutically acceptable salt thereof is administered to the subject starting at least two days before administering the checkpoint inhibitor to the subject. (Item 28) The method according to item 25, wherein the bosentan or a pharmaceutically acceptable salt thereof is administered to the subject starting at least 3 days before administering the checkpoint inhibitor to the subject. (Item 29) The method of item 25, wherein the bosentan or a pharmaceutically acceptable salt thereof is administered to the subject starting at least 5 days before administering the checkpoint inhibitor to the subject. (Item 30) The method according to any one of items 1 to 29, wherein the administration of the bosentan or a pharmaceutically acceptable salt thereof to the subject is maintained for at least a portion of the period during which the checkpoint inhibitor is administered to the subject. (Item 31) The method according to item 30, wherein the administration of the bosentan or a pharmaceutically acceptable salt thereof to the subject is maintained for the entire duration of the administration of the checkpoint inhibitor to the subject. (Item 32) The method according to any one of items 1 to 31, wherein one or more therapeutic effects in the subject are improved compared to baseline after administration of the bosentan or a pharmaceutically acceptable salt thereof and the checkpoint inhibitor. (Item 33) The method according to item 32, wherein the one or more therapeutic effects are selected from the group consisting of tumor size derived from cancer, objective response rate, duration of response, time to response, progression-free survival, and overall survival. (Item 34) The method according to any one of items 1 to 33, wherein the size of the tumor derived from the cancer is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% compared to the size of the tumor derived from the cancer before administration of the bosentan or a pharmaceutically acceptable salt thereof and the checkpoint inhibitor. (Item 35) The method according to any one of items 1 to 34, wherein the objective response rate is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%. (Item 36) The method according to any one of items 1 to 35, wherein the subject exhibits a progression-free survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of the bosentan or a pharmaceutically acceptable salt thereof and the checkpoint inhibitor. (Item 37) The method according to any one of items 1 to 36, wherein the subject exhibits an overall survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of the bosentan or a pharmaceutically acceptable salt thereof and the checkpoint inhibitor. (Item 38) The method according to any one of items 1 to 37, wherein the duration of response to the antibody-drug conjugate is at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of the bosentan or a pharmaceutically acceptable salt thereof and the checkpoint inhibitor. (Item 39) The method according to any one of items 1 to 38, wherein the solid tumor is selected from the group consisting of breast cancer, lung metastases of breast cancer, sarcoma, pancreatic cancer, ovarian cancer, liver metastases, prostate cancer, brain cancer, melanoma, mesothelioma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, lung cancer, head and neck squamous cell carcinoma, urothelial carcinoma, esophageal squamous cell carcinoma, gastric cancer, esophageal cancer, cervical cancer, Merkel cell carcinoma, endometrial cancer, and cutaneous squamous cell carcinoma. (Item 40) The method according to item 39, wherein the solid tumor is breast cancer. (Item 41) The method according to item 40, wherein the breast cancer is triple-negative breast cancer. (Item 42) The method described in any one of items 1 to 41, wherein the subject is a human. (Item 43) (a) an effective amount of bosentan or a pharmaceutically acceptable salt thereof; (b) an effective dose of a checkpoint inhibitor; and (c) Instructions for use of the bosentan or a pharmaceutically acceptable salt thereof and the checkpoint inhibitor in accordance with the method described in any one of items 1 to 42. A kit that includes this. (Item 44) A method for determining the effective amount of an agent that decompresses blood vessels in a subject with a solid tumor: (a) A step of measuring the blood flow and / or hardness of the solid tumor; (b) The step of administering an effective amount of an agent that decompresses the blood vessels to the subject; and (c) A method comprising the step of measuring the blood flow and / or hardness of a solid tumor after administration of a vascular decompressant, wherein an increase in blood flow and / or decrease in hardness after administration of the vascular decompressant to the subject indicates that the administered amount was an effective amount. (Item 45) A method for treating solid tumors in subjects requiring it: (a) A step of measuring the blood flow and / or hardness of the solid tumor; (b) The step of administering an effective amount of a vasodilator to the subject; (c) the step of measuring the blood flow and / or hardness of the solid tumor after administering an agent that decompresses the blood vessels; and (d) A step of administering a chemotherapeutic agent if the blood flow to the solid tumor increases and / or the hardness of the solid tumor decreases after administration of the vasoconstrictor. A method that includes this. (Item 46) A method for treating solid tumors in subjects requiring it: (a) A step of measuring the blood flow and / or hardness of the solid tumor; (b) The step of administering an effective amount of a vasodilator to the subject; (c) A step of measuring the blood flow and / or hardness of the solid tumor after administering an agent that decompresses the blood vessels; (d) A step of determining that the subject is responsive to a chemotherapeutic agent based on an increase in blood flow to the solid tumor or a decrease in the hardness of the solid tumor after administration of an agent that decompresses the blood vessels; and (e) The step of administering the chemotherapeutic agent to a subject that has been determined to be responsive to the chemotherapeutic agent based on an increase in blood flow to the solid tumor or a decrease in the hardness of the solid tumor after administration of the vasodilator. A method that includes this. (Item 47) A method for predicting the response to treatment with chemotherapeutic agents: (a) A step of measuring the blood flow and / or hardness of a solid tumor; (b) A step of administering an effective dose of a vasodilator to the target; (c) A step of measuring the blood flow and / or hardness of the solid tumor after administering an agent that decompresses the blood vessels. A method comprising, wherein an increase in blood flow to the solid tumor or a decrease in the hardness of the solid tumor after administration of the vasodilator indicates that the subject is likely to respond to treatment with the chemotherapeutic agent. (Item 48) The method according to any one of items 45 to 47, wherein an effective amount of the vascular decompression agent is determined by measuring the change in blood flow and / or stiffness of the solid tumor after administration of the vascular decompression agent to the subject, and an increase in blood flow and / or decrease in stiffness after administration of the vascular decompression agent to the subject indicates that the administered amount was an effective amount. (Item 49) The method according to any one of items 44 to 48, wherein the method comprises the step of measuring the blood flow of the solid tumor, and the blood flow of the solid tumor increases after administration of an agent that decompresses the blood vessels. (Item 50) The method according to any one of items 44 to 48, wherein the method comprises the step of measuring the hardness of the solid tumor, and the hardness of the solid tumor decreases after administration of an agent that decompresses the blood vessels. (Item 51) The method according to any one of items 44 to 50, wherein the vasodilator is administered for at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days prior to the administration of the chemotherapeutic agent. (Item 52) The method according to any one of items 44 to 51, wherein the vasodilator is administered in a dose that increases blood flow to the solid tumor and / or reduces the hardness of the solid tumor. (Item 53) The method according to any one of items 44 to 52, wherein the decompressant agent is bosentan or a pharmaceutically acceptable salt thereof. (Item 54) The method according to any one of items 44 to 53, wherein the blood flow and / or hardness of the solid tumor is measured using ultrasound. (Item 55) The method according to any one of items 44 to 53, wherein the blood flow of the solid tumor is measured using histological techniques for measuring hypoxia. (Item 56) The method according to any one of items 45 to 55, wherein the chemotherapeutic agent is a checkpoint inhibitor. (Item 57) The method according to item 56, wherein the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, BMA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof. (Item 58) The method according to item 57, wherein the checkpoint inhibitor is a CTLA-4, PD-L1, PD-L2, or PD-1 inhibitor. (Item 59) The method according to item 57, wherein the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. (Item 60) The method according to item 57, wherein the checkpoint inhibitor is selected from the group consisting of MEDI0680, AMP-224, nivolumab, pembrolizumab, pidilizumab, MEDI4736, atezolizumab, ipilimumab, tremelimumab, and BMS-936559. (Item 61) The method according to any one of items 57 to 60, wherein the checkpoint inhibitor is a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody. (Item 62) The method according to any one of items 44 to 61, wherein the solid tumor is selected from the group consisting of breast cancer, lung metastases of breast cancer, sarcoma, pancreatic cancer, ovarian cancer, liver metastases, prostate cancer, brain cancer, melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, lung cancer, head and neck squamous cell carcinoma, urothelial carcinoma, esophageal squamous cell carcinoma, gastric cancer, esophageal cancer, cervical cancer, Merkel cell carcinoma, endometrial cancer, mesothelioma, and cutaneous squamous cell carcinoma. (Item 63) The method according to item 62, wherein the solid tumor is breast cancer. (Item 64) The method according to item 63, wherein the breast cancer is triple-negative breast cancer. (Item 65) The method described in any one of items 44 to 64, wherein the subject is a human. [Brief explanation of the drawing]

[0017] [Figure 1-1]Figures 1A–1L are a series of images and graphs showing that bosentan normalizes the tumor's organic microenvironment. Figure 1A: Heatmap of ultrasound elastography 10 days post-treatment of E0771 tumors treated with control (top) and 1 mg / kg bosentan, where low kPa indicates flexible tissue and high kPa indicates rigid tissue. The dashed black line indicates the tumor boundary. Figure 1B: Quantification of longitudinal elasticity of E0771 tumor. Symbols indicate P<0.05. Figure 1C: Quantification of longitudinal elasticity in 4T1 tumor. Symbols indicate P<0.05. Figure 1D: Quantification of Young's modulus by atomic force microscopy measurement in E0771 tumor. Symbols indicate P<0.05. Figure 1E: Representative atomic force microscopy hardness fingerprint histogram of E0771 tumors treated with control. The peak on the left of the graph represents the involvement of flexible cancer cells, while the tail portion within the frame represents the involvement of harder components such as collagen. Figure 1F: Representative atomic force microscopy hardness fingerprint histogram of E0771 tumors treated with 1 mg / kg bosentan. Figure 1G: Representative atomic force microscopy hardness fingerprint histogram of E0771 tumors treated with 10 mg / kg bosentan. Figure 1H: Quantification of interstitial fluid pressure in E0771 tumors. Symbols indicate P<0.05. Figure 1I: Representative images of pico-sirius red staining, α-SMA immunofluorescence staining, and hyaluronan-binding protein (HABP) immunofluorescence staining in E0771 tumors. Figure 1J: Quantification of pico-sirius red in E0771 tumors. Symbols indicate P<0.05. Figure 1K: Quantification of α-SMA staining in 4T1 tumors. Symbols indicate P<0.05. Figure 1L shows the quantitative analysis of HABP staining in E0771 tumors. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above.

[0018] [Figure 2-1]Figures 2A-2F are a series of images and graphs showing that bosentan reduces hypoxia and increases the association between blood vessels and T cells. Figure 2A: Representative images of pimonidazole (hypoxia) staining (upper box) and colocalization of CD3+ T cells and CD31+ endothelial cells (lower box) in an E0771 tumor. Figure 2B: Quantification of hypoxic region fraction in a 4T1 tumor. Symbols indicate P<0.05. Figure 2C: Quantification of proximity between CD3+ T cells and CD31+ endothelial cells in a 4T1 tumor. Symbols indicate P<0.05. Figure 2D: Quantification of CD3+ region fraction in a 4T1 tumor. Symbols indicate P<0.05. Figure 2E: Quantification of CD31+ region fraction in a 4T1 tumor. Symbols indicate P<0.05. Figure 2F: Quantification of mRNA expression in a 4T1 tumor. Symbols indicate P<0.05. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0019] [Figure 3-1]Figures 3A–3E are a series of graphs showing that bosentan enhances the efficacy of immune checkpoint blockade (ICB) in triple-negative breast cancer (TNBC). Figure 3A: Tumor growth curves for E0771 tumors. Mice were treated with control (black), TME-normalizing 1 mg / kg bosentan monotherapy (purple), an anti-PD-1 and anti-CTLA-4 ICB cocktail (green), or a combination (orange). All of these significantly slowed tumor growth. n=10. Symbols indicate P<0.05. Figure 3B: Tumor growth curves for 4T1 tumors. Only the combination significantly slowed tumor growth. n=8–10. Symbols indicate P<0.05. Figure 3C: Kaplan-Meier survival curves for mice with spontaneously occurring E0771 metastases arising from surgically resected primary tumors. All mice except 80% of the combination-treated mice died before 80 days post-inoculation. n=10. Symbols indicate P<0.05. Figure 3D: Kaplan-Meier survival curves for mice with spontaneously occurring 4T1 metastases arising from surgically resected primary tumors. Only mice treated with the combination therapy had a longer median overall survival. n=8-10. The symbol indicates P<0.05. Figure 3E: Tumor growth curves comparing surviving mice rechallenged with E0771 cancer cells with control mice naive to E0771 cancer cells. [Figure 3-2] Same as above.

[0020] [Figure 4] Figures 4A-4B are a series of graphs showing the correlation between stiffness and tumor response to ICB. Figure 4A: Correlation between elastic Young's modulus before ICB treatment and tumor volume after treatment in mice with E0771 tumors (n=5-6 mice / group) treated with either the ICB cocktail alone or the combination therapy of bosentan and ICB (R2=0.9657, p<0.0001). Figure 4B: Correlation between elastic Young's modulus before ICB treatment and tumor volume after treatment in mice with 4T1 tumors (n=5-6 mice / group) treated with either the ICB cocktail alone or the combination therapy of bosentan and ICB (R2=0.9387, p<0.0001).

[0021] [Figure 5-1] Figures 5A-5C show mouse tumor models treated with bosentan plus anti-PD-1 / anti-CTLA-4 therapy or anti-PD-1 / anti-CTLA-4 therapy alone. Figure 5A: Schematic diagram of the study. Figure 5B: Effect of bosentan combined with antibody therapy or antibody therapy alone compared to the control, as evaluated by tumor volume over time. Figure 5C: Effect of bosentan plus antibody therapy or antibody therapy alone in the mouse model, as evaluated by elastic modulus. [Figure 5-2] Same as above.

[0022] [Figure 6] Figures 6A and 6B show the mean transit time (Figure 6A) and rise time (Figure 6B) calculated from time-intensity curves created during dynamic contrast-enhanced ultrasound measurements of mice with 4T1 tumors. Anti-PD-1 / anti-CTLA-4 (ICB) and bosentan plus ICB (Bos+ICB) were compared to controls.

[0023] [Figure 7] Figures 7A-7B show the measurement of the effect of ketotifen monotherapy in mice transplanted with MCA205 tumors (Figure 7A) or K7M2wt tumors (Figure 7B). All data are expressed as the mean + / - mean standard error (mouse n=5-7 / treatment group).

[0024] [Figure 8] Figure 8 shows IFP levels in untreated mice with MCA205 tumors and mice treated daily with ketotifen for 7 days (mouse n=7 / treatment group).

[0025] [Figure 9] Figure 9 shows the longitudinal axis measurement of macroscopic Young's modulus at the histological level of MCA205 tumors in mice or control mice treated with the indicated doses of ketotifen.

[0026] [Figure 10-1]Figures 10A–10D are a series of graphs showing the effects of ketotifen on vascular perfusion or functional perfusion areas in mice with MCA205 or K7M2wt tumors. Figures 10A and 10B show the effects on MCA205 tumors. Figures 10C and 10D show the effects on K7M2wt tumors. [Figure 10-2] Same as above.

[0027] [Figure 11] Figures 11A and 11B show the effects of the indicated monotherapy and combination therapy on mice with MCA205 tumors (Figure 11A) or K7M2wt tumors (Figure 11B).

[0028] [Figure 12] Figures 12A-12B show schematic diagrams of treatment with tranilast and anti-PD-L1 antibody in mice with MCA205 tumors (Figure 12A) or E0771 tumors (Figure 12B).

[0029] [Figure 13] Figure 13 shows the results of treating mice with MCA205 tumors with anti-PD-L1 therapy, along with control, anti-PD-L1 antibody, or tranilast pretreatment at concentrations indicated.

[0030] [Figure 14] Figure 14 shows the results of treating mice with E0771 tumors with a control, anti-PD-L1 antibody, or anti-PD-L1 therapy with tranilast pretreatment at concentrations indicated.

[0031] [Figure 15]Figures 15A–15E are a series of graphs showing the correlations between elastic modulus and relative tumor volume (Figure 15A), mean transit time and relative tumor volume (Figure 15B), rise time and relative tumor volume (Figure 15C), elastic modulus and mean transit time (Figure 15D), and elastic modulus and rise time (Figure 15E) for mice treated with bosentan or tranilast, or mice pre-treated with bosentan or tranilast and then treated with immunotherapy. The correlations were evaluated by measurements at the start and end of the immunotherapy experiment.

[0032] [Figure 16] Figures 16A–16E are a series of graphs showing the correlations between elastic modulus and relative tumor volume (Figure 16A), inflow velocity and relative tumor volume (Figure 16B), time to peak brightness and relative tumor volume (Figure 16C), elastic modulus and inflow velocity (Figure 16D), and elastic modulus and time to peak brightness (Figure 16E) for mice with MCA205 tumors treated with control, anti-PD-L1 alone, or anti-PD-L1 immunotherapy after tranilast pretreatment.

[0033] [Figure 17-1] Figure 17 shows the results of treating mice with MCA205 tumors with the treatment described above. [Figure 17-2] Same as above.

[0034] [Figure 18] Figures 18A–18E are a series of graphs showing the correlations between elastic modulus and relative tumor volume (Figure 18A), inflow velocity and relative tumor volume (Figure 18B), time to peak brightness and relative tumor volume (Figure 18C), elastic modulus and inflow velocity (Figure 18D), and elastic modulus and time to peak brightness (Figure 18E) for mice with E0771 tumors treated with control, anti-PD-L1 alone, or anti-PD-L1 immunotherapy after tranilast pretreatment.

[0035] [Figure 19-1] Figure 19 shows the results of treatment of mice with E0771 tumors. [Figure 19-2] Same as above.

[0036] [Figure 20-1] Figures 20A–20E are a series of graphs showing the correlations between elastic modulus and relative tumor volume (Figure 20A), inflow velocity and relative tumor volume (Figure 20B), time to peak brightness and relative tumor volume (Figure 20C), elastic modulus and time to peak brightness (Figure 20D), and elastic modulus and inflow velocity (Figure 20E) in mice with MCA205 or E0771 tumors treated with anti-PD-L1 monotherapy, or anti-PD-L1 immunotherapy after tranilast pretreatment. [Figure 20-2] Same as above. [Modes for carrying out the invention]

[0037] Detailed explanation I. Definition To make the present invention more easily understandable, certain scientific and technical terms are defined below. Unless otherwise specifically defined elsewhere in this text, all other scientific and technical terms used herein have the meanings generally understood by those skilled in the art to which the present invention belongs.

[0038] As used herein, including in the attached claims, the singular forms of terms, such as “a,” “an,” and “the,” include their corresponding plural forms unless the context specifically indicates otherwise.

[0039] A composition or method “containing” one or more of the detailed elements may also contain other elements not specifically described. For example, a composition containing an antibody may contain the antibody alone or in combination with other components.

[0040] The aspects and embodiments of the present invention described herein are understood to include "including," "consisting of," and "essentially consisting of."

[0041] Specifying a range of values ​​includes all integers within or defining the range.

[0042] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art to whom this disclosure relates. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd ed., 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd ed., 1999, Academic Press; and *the Oxford Dictionary of Biochemistry and Molecular Biology*, Revised, 2000, Oxford University Press provide many common dictionaries of the terms used herein.

[0043] Units, prefixes, and symbols are shown in the form permitted by their International System of Units (SI). Numerical ranges include the digit defining the range. The headings provided herein are not limitations on the various aspects of this disclosure that can be obtained by referring to this specification in whole. Thus, the terms defined immediately below are more fully defined by referring to this specification in its entirety.

[0044] When referred to herein, the term "weight-based dosage" means that the dosage administered to a subject is calculated based on the subject's body weight. For example, if a subject weighing 60 kg requires 2.0 mg / kg of bosentan or a checkpoint inhibitor, the inventors can calculate and use an appropriate amount of bosentan or a checkpoint inhibitor (i.e., 120 mg) to administer to the subject.

[0045] With respect to the methods and dosages described herein, the term “fixed dose” means the dose administered to a subject regardless of their body weight or body surface area (BSA). Therefore, the fixed dose is provided as an absolute amount of the agonist (e.g., bosentan and / or checkpoint inhibitor), rather than as a mg / kg dose. For example, a subject weighing 60 kg and a subject weighing 100 kg would be administered the same dose of bosentan or checkpoint inhibitor.

[0046] "Cancer" refers to a broad range of diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancer tissue" can include tumors. Uncontrolled cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distal parts of the body through the lymphatic system or bloodstream. After metastasis, a distal tumor can be said to "originate" from the tumor that was present before metastasis. For example, a "tumor originating from breast cancer" refers to a tumor that is a result of metastatic breast cancer.

[0047] "Administer" or "dosage" refers to the physical introduction of a therapeutic agent into a target using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for bosentan and / or checkpoint inhibitors include the intestinal route and intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, or other parenteral routes, such as injection or infusion (e.g., intravenous infusion). The term "parenteral administration," as used herein, typically means, but is not limited to, intestinal and topical administration by injection, but includes intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intracisional injections and infusions, as well as in vivo electroporation. The therapeutic agent may be administered via parenteral routes or orally. Other parenteral routes include topical, cutaneous, or mucosal administration routes, such as intranasal, intravaginal, intrarectal, sublingual, or topical. Administration can also be carried out, for example, once, multiple times, and / or one or more long-term periods.

[0048] In antibodies or other proteins described herein, references to amino acid residues corresponding to residues specified by sequence numbers include post-translational modifications of such residues.

[0049] The term "antibody" refers to an immunoglobulin protein produced by the body in response to the presence of an antigen and bound to that antigen, as well as its antigen-binding fragments and engineered variants. Therefore, the term "antibody" includes, for example, intact monoclonal antibodies (e.g., antibodies produced using hybridoma technology) and antigen-binding antibody fragments, such as F(ab')2, Fv fragments, diabodies, single-chain antibodies, scFv fragments, or scFv-Fc. Generally, it also includes genetically engineered intact antibodies and fragments, such as chimeric antibodies, humanized antibodies, single-chain Fv fragments, single-chain antibodies, diabodies, minibodies, linear antibodies, and multivalent or multispecific (e.g., bispecific) hybrid antibodies. Thus, the term "antibody" is broadly used to include any protein that contains an antigen-binding site and is capable of specifically binding to that antigen.

[0050] The term "antibody or its antigen-binding fragment" includes "conjugated" antibodies or their antigen-binding fragments, or "antibody-drug conjugates (ADCs)," in which the antibody or its antigen-binding fragment is covalently or noncovalently bound to a drug, such as a cytotoxic agent or cytotoxic drug.

[0051] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a particular species (e.g., human) or belongs to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody from another species (e.g., mouse) or belongs to another antibody class or subclass, as well as fragments of such antibodies insofar as they exhibit the desired biological activity.

[0052] The "antigen-binding site of an antibody" is the portion of the antibody that is sufficient to bind to that antigen. The smallest such region is typically a variable domain or a genetically modified variant thereof. Single-domain antibodies can be produced by generating a single-domain binding site from a camelid antibody (Muyldermans and Lauwereys, Mol. Recog. 12: 131-140, 1999; Nguyen et al., EMBO J. 19:921-930, 2000) or a VH domain from another species ("dAb", Ward et al., Nature 341: 544-546, 1989; see U.S. Patent No. 6,248,516 to Winter et al.). Generally, the antigen-binding site of an antibody contains both a heavy-chain variable (VH) domain and a light-chain variable (VL) domain that bind to a common epitope. In the context of the present invention, an antibody may include, in addition to an antigen-binding site, one or more components, such as a second antigen-binding site of the antibody (which may bind to the same or different epitopes or the same or different antigens), a peptide linker, an immunoglobulin constant region, an immunoglobulin hinge, an amphiphilic helix (see Pack and Pluckthun, Biochem. 31: 1579-1584, 1992), a non-peptide linker, an oligonucleotide (see Chaudri et al., FEBS Letters 450:23-26, 1999), a cell suppressor or cytotoxic agent, and may be a monomer or a multimeric protein.Examples of molecules containing antibody antigen-binding sites are known in the art, including, for example, Fv, single-stranded Fv(scFv), Fab, Fab', F(ab')2, F(ab)c, diabody, minibody, nanobody, Fab-scFv fusion, bispecific (scFv)4-IgG, and bispecific (scFv)2-Fab (e.g., Hu et al., Cancer Res. 56:3055-3061, 1996; Atwell et al., Molecular Immunology 33: 1301-1312, 1996; Carter and Merchant, Curr. Op. Biotechnol. 8:449-454, 1997; Zuo et al., Protein Engineering 13:361-367, 2000; and Lu et al., J. Immunol. Methods 267:213-226). (See 2002).

[0053] The term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by an immunoglobulin gene(s). One form of immunoglobulin constitutes the basic structural unit of native (i.e., native or parental) antibodies in vertebrates. This form is a tetramer, consisting of two identical pairs of immunoglobulin chains, each having one light chain and one heavy chain. In each pair, the variable regions (VL and VH) of the light and heavy chains are both primarily involved in binding to the antigen, while the constant region is primarily involved in antibody effector function. In higher vertebrates, five classes of immunoglobulin proteins (IgG, IgA, IgM, IgD, and IgE) have been identified. IgG is the major class and is usually found as the second most abundant protein in plasma. In humans, IgG consists of four subclasses designated as IgG1, IgG2, IgG3, and IgG4. Each immunoglobulin heavy chain possesses a constant region consisting of constant region protein domains (CH1, hinge, CH2, and CH3; IgG3 also contains a CH4 domain) that are essentially invariant with respect to a given subclass in a species.

[0054] DNA sequences encoding human and non-human immunoglobulin chains are publicly known in the art. (e.g. Ellison et al, DNA 1: 11-18, 1981;Ellison et al, Nucleic Acids Res. 10:4071-4079, 1982;Kenten et al., Proc. Natl. Acad. Set USA 79:6661-6665, 1982;Seno et al., Nucl. Acids Res. 11 8:2055-2065, 1980;Rusconi and Kohler, Nature 314:330-334, 1985;Boss et al., Nucl. Acids Res. See also 12:3791-3806, 1984; Bothwell et al., Nature 298:380-382, 1982; van der Loo et al., Immunogenetics 42:333-341, 1995; Karlin et al., J. Mol. Evol. 22: 195-208, 1985; Kindsvogel et al., DNA 1:335-343, 1982; Breiner et al., Gene 18: 165-174, 1982; Kondo et al., Eur. J. Immunol. 23:245-249, 1993; and GenBank Accession No. J00228). For commentaries on immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol V, Academic Press, Inc., 49-140, 1987; and Padlan, Mol. Immunol. 31: 169-217, 1994. The term “immunoglobulin” is used herein in its general sense and, depending on the context, refers to an intact antibody, a chain of its constituents, or a fragment of a chain.

[0055] The full-length immunoglobulin "light chain" (approximately 25 kDa or 214 amino acids) is encoded at the amino terminus by a variable region gene (encoding approximately 110 amino acids) and at the carboxyl terminus by a kappa or lambda constant region gene. The full-length immunoglobulin "heavy chain" (approximately 50 kDa or 446 amino acids) is encoded by a variable region gene (encoding approximately 116 amino acids) and a gamma, mu, alpha, delta, or epsilon constant region gene (encoding approximately 330 amino acids), the latter defining the antibody isotype, e.g., IgG, IgM, IgA, IgD, or IgE, respectively. In the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 10 or more amino acids. (For general reference, see Fundamental Immunology (Paul, ed., Raven Press, NY, 2nd ed. 1989), Chapter 7).

[0056] The immunoglobulin light chain or heavy chain variable region (hereinafter also referred to herein as the “light chain variable domain” (“VL domain”) or the “heavy chain variable domain” (“VH domain”), respectively) consists of a “framework” region interrupted by three “complementarity-determining regions” or “CDRs.” The framework region acts to align the CDRs to specifically bind to the antigen’s epitope. Thus, the term “CDR” refers to the amino acid residue of the antibody primarily involved in antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0057] The assignment of amino acids to each variable region domain follows the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991). Kabat also provides a widely used numbering convention (Kabat numbering) in which the same number of corresponding residues are assigned between different heavy chain variable regions or between different light chain variable regions. The VL domains CDR1, 2, and 3 are also referred herein as CDR-L1, CDR-L2, and CDR-L3, respectively. The VH domains CDR1, 2, and 3 are also referred herein as CDR-H1, CDR-H2, and CDR-H3, respectively. Where referred to in this way, the assignment of CDRs may follow IMGT® (Lefranc et al., Developmental & Comparative Immunology 27:55-77; 2003) instead of Kabat.

[0058] The numbering of the heavy chain constant region is done via the EU index described in Kabat (Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991).

[0059] Unless otherwise specified in the context, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” may include antibodies derived from a single clone, including any eukaryotic cell, prokaryotic cell, or phage clone. In certain embodiments, the antibodies described herein are monoclonal antibodies.

[0060] A “human antibody” (HuMAb) refers to an antibody in which both the FR and CDR have variable regions derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also derives from a human germline immunoglobulin sequence. The human antibodies of this disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutations in vitro, or by somatic mutations in vivo). However, as used herein, the term “human antibody” is intended not to include antibodies in which a CDR sequence derived from the germline of another mammalian species, e.g., mouse, is grafted onto a human framework sequence. The terms “human antibody” and “complete human antibody” are used synonymously.

[0061] The terms “humanized VH domain” or “humanized VL domain” refer to an immunoglobulin VH or VL domain that contains some or all of the CDRs that are entirely or substantially derived from a non-human donor immunoglobulin (e.g., mouse or rat) and a variable domain framework sequence that is entirely or substantially derived from a human immunoglobulin sequence. The non-human immunoglobulin that provides the CDRs is called the “donor,” and the human immunoglobulin that provides the framework is called the “acceptor.” In some cases, humanized antibodies retain some non-human residues within the human variable domain framework region to enhance appropriate binding characteristics (e.g., mutations in the framework may be necessary to maintain binding affinity if the antibody is humanized).

[0062] A "humanized antibody" is an antibody that contains one or both of the humanized VH domain and / or humanized VL domain. The immunoglobulin constant region(s) are not required to be present, but if present, they are entirely or substantially derived from the human immunoglobulin constant region.

[0063] Humanized antibodies are genetically engineered antibodies in which a CDR from a non-human "donor" antibody is grafted onto a human "acceptor" antibody sequence (see, for example, Queen, U.S. Patents 5,530,101 and 5,585,089; Winter, U.S. Patent 5,225,539; Carter, U.S. Patent 6,407,213; Adair, U.S. Patent 5,859,205; and Foote, U.S. Patent 6,881,557). The acceptor antibody sequence may be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence.

[0064] Hi The acceptor sequence can be selected for a high degree of sequence identity in the variable region framework having the donor sequence to match the typical form between the acceptor and donor CDR, among other criteria. Thus, a humanized antibody is an antibody having a CDR that is entirely or substantially derived from the donor antibody, and, if present, a variable region framework sequence and constant region that are entirely or substantially derived from the human antibody sequence. Similarly, a humanized heavy chain typically has all three CDRs that are entirely or substantially derived from the donor antibody heavy chain, and, if present, a heavy chain variable region framework sequence and heavy chain constant region that are substantially derived from the human heavy chain variable region framework and constant region sequences. Likewise, a humanized light chain typically has all three CDRs that are entirely or substantially derived from the donor antibody light chain, and, if present, a light chain variable region framework sequence and light chain constant region that are substantially derived from the human light chain variable region framework and constant region sequences.

[0065] In humanized antibodies, CDRs are substantially derived from the corresponding CDRs of non-human antibodies if at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the corresponding residues (defined by Kabat numbering), or approximately 100% of the corresponding residues (defined by Kabat numbering), are identical among the respective CDRs. The variable region framework sequence or the constant region of an antibody chain substantially originates from a human variable region framework sequence or human constant region, respectively, if at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the corresponding residues (defined by Kabat numbering for the variable region and by EU numbering for the constant region) are identical, or if about 100% of the corresponding residues (defined by Kabat numbering for the variable region and by EU numbering for the constant region) are identical.

[0066] Humanized antibodies often incorporate all six CDRs (preferably defined by Kabat or IMGT®) of a mouse antibody, but they can also be constructed from fewer than six CDRs from a mouse antibody (e.g., at least three, four, or five CDRs) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology, 164: 1432-1441, 2000).

[0067] In a humanized antibody, a CDR is "substantially derived" from the corresponding CDR of a non-human antibody if at least 60%, at least 85%, at least 90%, at least 95%, or 100% of the corresponding residues (defined by Kabat (or IMGT)) are identical among the respective CDRs. In certain variations of a humanized VH or VL domain where the CDR is substantially derived from a non-human immunoglobulin, the CDR of the humanized VH or VL domain has six or fewer amino acid substitutions (preferably conservative substitutions) for all three CDRs compared to the corresponding non-human VH or VL CDR (e.g., five or fewer, four or fewer, three or fewer, two or fewer, or one or fewer). The variable region framework sequence of the antibody VH or VL domain, or the immunoglobulin constant region, if present, is "substantially derived" from the human VH or VL framework sequence or human constant region, respectively, if at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the corresponding residues (defined by Kabat numbering for the variable region and by EU numbering for the constant region) are identical, or if about 100% of the corresponding residues (defined by Kabat numbering for the variable region and by EU numbering for the constant region). Thus, all portions of humanized antibodies, excluding the CDR, are typically entirely or substantially derived from the corresponding portions of the natural human immunoglobulin sequence.

[0068] Antibodies are typically supplied in an isolated form. This means that the antibody is at least about 50% by weight pure of the interfering protein and other impurities resulting from its production or purification, but does not rule out the possibility that the antibody may be combined with an excess amount of a pharmaceutically acceptable carrier or other medium intended to facilitate its use. Sometimes, the antibody is at least about 60% by weight, about 70% by weight, about 80% by weight, about 90% by weight, about 95% by weight, or about 99% by weight pure of the interfering protein and impurities from its production or purification. Antibodies containing isolated antibodies can be conjugated with cytotoxic agents and can be supplied as antibody-drug conjugates.

[0069] The specific binding of an antibody to its target antigen typically occurs at least approximately 10 times. 6 , about 10 7 , about 10 8 , about 10 9 , or about 10 10 M -1 This refers to affinity. Specific bonds are of a detectably high order and are distinguishable from nonspecific bonds that occur at at least one nonspecific target. Specific bonds may result from the formation of bonds between specific functional groups or specific spatial fits (e.g., lock and key types), while nonspecific bonds are typically the result of van der Waals forces.

[0070] The term "epitope" refers to the site on an antigen to which an antibody binds. Epitopes can be formed from a sequence of amino acids or from adjacent discontinuous amino acids through the three-dimensional folding of one or more proteins. Epitopes formed from a sequence of amino acids are typically retained even when exposed to denaturants, such as solvents, while epitopes formed by three-dimensional folding are typically lost upon treatment with denaturants, such as solvents. Epitopes typically have a unique spatial structure and contain at least about 3, and more commonly at least about 5, at least about 6, at least about 7, or about 8–10 amino acids. Methods for determining the spatial structure of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0071] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay demonstrating the ability of one antibody to compete for binding to a target antigen with that of another antibody. Antibody epitopes can also be defined by X-ray crystallography of the antibody bound to its antigen to identify contact residues.

[0072] Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other (provided that such mutations do not result in an overall change in the antigenic structure). Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody.

[0073] Antibody competition can be determined by assays in which the test antibody inhibits the specific binding of the reference antibody to a common antigen (see, for example, Junghans et al., Cancer Res. 50: 1495, 1990). The test antibody competes with the reference antibody if an excess amount of the test antibody inhibits the binding of the reference antibody.

[0074] Antibodies identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to an adjacent epitope sufficiently proximal to the epitope to which the reference antibody binds, causing steric interference. Antibodies identified by competitive assays also include antibodies that indirectly compete with the reference antibody by causing a conformational change in the target protein, thereby preventing the reference antibody from binding to an epitope different from the epitope to which the test antibody binds.

[0075] Antibody effector function refers to functions involving the Fc region of Ig. Such functions may include, for example, antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), or complement-dependent cytotoxicity (CDC). Such functions may be influenced, for example, by the binding of the Fc region to Fc receptors on immune cells having phagocytic or lytic activity, or by the binding of the Fc region to components of the complement system. Typically, actions mediated by Fc-binding cells or complement components result in inhibition and / or depletion of LIV1-targeted cells. The Fc region of an antibody can recruit Fc receptor (FcR)-expressing cells and juxtapose them with antibody-coated target cells. Cells expressing IgG surface FcRs, including FcγRIII(CD16), FcγRII(CD32), and FcγRIII(CD64), can act as effector cells to destroy IgG-coated cells. Such effector cells include monocytes, macrophages, natural killer (NK) cells, neutrophils, and eosinophils. When FcγR engages with IgG, it activates ADCC or ADCP. ADCC is mediated by CD16+ effector cells through the secretion of pore-forming proteins and proteases, while phagocytosis is mediated by CD32+ and CD64+ effector cells (Fundamental Immunology, 4). th See Paul ed., Lippincott-Raven, NY, 1997, Chapters 3, 17 and 30; Uchida et al., J. Exp. Med. 199:1659-69, 2004; Akewanlop et al., Cancer Res. 61:4061-65, 2001; Watanabe et al., Breast Cancer Res. Treat. 53: 199-207, 1999).

[0076] In addition to ADCC and ADCP, the Fc region of cell-binding antibodies can also activate the classical complement pathway that induces CDC. C1q proteins in the complement system bind to the Fc region of antibodies when they form complexes with antigens. When C1q binds to a cell-binding antibody, it can initiate a cascade of events involving the activation of C4 and C2 proteolytic activity, generating C3 convertase. Cleavage of C3 to C3b by C3 convertase enables the activation of terminal complement components, including C5b, C6, C7, C8, and C9. Collectively, these proteins form membrane attack complex pores on antibody-coated cells. These pores disrupt the integrity of the cell membrane, killing the target cell (Immunobiology, 6). th See Chapter 2 of Janeway et al., Garland Science, NY, 2005.

[0077] The term “antibody-dependent cytotoxicity” or “ADCC” refers to a mechanism that induces cell death dependent on the interaction between antibody-coated target cells and immune cells (also called effector cells) that possess lytic activity. Such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. Effector cells bind to the Fc region of Ig bound to target cells via their antigen-binding site. As a result of effector cell activity, death of antibody-coated target cells occurs. In certain exemplary embodiments, the anti-LIV1 IgG1 antibody of the present invention mediates ADCC equal to or increased compared to the parent antibody and / or compared to the anti-LIV1 IgG3 antibody.

[0078] The term “antibody-dependent cell phagocytosis” or “ADCP” refers to the process by which antibody-coated cells are whole or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and / or dendritic cells) that bind to the Fc region of Ig. In certain exemplary embodiments, the anti-LIV1 IgG1 antibody of the present invention mediates ADCP that is equal to or increased compared to the parent antibody and / or compared to the anti-LIV1 IgG3 antibody.

[0079] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism in which the Fc region of a target-binding antibody activates a series of enzymatic reactions to induce cell death by creating holes in the target cell membrane.

[0080] Typically, antigen-antibody complexes, such as antibody-coated complexes on target cells, bind to and activate complement component C1q, which in turn activates the complement cascade, leading to target cell death. Complement activation also results in the deposition of complement components on the target cell surface, which can facilitate ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0081] "Cytotoxic effect" refers to the depletion, removal, and / or killing of target cells. "Cytotoxic agent" refers to a compound that has a cytotoxic effect on cells, thereby mediating the depletion, removal, and / or killing of target cells. In certain embodiments, the cytotoxic agent is conjugated with an antibody or administered in combination with an antibody. Suitable cytotoxic agents are further described herein.

[0082] "Cell inhibitory effect" refers to the inhibition of cell proliferation. "Cell inhibitor" refers to a compound that has a cell inhibitory effect on cells, thereby mediating the inhibition of growth and / or enlargement of a particular cell type and / or subset of cells. Suitable cell inhibitors are further described herein.

[0083] As used herein, “sub-therapeutic dose” means a dose lower than the usual or typical dose of a therapeutic compound when administered alone for the treatment of a proliferative disease (e.g., cancer), and / or, in the case of bosentan, a dose lower than the usual or typical dose of a therapeutic compound (e.g., bosentan or a checkpoint inhibitor) used to treat its indicated disease (e.g., pulmonary hypertension).

[0084] For example, “anticancer drugs” promote the regression of cancer in a subject. In some embodiments, a therapeutically effective dose of a drug promotes cancer regression to the point of eliminating the cancer. “Promoting cancer regression” means that administering an effective dose of a drug alone or in combination with an anticancer drug results in a reduction of tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the number and duration of disease-asymptomatic periods, or prevention of functional or physical impairment due to the disease. In addition, the terms “effective” and “efficacy” in relation to a treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other harmful physiological effects (adverse effects) at the cellular, organ, and / or biological level resulting from the administration of a drug.

[0085] "Chemotherapy agents" are chemical compounds useful in the treatment of cancer. Examples of chemotherapy agents include alkylating agents, e.g., thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates, e.g., busulfan, improsulfan and pigosulfan; aziridines, e.g., benzodopa, carbocone, metsuredopa, and uredopa; ethyleneimines and methylamelamines, e.g., altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenes (especially bratacin and bratacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapacon; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; calistatin; CC-1065 (that This includes adzeresin, karzeresin, and bizeresin synthetic analogs; podophyllotoxin; podophyllic acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including synthetic analogs, KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodicin; spongstatin; nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine , ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobembicin, fenestrine, prednimustine, trophosfamide, uracil mustard; nitrosourea, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, e.g., engine antibiotics (e.g., calichemycin, especially calichemycin gamma II and calichemycin omega II (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994));CDP323, oral alpha-4 integrin inhibitors; dynemicins, e.g., dynemicin A; esperamicin; and neocardinostatin chromophores and related chromoprotein enediin antibiotics); acrasinomycin, actinomycin, ausuramycin, azaserin, bleomycin, kactinomycin, carabicin, kaminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (e.g., ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposomal injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), PEGylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin; antimetabolites, e.g., methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epotilon, and 5-f Luoruracil (5-FU); Combretastatin; Folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; Purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; Androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; Antiadrenal agents, e.g., aminoglutethimide, mitotane, trilostane; Folic acid supplements, e.g., floric acid; Aceglatone; Aldophosphamide glycoside;Aminolevulinic acid; Enyluracil; Amsacrin; Bestrabusil; Bisanthren; Edatrexate; Defofamine; Demecolsin; Diadicone; Elformithine; Erliptinium acetate; Epotilon; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Ronidynin; Maytansinoids, e.g., Maytansine and Ansamitosine; Mitoguazone; Mitoxanthrone; Mopidanmol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Rosoxanthrone; 2-Ethylhydrazide; Procarbazine; PSK (Registered Trademark) Polysaccharide Complex (JHS Natural) Products, Eugene, Oreg.); Lazoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic Acid; Triadicone; 2,2',2'-Trichlorotriethylamine; Trichothecene (especially T-2 toxin, Beraclin A, Loridine A and Anguidin); Urethane; Vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Thiotepa; Taxoids, e.g., Paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), Albumin-Modified Nanoparticle Formulations of Paclitaxel (ABRAXANE®), and Docetaxel (TAXOTERE®, Rhome-Poulene) Rorer, Antony, France; chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents, e.g., cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vinca, e.g., vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®), which prevent tubulin polymerization from forming microtubules; etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin;Aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid, e.g., bexarotene (TARGRETIN®); bisphosphonates, e.g., clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pa Midronate (AREDIA®), Childronate (SKELID®), or Risedronate (ACTONEL®); Troxacitabine (1,3-dioxolane nucleoside cytosine analog); Antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R) (e.g., Erlotinib (Tarceva®)); and Cell proliferation-reducing agents such as VEGF-A; vaccines, e.g., THERATOOPE® vaccine and gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 sunitinib, SUTENT®, Pfizer) Perifosin, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); olafenib, ABT510; Bcl-2 inhibitors, e.g., oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors; tyrosine kinase inhibitors; serine-threonine kinase inhibitors, e.g., rapamycin (sirolimus, RAPAMUNE®);Farnesyltransferase inhibitors, such as ronafarnib (SCH Examples include 6636, SARASAR®; checkpoint inhibitors (e.g., inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, BMA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, or B-7 family ligands); and any pharmaceutically acceptable salts, acids, or derivatives of any of the above; and two or more combinations of the above, e.g., CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for treatment regimens with oxaliplatin (ELOXATIN®) in combination with 5-FU and leucovorin; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above; and two or more combinations of the above.

[0086] Chemotherapy agents as defined herein include “anti-hormone agents” or “endocrine therapeutic agents” that act to modulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth. These include hormones themselves, such as, but not limited to, anti-estrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (including NOLVADEX® tamoxifen), raloxifen, doroxifen, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and FARESTON® toremifene; aromatase inhibitors that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal gland, such as 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozol, RIVISOR® borozole, FEMARA® letrozole, and ARIMIDESEX® anastrozole; and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and go Sererin; and troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, e.g., PKC-alpha, Raf, and H-Ras; ribozymes, e.g., VEGF expression inhibitors (e.g., ANGIOZYME® ribozymes) and HER2 expression inhibitors; vaccines, e.g., gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rlL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; vinorelbine and esperamicin (see U.S. Patent No. 4,675,187), and any pharmaceutically acceptable salts, acids, or derivatives of any of the above; and any combination of two or more of the above.

[0087] The terms “baseline” or “baseline value” are used interchangeably herein and may refer to the measurement or characterization of symptoms before administration of treatment (e.g., bosentan or its pharmaceutically acceptable salts as described herein and / or checkpoint inhibitors as described herein) or at the start of treatment. Baseline values ​​may be compared to reference values ​​to determine reduction or improvement of symptoms of a disease such as cancer. The terms “reference” or “reference value” are used interchangeably herein and may refer to the measurement or characterization of symptoms after administration of treatment (e.g., bosentan or its pharmaceutically acceptable salts as described herein and / or checkpoint inhibitors as described herein). Reference values ​​may be measured once or multiple times during a drug regimen or treatment cycle, or at the completion of a drug regimen or treatment cycle. A “reference value” may be an absolute value, a relative value, a value with upper and / or lower limits, a range of values, an average value, a median, a mean value, or a value compared to a baseline value.

[0088] Similarly, the “baseline value” may be an absolute value, a relative value, a value with upper and / or lower limits, a range of values, an average value, a median, a mean value, or a value compared to a reference value. The reference value and / or baseline value may be obtained from one individual, from two different individuals, or from a group of individuals (e.g., a group of two, three, four, five, or more individuals).

[0089] "Sustained response" refers to a sustained effect on reducing tumor growth after discontinuation of treatment. For example, tumor size may remain the same as or smaller than the size at the start of the administration phase. In some embodiments, the sustained response has a duration at least the same as the treatment period, or at least 1.5, 2.0, 2.5, or 3 times longer than the treatment period.

[0090] As used herein, “complete response” or “CR” means the disappearance of all target lesions; “partial response” or “PR” means a reduction of at least 30% in the total sum of the longest diameters (SLD) of target lesions, with reference to baseline SLD; and “stable disease” or “SD” means that there is no reduction in target lesions sufficient to meet the criteria for PR, and no increase sufficient to meet the criteria for PD, with reference to the smallest SLD since the start of treatment.

[0091] As used herein, “progression-free survival” or “PFS” refers to the period during and after treatment in which the treated disease (e.g., cancer) does not worsen. Progression-free survival may include the length of time the patient experienced a complete or partial response, as well as the length of time the patient experienced stable disease.

[0092] As used herein, “Objective Response Rate” or “ORR” refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0093] As used herein, “overall survival” or “OS” refers to the percentage of individuals in a group that is likely to survive after a particular period of time.

[0094] The terms “patient” or “subject” include human and other mammalian subjects, such as non-human primates, rabbits, rats, mice, etc., and their transgenic species, who are receiving either prophylactic or therapeutic treatment.

[0095] In the context of the treatment of solid tumors with the administration of bosentan and / or checkpoint inhibitors described herein, the term “effective dose” refers to the amount of such bosentan and / or checkpoint inhibitor that is sufficient to inhibit or improve the development of one or more symptoms of the solid tumor. The effective dose of the antibody is administered in an “effective regimen.” The term “effective regimen” refers to the combination of the amount of bosentan and / or checkpoint inhibitor administered and the dosing frequency that is appropriate to achieve prophylactic or therapeutic treatment of the disorder (e.g., prophylactic or therapeutic treatment of a solid tumor).

[0096] The term "pharmaceutically acceptable" means that it is approved or eligible for approval by a federal or state regulatory authority for use in animals, more specifically in humans, or is listed in the United States Pharmacopoeia or other generally accepted pharmacopoeias. The term "pharmaceutically acceptable ingredient" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or medium used to formulate bosentan or a checkpoint inhibitor.

[0097] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt. Exemplary salts include sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, hydrogen sulfates, phosphates, superphosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, acid tartrates, ascorbic acid, succinates, maleates, gentisinates, fumarates, glucons, glucurons, saccharates, formates, benzoates, glutamates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, and pamoates (i.e., 1,1' -Methylenebis-(2-hydroxy-3-naphthoate) is an example. A pharmaceutically acceptable salt may also contain additional molecules, such as acetate ions, succinate ions, or other counterions. A counterion can be any organic or inorganic part that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have one or more charged atoms in its structure. An example of a pharmaceutically acceptable salt having multiple charged atoms may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0098] The use of alternatives (e.g., "or") should be understood to mean one of the alternatives, both, or any combination thereof. Where used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any detailed or enumerated components.

[0099] The term "and / or," as used herein, should be understood to refer to the specific disclosure of each of two designated features or components, with or without the other. Therefore, when used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when used in phrases such as "A, B, and / or C," the term "and / or" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0100] The terms “approximately” or “essentially including” refer to a value or composition that is within an acceptable margin of error for a particular value or composition as determined by those skilled in the art, and these depend in part on the method by which the value or composition is measured or determined, i.e., the limits of the measurement system. For example, “approximately” or “essentially including” may mean within one standard deviation or a standard deviation greater than one, according to the practice of the art. Alternatively, “approximately” or “essentially including” may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term may mean up to one order of magnitude, or up to five times the value. Where a particular value or composition is provided in this application and claims, unless otherwise stated, the meaning of “approximately” or “essentially including” should be assumed to be within an acceptable margin of error for that particular value or composition.

[0101] In the context of the present invention, a solvate is a form of the compound of the present invention that forms a complex in a solid or liquid state through coordination with a solvent molecule. A hydrate is one specific form of a solvate in which coordination occurs with water. In certain exemplary embodiments, a solvate in the context of the present invention is a hydrate.

[0102] The terms “inhibit” or “inhibit” mean to reduce by a measurable amount or to prevent completely. As used herein, the term “inhibit” may mean an inhibition or reduction of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.

[0103] The terms “treatment” or “to treat” refer to slowing, stopping, or reversing the progression of a disease or condition in a patient, as evidenced by a reduction or disappearance of the clinical or diagnostic symptoms of the disease or condition. Treatment may include, for example, a reduction in the severity of symptoms, the frequency of symptoms, or the frequency of relapses.

[0104] The term "prodrug," as used herein, refers to a compound that is converted to an active form upon in vivo administration. For example, the prodrug form of an active compound may be, but is not limited to, acylated (acetylated or otherwise) and ether derivatives, carboxylic acid esters or phosphate esters, and various salt forms of the active compound. Those skilled in the art will recognize how to readily modify the compounds of the present invention into prodrug forms to facilitate the delivery of the active compound to a target site in the host organism or patient. Those skilled in the art will also, where applicable, utilize the preferred pharmacokinetic parameters of the prodrug form to deliver the desired compound to a target site in the host organism or patient in order to maximize the intended effect of the compound in the treatment of cancer.

[0105] As used herein, the terms “synergistic” or “synergistic effect,” when used in connection with a description of the effectiveness of a combination of agonists, mean any measured effect of a combination that is greater than the effect predicted from the sum of the effects of the individual agonists.

[0106] As used herein, the terms “additive” or “additive effect,” when used in connection with a description of the efficacy of a combination of agonists, mean any measured effect of a combination that is similar to the effect predicted from the sum of the effects of the individual agonists.

[0107] The terms “approximately once a week,” “approximately once every two weeks,” or any other similar terms for dosing intervals used herein are approximate. “Approximately once a week” may include every 7 ± 1 days, i.e., every 6 to 8 days. “Approximately once every two weeks” may include every 14 ± 2 days, i.e., every 12 to 16 days. “Approximately once every three weeks” may include every 21 ± 3 days, i.e., every 18 to 24 days. Similar approximations would be, for example, once every 4 weeks, once every 5 weeks, once every 6 weeks, and once every 12 weeks. In some embodiments, dosing intervals of approximately once every 6 weeks or once every 12 weeks mean that the first dose can be administered on any day in the first week, and then subsequent doses can be administered on any day in the 6th or 12th week, respectively. In other embodiments, a dosing interval of approximately once every six weeks or once every twelve weeks means that the first dose is administered on a specific day in the first week (e.g., Monday), and then the next dose is administered on the same day in the sixth or twelfth week (i.e., Monday), respectively.

[0108] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the detailed range, and, unless otherwise specified, its decimal form (e.g., one-tenth and one-hundredth of an integer) where applicable.

[0109] Various aspects of this disclosure are described in further detail in the following subsections. II. Bosentan

[0110] The compound 4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidine-2-yl)pyrimidine-4-yl]benzenesulfonamide, also known as bosentan, is a biendothelin receptor antagonist with affinity for both endothelin ETA and ETB receptors, useful for treating or preventing endothelin receptor-mediated disorders, such as pulmonary arterial hypertension ("PAH") in individuals with World Health Organization functional class III or IV primary pulmonary hypertension, and secondary pulmonary hypertension in patients with scleroderma or congenital heart disease, or human immunodeficiency virus (HIV). Bosentan is described in U.S. Patent No. 5,292,740.

[0111] In some embodiments, bosentan, as used herein, refers to the compound described in U.S. Patent No. 5,292,740. In some embodiments, bosentan, as used herein, refers to a compound having the following formula: [ka]

[0112] In some embodiments, the following formula is used herein: [ka] A bosentan hydrate having the following properties is provided.

[0113] In some embodiments, pharmaceutically acceptable salts of bosentan are provided herein.

[0114] Preparations of bosentan are disclosed in the following patents: European Patent No. 0526708, Canadian Patent No. 2,071,193, U.S. Patent No. 5,292,740, Canadian Patent No. 2,397,258, and U.S. Patent No. 5,883,254. III. Checkpoint Inhibitors

[0115] Immune checkpoints refer to inhibitory pathways in the immune system that are involved in maintaining self-tolerance to minimize peripheral tissue damage and modulating the degree of the immune system's response. However, tumor cells can also activate immune system checkpoints to reduce the effectiveness of the immune response against tumor tissue ("blocking" the immune response). In contrast to many anticancer drugs, checkpoint inhibitors do not directly target tumor cells, but rather target lymphocyte receptors or their ligands to enhance the endogenous antitumor activity of the immune system (Pardoll, 2012, Nature Reviews Cancer 12:252-264). Treatment with antagonistic checkpoint-blocking antibodies against immune system checkpoints, such as CTLA4, PD1, and PD-L1, is one of the most promising novel approaches to immunotherapy for cancer and other diseases. Additional checkpoint targets, such as TIM-3, LAG-3, various B-7 ligands, CHK1 and CHK2 kinases, BTLA, A2aR, and others, are also being investigated. Checkpoint inhibitors include atezolizumab (Tecentriq®), PD-L1 inhibitors, ipilimumab (Yervoy®), CTLA-4 inhibitors, and pembrolizumab (Keytruda®) and nivolumab (Opdivo®), both of which are PD-1 inhibitors.

[0116] Recent data suggest a possible secondary mechanism of anti-CTLA-4 antibodies occurring within the tumor itself. CTLA-4 has been found to be expressed at high levels in regulatory T cells (also known as "Treg cells") compared to intratumor effector T cells (also known as "Teff cells") in tumors, leading to the hypothesis that anti-CTLA-4 preferentially affects Treg cells.

[0117] One mechanism by which checkpoint-blocking anti-CTLA-4 antibodies mediate the antitumor effect is by reducing regulatory T cells. Due to the distinct mechanisms of action of anti-CTLA-4 antibodies, they can be successfully combined with anti-PD-1 checkpoint-blocking antibodies that act to release inhibitory signals conferred to effector T cells. Combining these dual blockades improves the antitumor response in both preclinical (Proc Natl Acad Sci USA 2010, 107, 4275-4280) and clinical (N Engl J Med 2013, 369, 122-133; N Engl J Med 2015, 372, 2006-2017) settings.

[0118] In some embodiments, the checkpoint inhibitor inhibits a checkpoint protein or a combination thereof selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, BMA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein CTLA-4. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein PD-1. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein PD-L1. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein PD-L2. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein B7-H3. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein B7-H4. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein BMA. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein HVEM. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein TIM3. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein GAL9. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein LAG3. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein VISTA. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein KIR. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein 2B4. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein CD160. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein CGEN-15049. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein CHK1.In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein CHK2. In some embodiments, the checkpoint inhibitor inhibits the checkpoint protein A2aR. In some embodiments, the checkpoint inhibitor inhibits B-7 family ligands. In some embodiments, the checkpoint is an antibody. In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody. In some embodiments, the checkpoint inhibitor is an anti-PD-L2 antibody. In some embodiments, the checkpoint inhibitor is an anti-B7-H3 antibody. In some embodiments, the checkpoint inhibitor is an anti-B7-H4 antibody. In some embodiments, the checkpoint inhibitor is an anti-BMA antibody. In some embodiments, the checkpoint inhibitor is an anti-HVEM antibody. In some embodiments, the checkpoint inhibitor is an anti-TIM3 antibody. In some embodiments, the checkpoint inhibitor is an anti-GAL9 antibody. In some embodiments, the checkpoint inhibitor is an anti-LAG3 antibody. In some embodiments, the checkpoint inhibitor is an anti-VISTA antibody. In some embodiments, the checkpoint inhibitor is an anti-KIR antibody. In some embodiments, the checkpoint inhibitor is an anti-2B4 antibody. In some embodiments, the checkpoint inhibitor is an anti-CD160 antibody. In some embodiments, the checkpoint inhibitor is an anti-CGEN-15049 antibody. In some embodiments, the checkpoint inhibitor is an anti-CHK1 antibody. In some embodiments, the checkpoint inhibitor is an anti-CHK2 antibody. In some embodiments, the checkpoint inhibitor is an anti-A2aR antibody. In some embodiments, the checkpoint inhibitor is an anti-B7 family ligand antibody. In some embodiments, the checkpoint inhibitor described herein is a monoclonal antibody. In some embodiments, the checkpoint inhibitor described herein is a human antibody. In some embodiments, the checkpoint inhibitor described herein is a humanized antibody.In some embodiments, the checkpoint inhibitor described herein is a chimeric antibody. In some embodiments, the checkpoint inhibitor described herein is a full-length antibody. In some embodiments, the checkpoint inhibitor described herein is an antigen-binding fragment of an antibody. In some embodiments, the antigen-binding fragment is Fab, Fab’, and F(ab’)2, Fd, single-chain Fv (scFv), single-chain antibody, disulfide-bonded Fv (sdFv), as well as V. L or V HThe selection is made from a group consisting of fragments containing any of the domains. In some embodiments, the checkpoint inhibitors described herein are antibodies containing a complementation-determining region (CDR) of an antibody selected from the group consisting of MEDI0680, AMP-224, nivolumab, pembrolizumab, pidilizumab, MEDI4736, atezolizumab, ipilimumab, tremelimumab, and BMS-936559. In some embodiments, the CDR is the CDR of Kabat. Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme). In some embodiments, the checkpoint inhibitors described herein include the heavy chain variable region and / or light chain variable region of an antibody selected from the group consisting of MEDI0680, AMP-224, nivolumab, pembrolizumab, pizilizumab, MEDI4736, atezolizumab, ipilimumab, tremelimumab, and BMS-936559. In some embodiments, the checkpoint inhibitors described herein include the light chain variable region of an antibody selected from the group consisting of MEDI0680, AMP-224, nivolumab, pembrolizumab, pizilizumab, MEDI4736, atezolizumab, ipilimumab, tremelimumab, and BMS-936559. In some embodiments, the checkpoint inhibitors described herein include the heavy chain variable region and the light chain variable region of an antibody selected from the group consisting of MEDI0680, AMP-224, nivolumab, pembrolizumab, pizilizumab, MEDI4736, atezolizumab, ipilimumab, tremelimumab, and BMS-936559.In some embodiments, the checkpoint inhibitor described herein is an antibody selected from the group consisting of MEDI0680, AMP-224, nivolumab, pembrolizumab, pizilizumab, MEDI4736, atezolizumab, ipilimumab, tremelimumab, and BMS-936559. In some embodiments, the checkpoint inhibitor described herein is a bioanalyte of an antibody selected from the group consisting of MEDI0680, AMP-224, nivolumab, pembrolizumab, pizilizumab, MEDI4736, atezolizumab, ipilimumab, tremelimumab, and BMS-936559. In some embodiments, the checkpoint inhibitor described herein is MEDI0680. In some embodiments, the checkpoint inhibitor described herein is AMP-224. In some embodiments, the checkpoint inhibitor described herein is nivolumab. In some embodiments, the checkpoint inhibitor described herein is pembrolizumab. In some embodiments, the checkpoint inhibitor described herein is pizilizumab. In some embodiments, the checkpoint inhibitor described herein is MEDI4736. In some embodiments, the checkpoint inhibitor described herein is atezolizumab. In some embodiments, the checkpoint inhibitor described herein is ipilimumab. In some embodiments, the checkpoint inhibitor described herein is tremelimumab. In some embodiments, the checkpoint inhibitor described herein is BMS-936559. In some embodiments, the checkpoint inhibitor is a combination of an anti-PD-1 antibody and an anti-CTLA4 antibody. In some embodiments, the checkpoint inhibitor is a combination of nivolumab and ipilimumab. In some embodiments, the checkpoint inhibitor is a combination of pembrolizumab and ipilimumab. In some embodiments, the checkpoint inhibitor is a combination of an anti-PD-L1 antibody and an anti-CTLA4 antibody. In some embodiments, the checkpoint inhibitor is a combination of atezolizumab and ipilimumab. IV. Method A. Treatment of solid tumors

[0119] One aspect of the present invention provides a method for treating a solid tumor in a subject requiring such treatment, comprising administering an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with a checkpoint inhibitor to the subject. Another aspect of the present invention provides a method for inducing, enhancing, or prolonging the effect of a checkpoint inhibitor in a subject requiring such treatment, or enabling the subject to respond to a checkpoint inhibitor, comprising administering an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with a checkpoint inhibitor to the subject, wherein the subject has a solid tumor. Another aspect of the present invention provides a method for enhancing the effect of a checkpoint inhibitor in a subject requiring such treatment, comprising administering an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with a checkpoint inhibitor to the subject, wherein the subject has a solid tumor. A method for increasing blood flow to a solid tumor in a subject is also provided herein, comprising administering an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with a checkpoint inhibitor to the subject, wherein increasing blood flow to the solid tumor enhances the effect of the checkpoint inhibitor. In some embodiments, blood flow in a solid tumor is determined using ultrasound-based blood flow measurement or histological techniques for measuring hypoxia. In some embodiments, blood flow in a solid tumor is determined using ultrasound-based blood flow measurement. In some embodiments, blood flow in a solid tumor is determined using histological techniques for measuring hypoxia. In some embodiments, blood flow is measured using histological techniques for measuring hypoxia in a biopsy from a solid tumor. In another embodiment, the present invention provides a method for improving the delivery or efficacy of a checkpoint inhibitor in a subject, comprising administering an effective amount of bosentan or a pharmaceutically acceptable salt thereof in combination with a checkpoint inhibitor, wherein the subject has a solid tumor, and thereby the delivery or efficacy of the treatment in the subject is improved. In some embodiments, the subject is human.

[0120] In another aspect, the present invention provides a method for determining an effective dose of a vascular decompressant in a subject having a solid tumor, comprising: (a) measuring the blood flow and / or stiffness of the solid tumor; (b) administering an effective dose of the vascular decompressant to the subject; and (c) measuring the blood flow and / or stiffness of the solid tumor after administration of the vascular decompressant, wherein an increase in blood flow and / or decrease in stiffness after administration of the vascular decompressant to the subject indicates that the administered dose was an effective dose. In another aspect, the present invention provides a method for treating a solid tumor in a subject requiring such treatment, comprising: (a) measuring the blood flow and / or stiffness of the solid tumor; (b) administering an effective dose of the vascular decompressant to the subject; (c) measuring the blood flow and / or stiffness of the solid tumor after administration of the vascular decompressant; and (d) administering a chemotherapeutic agent if the blood flow to the solid tumor increases and / or the stiffness of the solid tumor decreases after administration of the vascular decompressant. In another aspect, the present invention provides a method for treating a solid tumor in a subject requiring such treatment, comprising: (a) measuring the blood flow and / or stiffness of the solid tumor; (b) administering an effective amount of a vasodilator to the subject; (c) measuring the blood flow and / or stiffness of the solid tumor after administration of the vasodilator; (d) determining that the subject is responsive to a chemotherapy agent based on an increase in blood flow or a decrease in the stiffness of the solid tumor after administration of the vasodilator; and (e) administering a chemotherapy agent to a subject that has been determined to be responsive to a chemotherapy agent based on an increase in blood flow or a decrease in the stiffness of the solid tumor after administration of the vasodilator. In another aspect, the present invention provides a method for predicting a response to treatment with a chemotherapeutic agent, comprising: (a) measuring the blood flow and / or stiffness of a solid tumor; (b) administering an effective amount of a vasodilator to a subject; and (c) measuring the blood flow and / or stiffness of the solid tumor after administration of the vasodilator, wherein an increase in blood flow or a decrease in the stiffness of the solid tumor after administration of the vasodilator indicates that the subject is likely to respond to treatment with a chemotherapeutic agent.In some embodiments, the effective dose of a vascular decompressant is determined by measuring changes in blood flow and / or stiffness of a solid tumor after administration of the vascular decompressant to the target, and an increase in blood flow and / or decrease in stiffness after administration of the vascular decompressant indicates that the administered dose was an effective dose. In some embodiments, the method includes a step of measuring blood flow to a solid tumor, and the blood flow to the solid tumor increases after administration of the vascular decompressant. In some embodiments, the method includes a step of measuring the stiffness of a solid tumor, and the stiffness of the solid tumor decreases after administration of the vascular decompressant. In some embodiments, the vascular decompressant is administered for at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days prior to the administration of a chemotherapeutic agent. In some embodiments, the vascular decompressant is administered in a dose that increases blood flow to a solid tumor and / or decreases the stiffness of a solid tumor. In some embodiments, the vasodilator is selected from the group consisting of ketotifen inhibitors, endothelin ETA receptor inhibitors, endothelin ETB receptor inhibitors, inhibitors of both endothelin ETA and ETB receptors, angiotensin inhibitors, glucocorticoid steroids (e.g., dexamethasone), vitamin D receptor agonists (e.g., paricalcitol), tranilast, pirfenidone, CXCR4 inhibitors (e.g., prelixafor), metformin, and taxanes. In some embodiments, the vasodilator is an endothelin ETA receptor inhibitor. In some embodiments, the vasodilator is an endothelin ETB receptor inhibitor. In some embodiments, the vasodilator is an inhibitor of both endothelin ETA and endothelin ETB receptors. In some embodiments, the vasodilator is an angiotensin inhibitor. In some embodiments, the vasodilator is dexamethasone. In some embodiments, the vasodilator is a glucocorticoid inhibitor. In some embodiments, the decompressant is a vitamin D receptor agonist. In some embodiments, the decompressant is paricalcitol. In some embodiments, the decompressant is tranilast.In some embodiments, the decompressant is ketotifen. In some embodiments, the decompressant is pirfenidone. In some embodiments, the decompressant is a CXCR4 inhibitor. In some embodiments, the decompressant is prelixafor. In some embodiments, the decompressant is metformin. In some embodiments, the decompressant is a taxane. In some embodiments, the decompressant is bosentan or a pharmaceutically acceptable salt thereof. In some embodiments, the decompressant is losartan or a pharmaceutically acceptable salt thereof. In some embodiments, blood flow and / or stiffness of solid tumors are measured using ultrasound. In some embodiments, blood flow of solid tumors is measured using histological techniques to measure hypoxia. In some embodiments, the chemotherapeutic agent is a checkpoint inhibitor. In some embodiments, the subject is human.

[0121] In some embodiments of the models provided herein, administration of bosentan or a pharmaceutically acceptable salt thereof increases the number of antitumor T cells co-localizing with solid tumors. In some embodiments, the number of antitumor T cells co-localizing with solid tumors increases by at least 10%. In some embodiments, the number of antitumor T cells co-localizing with solid tumors increases by at least 25%. In some embodiments, the number of antitumor T cells co-localizing with solid tumors increases by at least 50%. In some embodiments, the number of antitumor T cells co-localizing with solid tumors increases by at least 100%. In some embodiments, the number of antitumor T cells co-localizing with solid tumors increases by at least 150%.

[0122] In some embodiments of the embodiments provided herein, administering an agent that decompresses blood vessels reduces the tissue stiffness of a solid tumor. In some embodiments of the embodiments provided herein, administering bosentan or a pharmaceutically acceptable salt thereof reduces the tissue stiffness of a solid tumor. In some embodiments, the tissue stiffness of the solid tumor is reduced by at least 10%. In some embodiments, the tissue stiffness of the solid tumor is reduced by at least 20%. In some embodiments, the tissue stiffness of the solid tumor is reduced by at least 25%. In some embodiments, the tissue stiffness of the solid tumor is reduced by at least 30%. In some embodiments, the tissue stiffness of the solid tumor is reduced by at least 40%. In some embodiments, the tissue stiffness of the solid tumor is reduced by at least 50%. In some embodiments, the tissue stiffness of the solid tumor is reduced by at least 60%. In some embodiments, the tissue stiffness of the solid tumor is reduced by at least 70%. In some embodiments, the tissue stiffness of the solid tumor is reduced by at least 75%. In some embodiments, the tissue stiffness of the solid tumor is measured using ultrasound elastography.

[0123] In some embodiments of the embodiments provided herein, administering an agent that decompresses blood vessels reduces the level of extracellular matrix proteins in solid tumors. In some embodiments of the embodiments provided herein, administering bosentan or a pharmaceutically acceptable salt thereof reduces the level of extracellular matrix proteins in solid tumors. In some embodiments, the level of extracellular matrix proteins in solid tumors is reduced by at least 10%. In some embodiments, the level of extracellular matrix proteins in solid tumors is reduced by at least 20%. In some embodiments, the level of extracellular matrix proteins in solid tumors is reduced by at least 25%. In some embodiments, the level of extracellular matrix proteins in solid tumors is reduced by at least 30%. In some embodiments, the level of extracellular matrix proteins in solid tumors is reduced by at least 40%. In some embodiments, the level of extracellular matrix proteins in solid tumors is reduced by at least 50%. In some embodiments, the level of extracellular matrix proteins in solid tumors is reduced by at least 60%. In some embodiments, the level of extracellular matrix proteins in solid tumors is reduced by at least 70%. In some embodiments, the level of extracellular matrix proteins in solid tumors is reduced by at least 75%. In some embodiments, the extracellular matrix protein is collagen I. In some embodiments, the extracellular matrix protein is hyaluronan-binding protein (HABP).

[0124] In some embodiments of the embodiments provided herein, administering an agent that decompresses blood vessels reduces hypoxia in solid tumors. In some embodiments of the embodiments provided herein, administering bosentan or a pharmaceutically acceptable salt thereof reduces hypoxia in solid tumors. In some embodiments, hypoxia is reduced by at least 10%. In some embodiments, hypoxia is reduced by at least 20%. In some embodiments, hypoxia is reduced by at least 25%. In some embodiments, hypoxia is reduced by at least 30%. In some embodiments, hypoxia is reduced by at least 40%. In some embodiments, hypoxia is reduced by at least 50%. In some embodiments, hypoxia is reduced by at least 60%. In some embodiments, hypoxia is reduced by at least 70%. In some embodiments, hypoxia is reduced by at least 75%.

[0125] In some embodiments of the embodiments provided herein, the solid tumor is selected from the group consisting of breast cancer, lung metastases of breast cancer, sarcoma, pancreatic cancer, ovarian cancer, liver metastases, prostate cancer, brain cancer, melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, lung cancer, head and neck squamous cell carcinoma, urothelial carcinoma, esophageal squamous cell carcinoma, gastric cancer, esophageal cancer, cervical cancer, Merkel cell carcinoma, endometrial cancer, mesothelioma, and cutaneous squamous cell carcinoma. In some embodiments, the solid tumor is breast cancer. In some embodiments, the breast cancer has higher tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the solid tumor is lung metastases of breast cancer. In some embodiments, the solid tumor is sarcoma. In some embodiments, the solid tumor is pancreatic cancer. In some embodiments, the solid tumor is ovarian cancer. In some embodiments, the solid tumor is liver metastases. In some embodiments, the liver metastases originate from colorectal cancer. In some embodiments, the solid tumor is prostate cancer. In some embodiments, prostate cancer has high tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, the solid tumor is brain cancer. In some embodiments, brain cancer has high tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, the solid tumor is melanoma. In some embodiments, the solid tumor is renal cell carcinoma. In some embodiments, the solid tumor is colorectal cancer. In some embodiments, colorectal cancer has high tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, colorectal cancer has low tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, colorectal cancer has high tumor endothelin-A receptor expression and low endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, the solid tumor is hepatocellular carcinoma. In some embodiments, the solid tumor is lung cancer. In some embodiments, lung cancer expresses endothelin-A receptor. In some embodiments, lung cancer expresses endothelin-B receptor. In some embodiments, lung cancer expresses both endothelin-A receptors and endothelin-B receptors.In some embodiments, lung cancer has higher tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, lung cancer has higher tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, lung cancer has higher tumor endothelin-A and endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, lung cancer is non-small cell lung cancer. In some embodiments, lung cancer is small cell lung cancer. In some embodiments, solid tumor is head and neck squamous cell carcinoma. In some embodiments, solid tumor is urothelial carcinoma. In some embodiments, solid tumor is esophageal squamous cell carcinoma. In some embodiments, solid tumor is gastric cancer. In some embodiments, solid tumor is esophageal cancer. In some embodiments, solid tumor is cervical cancer. In some embodiments, solid tumor is Merkel cell carcinoma. In some embodiments, solid tumor is endometrial cancer. In some embodiments, solid tumor is mesothelioma. In some embodiments, solid tumor is cutaneous squamous cell carcinoma. In some embodiments, the solid tumor is a cancer having compressed blood vessels and / or hypoperfusion. In some embodiments, the solid tumor is a cancer having compressed blood vessels. In some embodiments, the solid tumor is a cancer that is hypoperfusion. In some embodiments, the solid tumor having compressed blood vessels and / or hypoperfusion is selected from the group consisting of breast cancer, lung metastases of breast cancer, pancreatic cancer, ovarian cancer, and liver metastases. In some embodiments, the solid tumor having compressed blood vessels and / or hypoperfusion is breast cancer. In some embodiments, breast cancer has higher tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the solid tumor having compressed blood vessels and / or hypoperfusion is pancreatic cancer. In some embodiments, the solid tumor having compressed blood vessels and / or hypoperfusion is ovarian cancer. In some embodiments, the solid tumor having compressed blood vessels and / or hypoperfusion is liver metastases. In some embodiments, the liver metastases originate from colorectal cancer. In some embodiments, a solid tumor having compressed blood vessels and / or hypoperfusion is a lung metastasis.In some embodiments, liver metastases originate from breast cancer. In some embodiments, the solid tumor is a cancer that has endothelin receptor expression in tumor blood vessels and / or fibroblasts. In some embodiments, the solid tumor is a cancer that has endothelin receptor expression in tumor blood vessels. In some embodiments, the solid tumor is a cancer that has endothelin receptor expression in tumor fibroblasts. In some embodiments, the solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is selected from the group consisting of pancreatic cancer, ovarian cancer, lung cancer, prostate cancer, brain cancer, breast cancer, and colorectal cancer. In some embodiments, the solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is pancreatic cancer. In some embodiments, the solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is ovarian cancer. In some embodiments, the solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is lung cancer. In some embodiments, lung cancer expresses endothelin-A receptor. In some embodiments, lung cancer expresses endothelin-B receptor. In some embodiments, lung cancer expresses both endothelin-A and endothelin-B receptors. In some embodiments, lung cancer has higher tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, lung cancer has higher tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, lung cancer has higher tumor endothelin-A and endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, lung cancer is non-small cell lung cancer. In some embodiments, lung cancer is small cell lung cancer. In some embodiments, a solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is prostate cancer. In some embodiments, prostate cancer has higher tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, a solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is brain cancer. In some embodiments, brain cancer has higher tumor endothelin-A receptor expression compared to non-tumor tissue.In some embodiments, a solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is breast cancer. In some embodiments, breast cancer has higher tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, breast cancer is triple-negative breast cancer. In some embodiments, the solid tumor is a lung metastasis from breast cancer. In some embodiments, a solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is colorectal cancer. In some embodiments, colorectal cancer has higher tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, colorectal cancer has lower tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, colorectal cancer has higher tumor endothelin-A receptor expression and lower endothelin-B receptor expression compared to non-tumor tissue. B. Route of administration

[0126] The chemotherapeutic agents described herein may be administered by any preferred route and manner. Bosentan or its pharmaceutically acceptable salts, or the checkpoint inhibitors described herein, may be administered by any preferred route and manner. Preferred routes for administering the compounds or antibodies of the present invention are well known in the art and may be selected by those skilled in the art. In one embodiment, bosentan or its pharmaceutically acceptable salts, and / or the checkpoint inhibitors described herein, are administered parenterally. Parenteral administration refers to a mode of administration other than intestinal and topical administration, usually by injection, and includes epithelial, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrasternal, epidural, and intracisional injections and infusions. In some embodiments, the route of administration of the chemotherapeutic agent is intraperitoneal injection. In some embodiments, the route of administration of the chemotherapeutic agent is intravenous injection. In some embodiments, the route of administration of bosentan or a pharmaceutically acceptable salt thereof is intraperitoneal injection. In some embodiments, the route of administration of checkpoint inhibitors is intraperitoneal injection. In some embodiments, the route of administration of bosentan or a pharmaceutically acceptable salt thereof is intravenous injection. In some embodiments, the route of administration of checkpoint inhibitors is intravenous injection. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof, and / or checkpoint inhibitors described herein, are administered intestinally. In some embodiments, the route of administration of bosentan or a pharmaceutically acceptable salt thereof is intestinal. In some embodiments, the route of administration of bosentan or a pharmaceutically acceptable salt thereof is oral. In some embodiments, the route of administration of checkpoint inhibitors is intestinal. In some embodiments, the route of administration of checkpoint inhibitors is oral. In some embodiments, the route of administration of chemotherapeutic agents is intestinal. In some embodiments, the route of administration of chemotherapeutic agents is oral. C. Dosage and frequency

[0127] In one aspect, the present invention provides a method as described herein, comprising administering bosentan or a pharmaceutically acceptable salt thereof as described herein and a checkpoint inhibitor as described herein, wherein a subject is administered bosentan or a pharmaceutically acceptable salt thereof as described herein and a checkpoint inhibitor as described herein at a specific frequency. In another aspect, the present invention provides a method as described herein, comprising administering a vascular decompressant as described herein and a chemotherapeutic agent as described herein, wherein a subject is administered a vascular decompressant as described herein and a chemotherapeutic agent as described herein at a specific frequency.

[0128] In one embodiment of the methods, uses, or products for use provided herein, the vasodilator described herein is administered to a subject in a therapeutically effective dose. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a therapeutically effective dose. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a sub-therapeutic dose. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a dose sufficient to induce the effect of a checkpoint inhibitor. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a dose sufficient to enhance the effect of a checkpoint inhibitor. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a dose sufficient to prolong the effect of the checkpoint inhibitor. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a dose sufficient to enhance the effect of the checkpoint inhibitor. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a dose sufficient to improve the delivery of the checkpoint inhibitor to a solid tumor. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a dose sufficient to improve the potency of the checkpoint inhibitor.In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a dose sufficient to increase the number of antitumor T cells localizing to a solid tumor. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a dose sufficient to reduce the tissue stiffness of a solid tumor. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a dose sufficient to reduce the levels of extracellular matrix proteins in a solid tumor. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a dose sufficient to increase blood flow to a solid tumor. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a dose sufficient to reduce the levels of extracellular matrix proteins in a solid tumor and increase blood flow to the solid tumor. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof described herein is administered to a subject in a dose sufficient to reduce hypoxia in a solid tumor.

[0129] In some embodiments of the methods, uses, or products for use provided herein, the vasoconstricting agents described herein are administered to a subject in doses ranging from about 0.01 mg / kg to about 20 mg / kg of body weight. In some embodiments of the methods, uses, or products for use provided herein, bosentan or its pharmaceutically acceptable salts described herein are administered to a subject in doses ranging from about 0.01 mg / kg to about 20 mg / kg of body weight. In one embodiment, bosentan or its pharmaceutically acceptable salts described herein are administered in doses ranging from about 0.05 mg / kg to about 15 mg / kg of body weight. In one embodiment, bosentan or its pharmaceutically acceptable salts described herein are administered in doses ranging from about 0.01 mg / kg to about 0.1 mg / kg of body weight. In one embodiment, bosentan or its pharmaceutically acceptable salts described herein are administered in doses ranging from about 0.01 mg / kg to about 0.5 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 0.01 mg / kg to about 1.0 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 0.01 mg / kg to about 5 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 0.05 mg / kg to about 0.1 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 0.05 mg / kg to about 10 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 0.05 mg / kg to about 5 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 0.05 mg / kg to about 3 mg / kg of body weight. In another embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 0.25 mg / kg to about 10 mg / kg of body weight.In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 0.25 mg / kg to about 5 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 0.25 mg / kg to about 3 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 0.5 mg / kg to about 10 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 0.5 mg / kg to about 5 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 0.5 mg / kg to about 3 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 0.75 mg / kg to about 10 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 0.75 mg / kg to about 5 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 0.75 mg / kg to about 3 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 1 mg / kg to about 10 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 1 mg / kg to about 5.0 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 1 mg / kg to about 3 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 2 mg / kg to about 20 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 2 mg / kg to about 15 mg / kg of body weight.In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 2 mg / kg to about 10 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 2 mg / kg to about 5 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 4 mg / kg to about 20 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 4 mg / kg to about 15 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 4 mg / kg to about 10 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in doses ranging from about 4 mg / kg to about 5 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 5 mg / kg to about 20 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 0.01 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 0.05 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 0.1 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 0.15 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 0.16 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 0.2 mg / kg of body weight. In another embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 0.3 mg / kg of body weight.In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 0.4 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 0.5 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 0.6 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 0.7 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 0.8 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 0.9 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 1 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 1.2 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 1.4 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 1.6 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 1.8 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 2 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 2.2 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 2.4 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 2.6 mg / kg of body weight.In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 2.8 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 3 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 3.2 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 3.4 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 3.6 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 3.8 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 4 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 4.2 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 4.4 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 4.6 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 4.8 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 5 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 5.2 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 5.4 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 5.6 mg / kg of body weight.In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 5.8 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 6 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 6.5 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 7 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 7.5 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 7.5 mg / kg of body weight. The salt is administered at a dose of approximately 8 mg / kg of body weight. In one embodiment, bosentan or its pharmaceutically acceptable salt as described herein is administered at a dose of approximately 8.5 mg / kg of body weight. In one embodiment, bosentan or its pharmaceutically acceptable salt as described herein is administered at a dose of approximately 9 mg / kg of body weight. In one embodiment, bosentan or its pharmaceutically acceptable salt as described herein is administered at a dose of approximately 9.5 mg / kg of body weight. In one embodiment, bosentan or its pharmaceutically acceptable salt as described herein is administered at a dose of approximately 10 mg / kg of body weight. In one embodiment, bosentan or its pharmaceutically acceptable salt as described herein is administered at a dose of approximately 11 mg / kg of body weight. In one embodiment, bosentan or its pharmaceutically acceptable salt as described herein is administered at a dose of approximately 12 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 13 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 14 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 15 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 16 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 17 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 18 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of about 19 mg / kg of body weight. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered at a dose of approximately 20 mg / kg of body weight.

[0130] In some embodiments of the methods, uses, or products for use provided herein, the vasoconstricting agents described herein are administered to a subject in doses ranging from about 10 mg to about 1250 mg. In some embodiments of the methods, uses, or products for use provided herein, bosentan or its pharmaceutically acceptable salts described herein are administered to a subject in doses ranging from about 10 mg to about 1250 mg. In one embodiment, bosentan or its pharmaceutically acceptable salts described herein are administered in doses ranging from about 10 mg to about 150 mg. In one embodiment, bosentan or its pharmaceutically acceptable salts described herein are administered in doses ranging from about 10 mg to about 100 mg. In one embodiment, bosentan or its pharmaceutically acceptable salts described herein are administered in doses ranging from about 10 mg to about 50 mg. In one embodiment, bosentan or its pharmaceutically acceptable salts described herein are administered in doses ranging from about 25 mg to about 150 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 25 mg to about 100 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 25 mg to about 50 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 50 mg to about 150 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 50 mg to about 100 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 50 mg to about 75 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 75 mg to about 150 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 75 mg to about 100 mg. In another embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 100 mg to about 1200 mg.In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 10 mg to about 40 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 10 mg to about 30 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 10 mg to about 20 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 15 mg to about 40 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 20 mg to about 40 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 30 mg to about 40 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose ranging from about 10 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 15 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 20 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 25 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 30 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 35 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 40 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 45 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 50 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 55 mg. In another embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 60 mg.In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 62.5 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 65 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 70 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 75 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 80 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 85 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 90 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 95 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 100 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 105 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 110 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 115 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 120 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 125 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 130 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 135 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 140 mg.In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 145 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 150 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 175 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 200 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 250 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 300 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 350 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 400 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 450 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 500 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 550 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 600 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 650 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 700 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 750 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 800 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 850 mg.In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 900 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 950 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 1000 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 1050 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 1100 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 1150 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 1200 mg. In one embodiment, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered in a dose of about 1250 mg.

[0131] In one embodiment of the methods, uses, or products for use provided herein, a vasodilator is administered to a subject daily, twice daily, three times daily, or four times daily. In one embodiment of the methods, uses, or products for use provided herein, bosentan or a pharmaceutically acceptable salt thereof is administered to a subject daily, twice daily, three times daily, or four times daily. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to a subject every two days, about once a week, or about once every three weeks. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to a subject about once a day. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to a subject about twice a day. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to a subject once a day. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered to a subject twice a day. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof is administered orally to the subject.

[0132] In some embodiments of the methods, uses, or products for use provided herein, the chemotherapeutic agents described herein are administered to a subject in doses ranging from about 0.5 mg / kg to about 15 mg / kg of body weight. In some embodiments of the methods, uses, or products for use provided herein, the checkpoint inhibitors described herein are administered to a subject in doses ranging from about 0.5 mg / kg to about 15 mg / kg of body weight. In one embodiment, the checkpoint inhibitors described herein are administered in doses ranging from about 1 mg / kg to about 10 mg / kg. In one embodiment, the checkpoint inhibitors described herein are administered in doses ranging from about 1 mg / kg to about 10 mg / kg. In one embodiment, the checkpoint inhibitors described herein are administered in doses ranging from about 1 mg / kg of body weight. In one embodiment, the checkpoint inhibitors described herein are administered in doses ranging from about 2 mg / kg of body weight. In one embodiment, the checkpoint inhibitors described herein are administered in doses ranging from about 3 mg / kg of body weight. In one embodiment, the checkpoint inhibitors described herein are administered in doses ranging from about 4 mg / kg of body weight. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 5 mg / kg of body weight. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 6 mg / kg of body weight. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 7 mg / kg of body weight. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 8 mg / kg of body weight. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 9 mg / kg of body weight. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 10 mg / kg of body weight. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 11 mg / kg of body weight. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 12 mg / kg of body weight. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 13 mg / kg of body weight. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 14 mg / kg of body weight.In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 15 mg / kg of body weight. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 2 mg / kg, and the checkpoint inhibitor is pembrolizumab. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 1 mg / kg, and the checkpoint inhibitor is nivolumab. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 3 mg / kg, and the checkpoint inhibitor is nivolumab. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 1 mg / kg, and the checkpoint inhibitor is ipilimumab. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 3 mg / kg, and the checkpoint inhibitor is ipilimumab. In one embodiment, the checkpoint inhibitor described herein is administered at a dose of approximately 10 mg / kg, and the checkpoint inhibitor is ipilimumab.

[0133] In some embodiments of the methods, uses, or products for use provided herein, the chemotherapeutic agents described herein are administered to a subject in doses ranging from about 100 mg to about 2000 mg. In some embodiments of the methods, uses, or products for use provided herein, the checkpoint inhibitors described herein are administered to a subject in doses ranging from about 100 mg to about 2000 mg. In one embodiment, the checkpoint inhibitors described herein are administered in doses ranging from about 200 mg to about 1800 mg. In one embodiment, the checkpoint inhibitors described herein are administered in doses ranging from about 200 mg to about 400 mg. In one embodiment, the checkpoint inhibitors described herein are administered in doses ranging from about 400 mg to about 600 mg. In one embodiment, the checkpoint inhibitors described herein are administered in doses ranging from about 600 mg to about 1000 mg. In one embodiment, the checkpoint inhibitors described herein are administered in doses ranging from about 800 mg to about 1000 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose ranging from about 1000 mg to about 1800 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose ranging from about 1000 mg to about 1600 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose ranging from about 1000 mg to about 1300 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose ranging from about 140 mg to about 1800 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose ranging from about 1600 mg to about 1800 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of about 100 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of about 200 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of about 240 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of about 300 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 360 mg.In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 400 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 480 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 500 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 600 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 700 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 800 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 840 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 900 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 1000 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 1100 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 1200 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 1300 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 1400 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 1500 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 1600 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 1700 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 1800 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 1900 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 2000 mg. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 200 mg, and the checkpoint inhibitor is pembrolizumab.In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 400 mg, and the checkpoint inhibitor is pembrolizumab. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 240 mg, and the checkpoint inhibitor is nivolumab. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 480 mg, and the checkpoint inhibitor is nivolumab. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 360 mg, and the checkpoint inhibitor is nivolumab. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 840 mg, and the checkpoint inhibitor is atezolizumab. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 1200 mg, and the checkpoint inhibitor is atezolizumab. In one embodiment, the checkpoint inhibitor described herein is administered in a dose of approximately 1680 mg, and the checkpoint inhibitor is atezolizumab.

[0134] In one embodiment of the methods, uses, or products for use provided herein, the chemotherapeutic agents described herein are administered to a subject daily, twice daily, three times daily, or four times daily. In one embodiment of the methods, uses, or products for use provided herein, the checkpoint inhibitors described herein are administered to a subject daily, twice daily, three times daily, or four times daily. In some embodiments, the checkpoint inhibitors described herein are administered approximately once a week to approximately once every eight weeks. In some embodiments, the checkpoint inhibitors described herein are administered approximately once every week. In some embodiments, the checkpoint inhibitors described herein are administered approximately once every two weeks. In some embodiments, the checkpoint inhibitors described herein are administered approximately once every three weeks. In some embodiments, the checkpoint inhibitors described herein are administered approximately once every four weeks. In some embodiments, the checkpoint inhibitors described herein are administered approximately once every five weeks. In some embodiments, the checkpoint inhibitors described herein are administered approximately once every six weeks. In some embodiments, the checkpoint inhibitor described herein is administered once every approximately 7 weeks. In some embodiments, the checkpoint inhibitor described herein is administered once every approximately 8 weeks. In some embodiments, the checkpoint inhibitor described herein is administered once every approximately 3 weeks, and the checkpoint inhibitor is pembrolizumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 200 mg once every approximately 3 weeks, and the checkpoint inhibitor is pembrolizumab. In some embodiments, the checkpoint inhibitor described herein is administered once every approximately 6 weeks, and the checkpoint inhibitor is pembrolizumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 400 mg once every approximately 6 weeks, and the checkpoint inhibitor is pembrolizumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 2 mg / kg of target body weight once every approximately 3 weeks, and the checkpoint inhibitor is pembrolizumab.In some embodiments, the checkpoint inhibitor described herein is administered approximately once every two weeks, and the checkpoint inhibitor is nivolumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 240 mg approximately once every two weeks, and the checkpoint inhibitor is nivolumab. In some embodiments, the checkpoint inhibitor described herein is administered approximately once every three weeks, and the checkpoint inhibitor is nivolumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 360 mg approximately once every three weeks, and the checkpoint inhibitor is nivolumab. In some embodiments, the checkpoint inhibitor described herein is administered approximately once every four weeks, and the checkpoint inhibitor is nivolumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 480 mg approximately once every four weeks, and the checkpoint inhibitor is nivolumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 1 mg / kg approximately once every three weeks, and the checkpoint inhibitor is nivolumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 3 mg / kg once every two weeks, and the checkpoint inhibitor is nivolumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 3 mg / kg once every three weeks, and the checkpoint inhibitor is nivolumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 3 weeks, and the checkpoint inhibitor is ipilimumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 6 weeks, and the checkpoint inhibitor is ipilimumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 1 mg / kg once every three weeks, and the checkpoint inhibitor is ipilimumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 3 mg / kg once every three weeks, and the checkpoint inhibitor is ipilimumab.In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 10 mg / kg once every three weeks, and the checkpoint inhibitor is ipilimumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 10 mg / kg once every twelve weeks, and the checkpoint inhibitor is ipilimumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 1 mg / kg once every six weeks, and the checkpoint inhibitor is ipilimumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 2 weeks, and the checkpoint inhibitor is atezolizumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 840 mg once every two weeks, and the checkpoint inhibitor is atezolizumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately three weeks, and the checkpoint inhibitor is atezolizumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 1200 mg once every three weeks, and the checkpoint inhibitor is atezolizumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 14 weeks, and the checkpoint inhibitor is atezolizumab. In some embodiments, the checkpoint inhibitor described herein is administered at a dose of approximately 1680 mg once every four weeks, and the checkpoint inhibitor is atezolizumab. In some embodiments, the checkpoint inhibitor described herein is administered to the subject by intravenous infusion. D. Outcome of treatment

[0135] In one embodiment, a method of treating cancer with the vasodilator and chemotherapeutic agents described herein results in one or more improvements in therapeutic effect in a subject after administration compared to baseline. In one embodiment, a method of treating cancer with bosentan or its pharmaceutically acceptable salts described herein and checkpoint inhibitors described herein results in one or more improvements in therapeutic effect in a subject after administration compared to baseline. In some embodiments, one or more therapeutic effects are tumor size derived from cancer (e.g., solid tumor), objective response rate, duration of response, time to response, progression-free survival, overall survival, or any combination thereof. In one embodiment, one or more therapeutic effects are tumor size derived from cancer. In one embodiment, one or more therapeutic effects are a reduction in tumor size. In one embodiment, one or more therapeutic effects are stable disease. In one embodiment, one or more therapeutic effects are partial response. In one embodiment, one or more therapeutic effects are complete response. In one embodiment, one or more therapeutic effects are objective response rate. In one embodiment, one or more therapeutic effects are duration of response. In one embodiment, one or more therapeutic effects are the time to response. In one embodiment, one or more therapeutic effects are progression-free survival. In one embodiment, one or more therapeutic effects are overall survival. In one embodiment, one or more therapeutic effects are cancer regression.

[0136] In one embodiment of the methods, uses, or products for use provided herein, the response to treatment with the vascular decompression agents and chemotherapeutic agents described herein may include RECIST Criteria 1.1. In one embodiment of the methods, uses, or products for use provided herein, the response to treatment with bosentan or its pharmaceutically acceptable salts described herein and checkpoint inhibitors described herein may include RECIST Criteria 1.1. RECIST Criteria 1.1 is as follows: Table 1

[0137] In one embodiment of the methods, uses, or products for use provided herein, the efficacy of treatment with the vascular decompression agents and chemotherapeutic agents described herein is evaluated by measuring the objective response rate. In one embodiment of the methods, uses, or products for use provided herein, the efficacy of treatment with bosentan or its pharmaceutically acceptable salts described herein and checkpoint inhibitors described herein is evaluated by measuring the objective response rate. In some embodiments, the objective response rate is the percentage of patients in the minimum time having a predetermined amount of tumor size reduction. In some embodiments, the objective response rate is based on RECIST v1.1. In one embodiment, the objective response rate is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%. In one embodiment, the objective response rate is at least about 20% to 80%. In one embodiment, the objective response rate is at least about 30% to 80%. In one embodiment, the objective success rate is at least about 40% to 80%. In one embodiment, the objective success rate is at least about 50% to 80%. In one embodiment, the objective success rate is at least about 60% to 80%. In one embodiment, the objective success rate is at least about 70% to 80%. In one embodiment, the objective success rate is at least about 80%. In one embodiment, the objective success rate is at least about 85%. In one embodiment, the objective success rate is at least about 90%. In one embodiment, the objective success rate is at least about 95%. In one embodiment, the objective success rate is at least about 98%. In one embodiment, the objective success rate is at least about 99%. In one embodiment, the objective success rate is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, or at least 80%. In one embodiment, the objective success rate is at least 20% to 80%. In one embodiment, the objective success rate is at least 30% to 80%.In one embodiment, the objective success rate is at least 40% to 80%. In one embodiment, the objective success rate is at least 50% to 80%. In one embodiment, the objective success rate is at least 60% to 80%. In one embodiment, the objective success rate is at least 70% to 80%. In one embodiment, the objective success rate is at least 80%. In one embodiment, the objective success rate is at least 85%. In one embodiment, the objective success rate is at least 90%. In one embodiment, the objective success rate is at least 95%. In one embodiment, the objective success rate is at least 98%. In one embodiment, the objective success rate is at least 99%. In one embodiment, the objective success rate is 100%.

[0138] In one embodiment of the methods, uses, or products for use provided herein, the response to treatment with the vascular decompression agents and / or chemotherapeutic agents described herein is evaluated by measuring the size of tumors derived from cancer (e.g., solid tumors). In one embodiment of the methods, uses, or products for use provided herein, the response to treatment with bosentan or its pharmaceutically acceptable salts described herein and / or checkpoint inhibitors described herein is evaluated by measuring the size of tumors derived from cancer (e.g., solid tumors). In one embodiment, the size of tumors derived from cancer is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% compared to the size of tumors derived from cancer before administration of bosentan or its pharmaceutically acceptable salts and / or checkpoint inhibitors described herein. In one embodiment, the size of tumors derived from cancer is reduced by at least about 10% to 80%. In one embodiment, the size of tumors originating from cancer is reduced by at least approximately 20% to 80%. In one embodiment, the size of tumors originating from cancer is reduced by at least approximately 30% to 80%. In one embodiment, the size of tumors originating from cancer is reduced by at least approximately 40% to 80%. In one embodiment, the size of tumors originating from cancer is reduced by at least approximately 50% to 80%. In one embodiment, the size of tumors originating from cancer is reduced by at least approximately 60% to 80%. In one embodiment, the size of tumors originating from cancer is reduced by at least approximately 70% to 80%. In one embodiment, the size of tumors originating from cancer is reduced by at least approximately 80%. In one embodiment, the size of tumors originating from cancer is reduced by at least approximately 85%. In one embodiment, the size of tumors originating from cancer is reduced by at least approximately 90%. In one embodiment, the size of tumors originating from cancer is reduced by at least approximately 95%. In one embodiment, the size of tumors originating from cancer is reduced by at least approximately 98%.In one embodiment, the size of cancer-derived tumors is reduced by at least about 99%. In one embodiment, the size of cancer-derived tumors is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, or at least 80% compared to the size of cancer-derived tumors before administration of bosentan or its pharmaceutically acceptable salts described herein and / or checkpoint inhibitors described herein. In one embodiment, the size of cancer-derived tumors is reduced by at least 10% to 80%. In one embodiment, the size of cancer-derived tumors is reduced by at least 20% to 80%. In one embodiment, the size of cancer-derived tumors is reduced by at least 30% to 80%. In one embodiment, the size of cancer-derived tumors is reduced by at least 40% to 80%. In one embodiment, the size of cancer-derived tumors is reduced by at least 50% to 80%. In one embodiment, the size of cancer-derived tumors is reduced by at least 60% to 80%. In one embodiment, the size of cancer-derived tumors is reduced by at least 70% to 80%. In one embodiment, the size of cancer-derived tumors is reduced by at least 80%. In one embodiment, the size of cancer-derived tumors is reduced by at least 85%. In one embodiment, the size of cancer-derived tumors is reduced by at least 90%. In one embodiment, the size of cancer-derived tumors is reduced by at least 95%. In one embodiment, the size of cancer-derived tumors is reduced by at least 98%. In one embodiment, the size of cancer-derived tumors is reduced by at least 99%. In one embodiment, the size of cancer-derived tumors is reduced by 100%. In one embodiment, the size of cancer-derived tumors is measured by magnetic resonance imaging (MRI). In one embodiment, the size of cancer-derived tumors is measured by computed tomography (CT). In some embodiments, the size of cancer-derived tumors is reduced compared to the size of the tumor before administration of bosentan or its pharmaceutically acceptable salts as described herein and checkpoint inhibitors as described herein.In some embodiments, the size of cancer-derived tumors is reduced compared to the size of tumors before administration of bosentan or a pharmaceutically acceptable salt thereof as described herein. In some embodiments, the size of cancer-derived tumors is reduced compared to the size of tumors before administration of a checkpoint inhibitor as described herein.

[0139] In one embodiment of the methods, uses, or products for use provided herein, the response to treatment with a vasodilator and / or a chemotherapeutic agent described herein promotes regression of tumors originating from cancer (e.g., solid tumors). In one embodiment of the methods, uses, or products for use provided herein, the response to treatment with bosentan or a pharmaceutically acceptable salt thereof described herein and a checkpoint inhibitor described herein promotes regression of tumors originating from cancer (e.g., solid tumors). In one embodiment, a tumor originating from cancer regresses by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% relative to the size of the tumor originating from cancer before administration of bosentan or a pharmaceutically acceptable salt thereof described herein and / or a checkpoint inhibitor described herein. In one embodiment, a tumor originating from cancer regresses by at least about 10% to about 80%. In one embodiment, a tumor originating from cancer regresses by at least approximately 20% to approximately 80%. In one embodiment, a tumor originating from cancer regresses by at least approximately 30% to approximately 80%. In one embodiment, a tumor originating from cancer regresses by at least approximately 40% to approximately 80%. In one embodiment, a tumor originating from cancer regresses by at least approximately 50% to approximately 80%. In one embodiment, a tumor originating from cancer regresses by at least approximately 60% to approximately 80%. In one embodiment, a tumor originating from cancer regresses by at least approximately 70% to approximately 80%. In one embodiment, a tumor originating from cancer regresses by at least approximately 80%. In one embodiment, a tumor originating from cancer regresses by at least approximately 85%. In one embodiment, a tumor originating from cancer regresses by at least approximately 90%. In one embodiment, a tumor originating from cancer regresses by at least approximately 95%. In one embodiment, a tumor originating from cancer regresses by at least approximately 98%. In one embodiment, a tumor originating from cancer regresses by at least approximately 99%.In one embodiment, a cancer-derived tumor regresses by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, or at least 80% relative to the size of the cancer-derived tumor before administration of bosentan or any pharmaceutically acceptable salt thereof as described herein and / or a checkpoint inhibitor as described herein. In one embodiment, a cancer-derived tumor regresses by at least 10% to 80%. In one embodiment, a cancer-derived tumor regresses by at least 20% to 80%. In one embodiment, a cancer-derived tumor regresses by at least 30% to 80%. In one embodiment, a cancer-derived tumor regresses by at least 40% to 80%. In one embodiment, a cancer-derived tumor regresses by at least 50% to 80%. In one embodiment, a cancer-derived tumor regresses by at least 60% to 80%. In one embodiment, a cancer-derived tumor regresses by at least 70% to 80%. In one embodiment, the cancer-derived tumor regresses by at least 80%. In one embodiment, the cancer-derived tumor regresses by at least 85%. In one embodiment, the cancer-derived tumor regresses by at least 90%. In one embodiment, the cancer-derived tumor regresses by at least 95%. In one embodiment, the cancer-derived tumor regresses by at least 98%. In one embodiment, the cancer-derived tumor regresses by at least 99%. In one embodiment, the cancer-derived tumor regresses by 100%. In one embodiment, tumor regression is determined by measuring the size of the tumor by magnetic resonance imaging (MRI). In one embodiment, tumor regression is determined by measuring the size of the tumor by computed tomography (CT). In some embodiments, the cancer-derived tumor regresses compared to the tumor size before administration of bosentan or its pharmaceutically acceptable salt as described herein and the checkpoint inhibitor as described herein. In some embodiments, the cancer-derived tumor regresses compared to the tumor size before administration of bosentan or its pharmaceutically acceptable salt as described herein.In some embodiments, cancer-derived tumors regress compared to their size before administration of the checkpoint inhibitors described herein.

[0140] In one embodiment of the methods, uses, or products for use described herein, the response to treatment with the vascular decompressant and / or chemotherapeutic agents described herein is evaluated by measuring progression-free survival after administration of the vascular decompressant and / or chemotherapeutic agents described herein. In one embodiment of the methods, uses, or products for use described herein, the response to treatment with bosentan or a pharmaceutically acceptable salt thereof described herein and / or a checkpoint inhibitor described herein is evaluated by measuring progression-free survival after administration of bosentan or a pharmaceutically acceptable salt thereof described herein and / or a checkpoint inhibitor described herein. In some embodiments, subjects demonstrate progression-free survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects demonstrate progression-free survival of at least about 6 months after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects demonstrate progression-free survival of at least about 1 year after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects exhibit a progression-free survival of at least approximately two years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein.In some embodiments, subjects exhibit a progression-free survival of at least approximately 3 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects exhibit a progression-free survival of at least approximately 4 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects exhibit a progression-free survival of at least approximately 5 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects exhibit a progression-free survival of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects demonstrate a progression-free survival of at least 6 months after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects demonstrate a progression-free survival of at least 1 year after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects demonstrate a progression-free survival of at least 2 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects demonstrate a progression-free survival of at least 3 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein.In some embodiments, subjects exhibit a progression-free survival of at least 4 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects exhibit a progression-free survival of at least 5 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, the response to treatment is evaluated by measuring progression-free survival after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein and a checkpoint inhibitor as described herein. In some embodiments, the response to treatment is evaluated by measuring progression-free survival after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein. In some embodiments, the response to treatment is evaluated by measuring progression-free survival after administration of a checkpoint inhibitor as described herein.

[0141] In one embodiment of the methods, uses, or products for use described herein, the response to treatment with the vascular decompressant and / or chemotherapeutic agents described herein is evaluated by measuring overall survival after administration of the vascular decompressant and / or chemotherapeutic agents described herein. In one embodiment of the methods, uses, or products for use described herein, the response to treatment with bosentan or a pharmaceutically acceptable salt thereof described herein and / or a checkpoint inhibitor described herein is evaluated by measuring overall survival after administration of bosentan or a pharmaceutically acceptable salt thereof described herein and / or a checkpoint inhibitor described herein. In some embodiments, subjects exhibit an overall survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects exhibit an overall survival of at least about 6 months after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects exhibit an overall survival of at least about 1 year after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects exhibit an overall survival of at least approximately 2 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects exhibit an overall survival of at least approximately 3 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein.In some embodiments, subjects exhibit an overall survival of at least about 4 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects exhibit an overall survival of at least about 5 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects exhibit an overall survival of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least about 12 months, at least 18 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects exhibit an overall survival of at least 6 months after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects demonstrate an overall survival of at least one year after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects demonstrate an overall survival of at least two years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects demonstrate an overall survival of at least three years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, subjects demonstrate an overall survival of at least four years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein.In some embodiments, subjects exhibit overall survival of at least 5 years after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein, and / or a checkpoint inhibitor as described herein. In some embodiments, the response to treatment is evaluated by measuring the time of overall survival after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein and a checkpoint inhibitor as described herein. In some embodiments, the response to treatment is evaluated by measuring the time of overall survival after administration of bosentan or a pharmaceutically acceptable salt thereof as described herein. In some embodiments, the response to treatment is evaluated by measuring the time of overall survival after administration of a checkpoint inhibitor as described herein.

[0142] In one embodiment of the methods, uses, or products for use described herein, the response to treatment with the vascular decompressant and / or chemotherapeutic agents described herein is evaluated by measuring the duration of response to the vascular decompressant and / or chemotherapeutic agents described herein after administration of the vascular decompressant and / or chemotherapeutic agents described herein. In one embodiment of the methods, uses, or products for use described herein, the response to treatment with bosentan or its pharmaceutically acceptable salts described herein and checkpoint inhibitors described herein is evaluated by measuring the duration of response to bosentan or its pharmaceutically acceptable salts described herein and checkpoint inhibitors described herein after administration of the pharmaceutically acceptable salts described herein and / or checkpoint inhibitors described herein. In some embodiments, the duration of response to bosentan or its pharmaceutically acceptable salts described herein and the checkpoint inhibitors described herein is at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of bosentan or its pharmaceutically acceptable salts described herein and the checkpoint inhibitors described herein.In some embodiments, the duration of response to bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein is at least about one year after administration of bosentan or its pharmaceutically acceptable salts as described herein and / or the checkpoint inhibitors as described herein. In some embodiments, the duration of response to bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein is at least about two years after administration of bosentan or its pharmaceutically acceptable salts as described herein and / or the checkpoint inhibitors as described herein. In some embodiments, the duration of response to bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein is at least about three years after administration of bosentan or its pharmaceutically acceptable salts as described herein and / or the checkpoint inhibitors as described herein. In some embodiments, the duration of response to bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein is at least about 4 years after administration of bosentan or its pharmaceutically acceptable salts as described herein and / or the checkpoint inhibitors as described herein. In some embodiments, the duration of response to bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein is at least about 5 years after administration of bosentan or its pharmaceutically acceptable salts as described herein and / or the checkpoint inhibitors as described herein.In some embodiments, the duration of response to bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein is at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years after administration of bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein. In some embodiments, the duration of response to bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein is at least one year after administration of bosentan or its pharmaceutically acceptable salts as described herein and / or the checkpoint inhibitors as described herein. In some embodiments, the duration of response to bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein is at least two years after administration of bosentan or its pharmaceutically acceptable salts as described herein and / or the checkpoint inhibitors as described herein. In some embodiments, the duration of response to bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein is at least three years after administration of bosentan or its pharmaceutically acceptable salts as described herein and / or the checkpoint inhibitors as described herein.In some embodiments, the duration of response to bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein is at least 4 years after administration of bosentan or its pharmaceutically acceptable salts as described herein and / or the checkpoint inhibitors as described herein. In some embodiments, the duration of response to bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein is at least 5 years after administration of bosentan or its pharmaceutically acceptable salts as described herein and / or the checkpoint inhibitors as described herein. In some embodiments, the duration of response is measured after administration of bosentan or its pharmaceutically acceptable salts as described herein and the checkpoint inhibitors as described herein. In some embodiments, the duration of response is measured after administration of bosentan or its pharmaceutically acceptable salts as described herein. In some embodiments, the duration of response is measured after administration of the checkpoint inhibitors as described herein. V. Composition

[0143] In some embodiments, compositions (e.g., pharmaceutical compositions and therapeutic formulations) comprising a vascular decompressant and / or a chemotherapeutic agent as described herein are also provided herein. In some embodiments, compositions (e.g., pharmaceutical compositions and therapeutic formulations) comprising bosentan or a pharmaceutically acceptable salt thereof as described herein and / or a checkpoint inhibitor as described herein are also provided herein.

[0144] Therapeutic formulations are prepared for storage by mixing the active ingredient, having the desired degree of purity, with pharmaceutically acceptable carriers, excipients, or stabilizers as needed (Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wiklins, Pub., Gennaro Ed., Philadelphia, Pa. 2000).

[0145] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffering agents, antioxidants such as ascorbic acid, methionine, vitamin E, sodium pyrosulfite; preservatives, isotonic agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents such as EDTA and / or nonionic surfactants.

[0146] Particularly when stability is pH-dependent, buffering agents can be used to control the pH within a range that optimizes therapeutic efficacy. The buffering agent can be present at a concentration in the range of about 50 mM to about 250 mM. Buffering agents suitable for use with the present invention include both organic and inorganic acids and their salts. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. In addition, the buffering agent can consist of histidine and trimethylamine salts, such as Tris.

[0147] Preservatives can be added to prevent the growth of microorganisms, and typically the preservative is present in the range of about 0.2% to 1.0% (weight / volume). Suitable preservatives for use with the present invention include octadecyldimethylbenzylammonium chloride, hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol, resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0148] Isotonic agents, sometimes also known as “stabilizers,” may be present to adjust or maintain the isotonicity of a liquid in a composition. When used with large charged biomolecules, such as proteins and antibodies, they can interact with the charged groups of amino acid side chains, thereby weakening the potential for intermolecular and intramolecular interactions, and are therefore often referred to as “stabilizers.” Isotonic agents may be present in any amount between about 0.1% to about 25% by weight, or between about 1% to about 5% by weight, taking into account the relative amounts of other components. In some embodiments, the isotonic agent includes polyhydric sugar alcohols, trihydric alcohols, or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0149] Additional excipients include: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that may act as one or more of the following: denaturing agents or agents that prevent adhesion to the walls of containers. Such excipients include: polyhydric sugar alcohols (as listed above); amino acids, e.g., alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, e.g., sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitol (e.g., The compounds include inositol, polyethylene glycol, sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, and sucrose; trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0150] Nonionic surfactants or detergents (also known as "wetting agents") may be present to help solubilize therapeutic agents and to protect therapeutic proteins against agitation-induced aggregation, thereby allowing the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant is present in the range of about 0.05 mg / ml to about 1.0 mg / ml, or about 0.07 mg / ml to about 0.2 mg / ml. In some embodiments, the nonionic surfactant is present in the range of about 0.001 weight / volume % to about 0.1 weight / volume %, or about 0.01 weight / volume % to about 0.1 weight / volume %, or about 0.01 weight / volume % to about 0.025 weight / volume %.

[0151] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glyceryl monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0152] In some embodiments, the bosentan-containing formulation comprises bosentan hydrate dissolved in ddH2O containing DMSO, PEG300, and Tween® 80. In some embodiments, the bosentan-containing formulation comprises bosentan hydrate dissolved in ddH2O containing 2% DMSO, 30% PEG300, and 2% Tween® 80. In some embodiments, the bosentan-containing formulation comprises bosentan hydrate (S3051, Selleckchem) dissolved in ddH2O containing 2% DMSO (GK2245, Glentham Life Science), 30% PEG300 (S6704, Selleckchem), and 2% Tween® 80 (S6702, Selleckchem).

[0153] For use in vivo administration, the formulations must be sterilized. The formulations may be sterilized by filtration through a sterile filtration membrane. The therapeutic compositions described herein are typically placed in containers with sterile access ports, such as intravenous infusion bags or vials with stoppers that can be penetrated by a subcutaneous needle.

[0154] The route of administration may be known and accepted, for example, by injection or infusion via a single or multiple bolus or a preferred mode of administration over a prolonged period, such as subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, or intra-articular route, local administration, inhalation, or by sustained-release or continuous-release means.

[0155] The formulations described herein may also contain one or more active compounds necessary for the specific indication being treated, preferably compounds having complementary activities that do not adversely affect each other. Alternatively, the composition may also contain cytotoxic agents, cytokines, or growth inhibitors. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0156] In some embodiments, a composition comprising a vasodilator described herein is administered co-administered with a composition comprising a chemotherapeutic agent described herein. In some embodiments, a composition comprising bosentan or a pharmaceutically acceptable salt thereof described herein is administered co-administered with a composition comprising a checkpoint inhibitor described herein. In some embodiments, co-administration is simultaneous or sequential. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof described herein is administered simultaneously with a checkpoint inhibitor described herein. In some embodiments, simultaneous means that bosentan or a pharmaceutically acceptable salt thereof described herein and the checkpoint inhibitor described herein are administered to a subject with a separation of less than about one hour, for example, less than about 30 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes. In some embodiments, simultaneous means that bosentan or a pharmaceutically acceptable salt thereof described herein and the checkpoint inhibitor described herein are administered to a subject with a separation of less than one hour, for example, less than 30 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered sequentially with a checkpoint inhibitor as described herein.In some embodiments, continuous administration means that bosentan or a pharmaceutically acceptable salt thereof as described herein and a checkpoint inhibitor as described herein are administered at intervals of at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, and at least This means that the drugs are administered at intervals of 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 5 days, at least 7 days, at least 2 weeks, at least 3 weeks, or at least 4 weeks. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered before administration of the checkpoint inhibitor as described herein. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered to the subject starting at least 1 day before administration of the checkpoint inhibitor as described herein. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered to the subject starting at least 2 days before administration of the checkpoint inhibitor as described herein. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered to the subject at least three days prior to administration of the checkpoint inhibitor described herein. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered to the subject at least four days prior to administration of the checkpoint inhibitor described herein.In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered to the subject at least 5 days before administration of the checkpoint inhibitor described herein. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered to the subject at least 1 week before administration of the checkpoint inhibitor described herein. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered to the subject at least 2 weeks before administration of the checkpoint inhibitor described herein. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered to the subject at least 3 weeks before administration of the checkpoint inhibitor described herein. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered to the subject at least 4 weeks before administration of the checkpoint inhibitor described herein. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered to the subject, initiated before administering the checkpoint inhibitor as described herein, and the administration is maintained for at least a portion of the period during which the subject is administered the checkpoint inhibitor. In some embodiments, bosentan or a pharmaceutically acceptable salt thereof as described herein is administered to the subject, initiated before administering the checkpoint inhibitor as described herein, and the administration is maintained for the entire period during which the subject is administered the checkpoint inhibitor.

[0157] In some embodiments, a composition comprising bosentan or a pharmaceutically acceptable salt thereof as described herein and / or a checkpoint inhibitor as described herein is administered concurrently with one or more therapeutic agents. In some embodiments, the concurrent administration is simultaneous or sequential. VI. Products and Kits

[0158] In another embodiment, a product or kit is provided comprising a vascular decompressant agent and / or a chemotherapeutic agent as described herein. In yet another embodiment, a product or kit is provided comprising bosentan or a pharmaceutically acceptable salt thereof as described herein and / or a checkpoint inhibitor as described herein. The product or kit may further include instructions for use of bosentan or a pharmaceutically acceptable salt thereof as described herein and / or a checkpoint inhibitor as described herein in a method of the present invention. Thus, in a particular embodiment, the product or kit includes instructions for use of bosentan or a pharmaceutically acceptable salt thereof as described herein and / or a checkpoint inhibitor as described herein in a method of treating cancer (e.g., a solid tumor) in a subject, comprising administering an effective amount of bosentan or a pharmaceutically acceptable salt thereof as described herein and / or a checkpoint inhibitor as described herein to the subject. In some embodiments of the embodiments provided herein, the solid tumor is selected from the group consisting of breast cancer, lung metastases of breast cancer, sarcoma, pancreatic cancer, ovarian cancer, liver metastases, prostate cancer, brain cancer, melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, lung cancer, head and neck squamous cell carcinoma, urothelial carcinoma, esophageal squamous cell carcinoma, gastric cancer, esophageal cancer, cervical cancer, Merkel cell carcinoma, endometrial cancer, mesothelioma, and cutaneous squamous cell carcinoma. In some embodiments, the solid tumor is breast cancer. In some embodiments, the breast cancer has higher tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the solid tumor is lung metastases of breast cancer. In some embodiments, the solid tumor is sarcoma. In some embodiments, the solid tumor is pancreatic cancer. In some embodiments, the solid tumor is ovarian cancer. In some embodiments, the solid tumor is liver metastases. In some embodiments, the liver metastases originate from colorectal cancer. In some embodiments, the solid tumor is prostate cancer. In some embodiments, prostate cancer has higher tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, the solid tumor is brain cancer.In some embodiments, brain cancer has high tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, the solid tumor is melanoma. In some embodiments, the solid tumor is renal cell carcinoma. In some embodiments, the solid tumor is colorectal cancer. In some embodiments, colorectal cancer has high tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, colorectal cancer has low tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, colorectal cancer has high tumor endothelin-A receptor expression and low endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, the solid tumor is hepatocellular carcinoma. In some embodiments, the solid tumor is lung cancer. In some embodiments, lung cancer expresses endothelin-A receptor. In some embodiments, lung cancer expresses endothelin-B receptor. In some embodiments, lung cancer expresses both endothelin-A and endothelin-B receptors. In some embodiments, lung cancer has high tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, lung cancer has high tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, lung cancer has high tumor endothelin-A receptor and endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, lung cancer is non-small cell lung cancer. In some embodiments, lung cancer is small cell lung cancer. In some embodiments, solid tumor is head and neck squamous cell carcinoma. In some embodiments, solid tumor is urothelial carcinoma. In some embodiments, solid tumor is esophageal squamous cell carcinoma. In some embodiments, solid tumor is gastric cancer. In some embodiments, solid tumor is esophageal cancer. In some embodiments, solid tumor is cervical cancer. In some embodiments, solid tumor is Merkel cell carcinoma. In some embodiments, solid tumor is endometrial cancer. In some embodiments, solid tumor is mesothelioma. In some embodiments, solid tumor is cutaneous squamous cell carcinoma. In some embodiments, solid tumor is cancer with compressed blood vessels and / or hypoperfusion. In some embodiments, solid tumor is cancer with compressed blood vessels.In some embodiments, the solid tumor is a hypoperfused cancer. In some embodiments, the solid tumor having compressed blood vessels and / or being hypoperfused is selected from the group consisting of breast cancer, lung metastases of breast cancer, pancreatic cancer, ovarian cancer, and liver metastases. In some embodiments, the solid tumor having compressed blood vessels and / or being hypoperfused is breast cancer. In some embodiments, breast cancer has higher tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the solid tumor having compressed blood vessels and / or being hypoperfused is pancreatic cancer. In some embodiments, the solid tumor having compressed blood vessels and / or being hypoperfused is ovarian cancer. In some embodiments, the solid tumor having compressed blood vessels and / or being hypoperfused is a liver metastasis. In some embodiments, the liver metastasis originates from colorectal cancer. In some embodiments, the solid tumor having compressed blood vessels and / or being hypoperfused is a lung metastasis. In some embodiments, the liver metastasis originates from breast cancer. In some embodiments, the solid tumor is a cancer that has endothelin receptor expression in tumor blood vessels and / or fibroblasts. In some embodiments, the solid tumor is a cancer that has endothelin receptor expression in tumor blood vessels. In some embodiments, the solid tumor is a cancer that has endothelin receptor expression in tumor fibroblasts. In some embodiments, the solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is selected from the group consisting of pancreatic cancer, ovarian cancer, lung cancer, prostate cancer, brain cancer, breast cancer, and colorectal cancer. In some embodiments, the solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is pancreatic cancer. In some embodiments, the solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is ovarian cancer. In some embodiments, the solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is lung cancer. In some embodiments, lung cancer expresses endothelin-A receptor. In some embodiments, lung cancer expresses endothelin-B receptor.In some embodiments, lung cancer expresses both endothelin-A and endothelin-B receptors. In some embodiments, lung cancer has higher tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, lung cancer has higher tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, lung cancer has higher tumor endothelin-A and endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, lung cancer is non-small cell lung cancer. In some embodiments, lung cancer is small cell lung cancer. In some embodiments, a solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is prostate cancer. In some embodiments, prostate cancer has higher tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, a solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is brain cancer. In some embodiments, brain cancer has higher tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, a solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is breast cancer. In some embodiments, breast cancer has high tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, breast cancer is triple-negative breast cancer. In some embodiments, the solid tumor is a lung metastasis from breast cancer. In some embodiments, the solid tumor having endothelin receptor expression in tumor blood vessels and / or fibroblasts is colorectal cancer. In some embodiments, colorectal cancer has high tumor endothelin-A receptor expression compared to non-tumor tissue. In some embodiments, colorectal cancer has low tumor endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, colorectal cancer has high tumor endothelin-A receptor expression and low endothelin-B receptor expression compared to non-tumor tissue. In some embodiments, the subject is human.

[0159] The product or kit may further include a container. Suitable containers include, for example, bottles, vials (e.g., two-chamber vials), syringes (e.g., one-chamber or two-chamber syringes), and test tubes. In some embodiments, the container is a vial. The container may be made of a variety of materials, such as glass or plastic. The container holds the formulation.

[0160] The product or kit may further include a label or accompanying leaflet on or relating to the container that may provide instructions regarding the recombination and / or use of the formulation. The label or accompanying leaflet may further indicate that the formulation is useful for or intended for other modes of administration, such as intraperitoneal injection, subcutaneous injection, intravenous injection (e.g., intravenous infusion), or other modes of administration for treating cancer in a subject (e.g., solid tumors). The container holding the formulation may be a single-use vial or a multi-use vial that allows for repeated administration of the recombined formulation. The product or kit may further include a second container containing a suitable diluent. The product or kit may further include other materials desirable from a commercial, therapeutic, and user standpoint, including other buffers, diluents, expanders, needles, syringes, and accompanying leaflets with instructions for use.

[0161] The products or kits described herein further include a container containing a second pharmaceutical, where bosentan or a pharmaceutically acceptable salt thereof as described herein is the first pharmaceutical, and the products or kits further include instructions for use on a label or package insert for treating a subject with an effective dose of the second pharmaceutical. In some embodiments, the second pharmaceutical is a checkpoint inhibitor as described herein. In some embodiments, the label or package insert indicates that the first and second pharmaceuticals are administered sequentially or simultaneously as described herein.

[0162] In some embodiments, the vasodilator and / or chemotherapeutic agents described herein are present in the container as lyophilized powder. In some embodiments, bosentan or its pharmaceutically acceptable salts described herein, and / or checkpoint inhibitors described herein are present in the container as lyophilized powder. In some embodiments, the lyophilized powder is in a sealed container indicating the amount of the activator, such as a vial, ampoule, or sachet. When the pharmaceutical is administered by injection, for example, ampoules of sterile water or saline for injection may be provided as part of the kit, if necessary, so that the components can be mixed before administration. Such a kit may further include, if desired, one or more of various conventional pharmaceutical components, such as containers having one or more pharmaceutically acceptable carriers, additional containers readily apparent to those skilled in the art, etc. Printed instructions for use, either as a package insert or label, indicating the amount of components to be administered, guidelines for administration, and / or guidelines for mixing the components, may also be included in the kit.

[0163] The present invention will be better understood by reference to the following examples. However, the examples should not be construed as limiting the scope of the invention. The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in light thereof will be suggested to those skilled in the art, but these will also be understood to be within the spirit and scope of this application and the appended claims. [Examples]

[0164] (Example 1) Bosentan normalizes the organic tumor microenvironment in a dose-dependent manner. The effectiveness of cancer immunotherapy depends on whether T cells can migrate to the tumor, reach adjacent malignant cells, recognize them, and kill them. One barrier to T cell homing is the tumor's vascular wall, which inhibits T cell attachment and migration within it via the endothelin B receptor, but antagonism of this receptor has not yet led to clinically approved drugs. One reason may be hypoperfusion in the tumor, which can limit the surface area of ​​perfusion vessels to which anti-tumor T cells bind. If collapsed tumor vessels can be decompressed and reperfused by reducing mechanical compression (i.e., solid stress), then antagonism of the endothelin B receptor could increase the effectiveness of cancer immunotherapy. Furthermore, antagonism of the endothelin A receptor inhibits fibrosis in certain disease conditions. Bosentan, a non-selective endothelin receptor blocker, was tested herein to determine whether it can reduce fibrosis in cancer.

[0165] Mice with orthotopic triple-negative mammary cancer (TNBC) of the same lineage were treated with a range of therapeutic doses of bosentan, from 0.2 mg / kg to 10 mg / kg body weight. The orthotopic mouse mammary tumor model was divided into 5 × 10⁶ cells in 40 μl of serum-free medium. 44T1 or E0771 cancer cells were generated by transplanting them into the third mammary fat pad of 6-8 week old BALB / c and C57BL / 6 female mice, respectively. 4T1 (ATCC® CRL-2539®) and E0771 (94A001, CH3 BioSystems) mouse mammary cancer cell lines were purchased from ATCC and CH3 BioSystems, respectively. The cells were maintained at 37°C / 5% CO2 in Roswell Park Memorial Institute medium (RPMI-1640, LM-R1637, Biosera) supplemented with 10% fetal bovine serum (FBS, FB-1001H, biosera) and 1% antibiotic (A5955, Sigma). 0.2 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg of bosentan (bosentan hydrate (S3051, Selleckchem) dissolved in ddH2O containing 2% DMSO (GK2245, Glentham Life Science), 30% PEG300 (S6704, Selleckchem), and 2% Tween® 80 (S6702, Selleckchem)), or an equal volume of diluent (control group) was administered to a tumor with a volume of 100 mm². 3 Treatment was initiated when the average size reached 500 mm, and administered once daily for 10 days by intraperitoneal injection (IP). 3The tumor was resected when it reached its average size. Next, tissue stiffness was measured non-invasively in the longitudinal direction using ultrasound elastography. Shear wave imaging was applied using a Philips Epiq Elite Ultrasound system with a portable linear array (eL18-4) transducer. The method generates shear wave velocity via acoustic pulse waves and creates a color-mapped elastogram, where red indicates hard tissue and blue indicates soft tissue. A confidence indicator was also used as a criterion for the highest shear wave quality in the target user-defined region (ROI). Average elasticity measurements were obtained from the median elasticity values ​​of eight ROIs within the main region. The median for each ROI was automatically generated by the system under default scanner settings and expressed in kPa. For dose-response studies, shear wave imaging of 4T1 tumors was performed before bosentan treatment and on days 3, 6, and 9 post-treatment, while imaging of E0771 tumors was performed before bosentan administration and on days 3, 7, and 10 post-treatment. Ultrasonography was performed before any treatment and before tumor removal to evaluate the effects of 1 mg / kg bosentan and ICB on tumor elasticity. As shown in Figures 1A-1C, daily administration of a moderate dose of 1 mg / kg bosentan reduced tissue stiffness in both E0771 and 4T1 mammary gland tumors. As shown in Figure 1D, this result was confirmed using atomic force microscopy (AFM). In these experiments, the tumor size was 500 mm during the dose-response studies using bosentan. 3The tumor was resected when it reached the average size. The AFM study was conducted using appropriately modified versions of previously published protocols (such as Stylianou A, Lekka M, & Stylianopoulos T (2018) AFM for Assessing Nanomechanical FingerPrints for Cancer Grading and Early Diagnosis: from single cell to tissue level. Nanoscale 10:20930). More specifically, after tumor collection, tissue biopsies were obtained using an automated biopsy tool (16G, MEDAX), and the samples were immediately transferred to ice-cold PBS supplemented with a protease inhibitor cocktail (Complete Mini, Roce Dianostics GmbH, 1 tablet per 10 mL) (as in Plodinec M, et al. (2012) The nanomechanical signature of breast cancer. Nature Nanotechnology 7(11):757-765 and Tian M, et al. (2015) The nanomechanical signature of liver cancer tissues and its molecular origin. Nanoscale 7(30):12998-13010). Next, each specimen was fixed in a 35 mm plastic cell culture petri dish with a thin layer of two-component quick-drying epoxy resin. The petri dishes were filled with PBS supplemented with the protease inhibitor cocktail and stored at 4°C to avoid tissue degradation. AFM measurements were performed using a commercially available AFM system (Molecular Imaging-Agilent PicoPlus AFM) and were carried out 1 to 72 hours after tumor removal to prevent any alteration of the hardness profile. The measurements were performed using a silicon nitride cantilever (MLCT-Bio, Cantilever D, Bruker Company, cone surface half-opening angle θ approximately 20°, tip radius: 20 nm, air frequency: 15 kHz).The maximum applicable load force was set to 1.8 nN, the precise spring constant k of the cantilever was determined before each experiment using the temperature-synchronized method, and the deflection sensitivity was determined in fluid using a Petri dish as an infinitely rigid reference material (as in Stylianou A, Gkretsi V, Patrickios CS, & Stylianopoulos T (2017) Exploring the Nano-Surface of Collagenous and Other Fibrotic Tissues with AFM. Fibrosis: Methods and Protocols, ed Rittie L (Springer New York, New York, NY), pp 453-489). AFM measurements were performed on 10–15 different 20 × 20 μm samples per specimen. 2This was done by recording force maps (16×16 point grid), which correspond to 256 load-displacement curves with a pixel size of 1.25 μm per map (up to 3840 load-displacement curves per sample). Similarly, for higher spatial resolution, 32×32 force-volume maps (1024 load-displacement curves per map and a pixel size of 0.625 nm) were obtained. The collected force maps were analyzed using AtomicJ (as in Hermanowicz P, Sarna M, Burda K, & Gabrys H (2014) AtomicJ: An open source software for analysis of force curves. Review of Scientific Instruments 85(6):063703), and the Young's modulus of the samples was calculated using the Hertz model (Poisson's ratio v set to 0.5). Since the control tumors involved both cancer cells and collagen, the tissue's mechanical fingerprint varied with dose (Figure 1E). Tumors treated with 1 mg / kg showed a large involvement from cancer cells and a small involvement from collagen (Figure 1F), while tumors treated with 10 mg / kg showed heterogeneous collagen involvement (Figure 1G). As shown in Figure 1H, administration of 1 mg / kg bosentan resulted in a decrease in interstitial fluid pressure in E0771 tumors. Considering the changes in stiffness, the extracellular matrix molecules hyaluronan-binding protein (HABP) and type I collagen were directly evaluated (see Figure 1I). Bosentan administration resulted in a decrease in collagen levels (Figure 1J), but not in αSMA (Figure 1K) or HABP (Figure 1L). For these experiments, the amount of collagen present in E0771 tumor samples was evaluated via picrosilius red staining (ab150681, Abcam). In short, the fixed E0771 sample was dehydrated through a series of stepwise ethanol washes and embedded in paraffin. 7 μm thick paraffin sections were prepared using a microtome (Accu-Cut SRM 200 Rotary Microtome, SAKURA), flattened in water, and dried overnight at 37°C.Next, the sections were deparaffinized, washed in ddH2O, and incubated in picrosilius red stain at room temperature for 1 hour. Then, the tissue sections were rinsed twice with acetic acid, then twice with anhydrous ethanol, and finally mounted with DPX mounting medium (Sigma) for histology. Collagen fibers stained red, while the remaining tissue was pale yellow. For hyaluronic acid quantification, the E0771 paraffin tumor sections were deparaffinized, rehydrated, and the antigen was recovered (microwave heat treatment with trisodium citrate, pH 6 for 20 minutes). Next, the tissue sections were washed in 1×TBS / 0.025% Triton® X-100 (TBS-T), incubated in blocking serum at room temperature for 2 hours, and then immunostained overnight at 4°C with primary biotinylated hyaluronan-binding protein (b-HABP) (AMS.HKD-BC41, amsbio 1:100). Hyaluronan was detected after incubation with streptavidin-FITC conjugate (SA1001, Invitrogen 1:1000) at room temperature in the dark for 1 hour. Sections were then mounted on microscope slides using ProLong gold anti-fading mounting medium (Invitrogen) and covered with coverslips. Considering the reduction in stiffness and collagen I levels, we hypothesized that lymphatic vessel decompression would reduce the permeability coefficient and lead to a reduction in interstitial fluid pressure (IFP). Interstitial fluid pressure (IFP) was measured in vivo using the previously described wick-in-needle technique after anesthesia of mice with aveltin ip injection and before tumor resection.See Stylianopoulos T, et al. (2012) Causes, consequences, and remedies for growth-induced solid stress in murine and human tumors. Proc. Natl. Acad. Sci. USA 109(38):15101-15108 and Boucher Y, et al. (1990) Interstitial pressure gradients in tissue-isolated and subcutaneous tumors: implications for therapy. Cancer Res 50(15):4478-4484. As shown in Figure 1H, administration of 1 mg / kg bosentan resulted in a decrease in interstitial fluid pressure in E0771 tumors. (Example 2) Bosentan dose-dependently reduces hypoxia and increases T cell association with blood vessels.

[0166] We hypothesized that reducing collagen I levels and tissue stiffness would decompress blood vessels, thus reducing hypoxia, an indicator of reduced blood flow to tumors, by bosentan treatment. As can be seen in Figures 2A and 2B, 1 mg / kg reduced hypoxia in a mouse mammary tumor model. For hypoxia studies, mice with orthotopic E0771 or 4T1 mammary tumors were injected (intraperitoneally) with 60 mg / kg of pimonidazole hydrochloride 2 hours before tumor resection. Prior to tumor resection, the animals were anesthetized via aveltin (200 mg / kg, intraperitoneally). The primary tumor was then resected, fixed in 4% PFA, embedded in OCT, and processed accordingly for IHC. Hypoxic regions were detected using mouse anti-pimonidazole RED 549 conjugate antibody (HP7-100Kit, 1:100). Hypoxic region fractions for different treatment groups were normalized to DAPI staining. For fluorescence immunohistochemistry, tumors were excised, washed twice in 1×PBS for 10 minutes each, and incubated overnight at 4°C with 4% PFA. The fixative was aspirated, and the samples were washed twice in 1×PBS for 10 minutes each. The fixed tissues were embedded in an optimal cutting temperature compound in cryomold (Tissue-Tek) and completely frozen at -20°C. Tumor sections with a cross-sectional thickness of 30 μm were prepared using Tissue-Tek Cryo3 (SAKURA). Four tissue sections per tumor were joined using positively charged HistoBond microscope slides (Marienfeld). Next, tumor sections were incubated for 2 hours in blocking solution (10% fetal bovine serum, 3% donkey serum, 1×PBS) and immunostained overnight at 4°C with the following primary antibodies: rabbit anti-collagen I (ab4710, Abcam 1:100), rabbit anti-CD31 (ab28364, Abcam 1:50), rat anti-CD3 (17A2, BioLegend 1:50), and αSMA (ab5694, Abcam 1:50). Secondary antibodies (Invitrogen) against rabbit, mouse, or rat conjugated with Alexa Fluor488 or 647 were used at a 1:400 dilution. All samples were incubated for 2 hours in the dark at room temperature (RT) in a secondary antibody solution containing DAPI (Sigma, 1 mg / mL storage solution at 1:100).Sections were mounted on microscope slides using ProLong gold anti-fading mounting medium (Invitrogen) and covered with coverslips. Despite no change in the CD3+ and CD31+ stained regional fractions (Figures 2D and 2E), increased blood flow, determined by reduced hypoxia, resulted in more T cells co-localizing with endothelial cells (Figure 2C). To demonstrate the presence of T cells proximal to tumor vascular capillaries, frozen tissue sections from 4T1 and E0771 primary tumors were incubated overnight at 4°C with primary rabbit anti-CD31 (ab28364, Abcam 1:50) and rat anti-CD3 (17A2, BioLegend 1:50). CD31 signaling was detected by Alexa Fluor-488 anti-rabbit IgG (H+L), and CD3 signaling was detected by Alexa Fluor-647 anti-rat IgG (H+L) secondary antibody. Tumor-associated T cells and vascular contents were determined by normalized CD31 and CD3 region fractions for DAPI staining. Overall tumor RT-PCR mRNA expression levels supported these findings, as evidenced by reduced hypoxia-associated gene expression, increased endothelial adhesion molecule expression, and increased T cell activity (Figure 2F). For this experiment, total RNA was isolated from mammary gland tumors according to a standard Trizol-based protocol (Invitrogen), and cDNA synthesis was performed using reverse transcriptase III (RT-III) enzyme and random hexamer (Invitrogen). Real-time polymerase chain reaction was performed using Sybr Fast Universal Master Mix (KAPA). Specific mouse primers used for gene expression in 4T1 tumors are listed in the table below. The reaction was performed using a CFX-96 real-time PCR detection system (Biorad) under the following conditions: 2 minutes at 95°C, 2 seconds at 95°C, 20 seconds at 60°C, 1 second at 60°C, with steps 2-4 repeated for 39 cycles. Real-time PCR analysis and calculation of changes in gene expression between compared groups are performed using ΔΔ. Ct The method was used to perform the study. Relative gene expression was normalized based on β-actin and GAPDH expression. The primer sequences are shown in the table below:

Table 2

[0167] Bosentan monotherapy did not affect tumor growth or mouse body weight at any dose. (Example 3) Bosentan enhances the efficacy of immune checkpoint blockade (ICB) in triple-negative breast cancer (TNBC). To determine whether a regimen that reduces the hardness of bosentan and reduces the hypoxic state (increases blood flow) can enhance the efficacy of an ICB cocktail of anti-PD-1 (mouse monoclonal anti-PD-1 CD279, clone RMP1-14, BioXCell) and anti-CTLA-4 (mouse monoclonal anti-CTLA-4 CD152, clone 9D9, BioXCell) antibodies, mice with primary tumors were administered various treatments in the context of neoadjuvant chemotherapy. To assess mouse survival against spontaneous metastases that occurred during treatment, the treatment was administered before surgically excising the primary tumor. For the study of the combination treatment of bosentan and immunotherapy, 1 mg / kg bosentan or an equal volume of diluent (control group) was initiated when the tumor volume reached an average size of 5 mm in diameter and administered once daily by intraperitoneal injection (i.p.) for 14 days. Immunotherapy was administered as a cocktail of 10 mg / kg anti-PD-1 (CD279, clone RMP1-14, BioXCell) and 5 mg / kg anti-CTLA-4 (CD152, clone 9D9) after dilution in the recommended InVivoPure pH 7.0 dilution buffer (BioXCell). The immunotherapy cocktail was administered at three doses i.p. every 3 days when the tumors reached an average size of 3 200 mm. In the immunotherapy study, the animals were also treated with an isotype control antibody (BioXCell) that was not targeted. When the primary tumor reached 3When the average size was reached, the primary tumor was excised and the tissue was sutured for the study of metastatic tumors. The planar dimensions (x, y) of the tumor were monitored every 2-3 days using digital calipers, and the tumor volume was estimated from the volume of an ellipse, assuming that a third dimension z was equal to xy. For overall survival studies, the endpoint was the time until the mouse died. E0771 and 4T1 were resistant to ICB, but bosentan enhanced tumor growth inhibition (Figure 3A and 3B) and survival (Figure 3C and 3D). In the E0771 study, 8 out of 10 mice survived. Three mice were sacrificed, but no evidence of macroscopic metastasis was found in the lungs. The remaining 5 mice were re-challenged with a second inoculation of E0771 cells, and the tumor growth rate was compared to that of healthy, age-matched control mice. The tumors grew considerably slower in the mice that underwent a second attempt, and tumors appeared in only 2 out of 5 mice (Figure 3E). (Example 4) Ultrasound biomarkers correlate with response.

[0168] Next, we investigated whether ultrasound elasticity measurements correlated with the tumor response to ICB. The Young's modulus of tumors measured before the initiation of ICB treatment was found to be positively correlated with tumor size reduction in E0771 (Figure 4A) and 4T1 tumors (Figure 4B) compared to ICB cocktail monotherapy and the combination of bosentan and ICB cocktail treatment. The results suggest that a significant increase in tumor response to treatment occurs when stiffness values ​​are reduced to less than 20 kPa for the tumor models examined. (Example 5) Effects of bosentan and immune checkpoint blockade in mouse tumor models

[0169] Cell cultures, drugs, and reagents. 4T1 mouse mammary carcinoma cell lines were maintained at 37°C / 5% CO2 in RPMI-1640 (catalog number LM-R1637; Biosera) supplemented with 10% fetal bovine serum (catalog number FB-1001H; Biosera) and 1% antibody (catalog number A5955; Sigma). Bosentan hydrate (catalog number S3051; Selleckchem) was dissolved in ddH2O containing 2% DMSO, 30% PEG300, and 2% Tween® 80. The immune checkpoint inhibitors mouse monoclonal anti-PD-1 (CD279; clone RMP1-14) and mouse monoclonal anti-CTLA-4 (CD152; clone 9D9) antibodies were purchased from BioXCell.

[0170] Syngeneic tumor model and treatment protocol. Orthotopic mouse mammary gland tumor model was treated with 5 × 10⁶ cells in 40 μL of serum-free medium. 4 Four T1 cancer cells were generated by transplanting them into the third mammary fat pad of 6-8 week old BALB / c mice. The mice were purchased from the Cyprus Institute of Neurology and Genetics, and all in vivo experiments were conducted under licenses obtained and approved by the Cyprus Veterinary Services Committee, the Cypriot national authority for monitoring animal research in educational institutions, in accordance with the animal welfare regulations and guidelines of the Republic of Cyprus and the European Union (European Directive 2010 / 63 / EE, and the Cyprus Legislation for the protection and welfare of animals, Decree 1994-2013) (No CY / EXP / PR.L2 / 2018, CY / EXP / PR.L14 / 2019, CY / EXP / PR.L15 / 2019, CY / EXP / PR.L03 / 2020).

[0171] 200 mg / kg tranilast, 1 mg / kg bosentan, or an equivalent amount of diluent (control group) was initiated when the tumor volume reached an average size of 5 mm in diameter, and administered once daily by intraperitoneal injection (ip) for 14 days. Immunotherapy was administered as a cocktail of 10 mg / kg anti-PD-1 and 5 mg / kg anti-CTLA-4 after dilution in InVivoPure pH7.0 dilution buffer (BioXCell). 4T1 tumors reached 300 mm on days 19, 22, and 25. 3 When the average size reached a certain level, the immunotherapy cocktail was administered via IP. For immunotherapy studies, animals were also treated with a non-targeted isotype control antibody (BioXCell).

[0172] The planar dimensions (x, y) of the tumor were monitored every 2-3 days using digital calipers, and the tumor volume was estimated from the volume of an ellipse, assuming that a third dimension z was equal to the square root of xy. For overall survival studies, the endpoint was the time until the mouse died.

[0173] Ultrasound elastography. Shear wave elastography was performed using a Premier Philips EPIQ Elite Ultrasound system with a portable linear array (eL18-4) transducer. The method involved generating shear wave velocity via acoustic pulse waves and creating a color-mapped elastogram, with red indicating hard tissue and blue indicating soft tissue. Confidence indicators were also used as a benchmark for the highest shear wave quality in the target user-defined region (ROI). Average values ​​for the tumor region were automatically generated by the system under default scanner settings and expressed in kPa. The settings used were: frequency, 10 MHz; output, 52%; B-mode gain, 22 dB; dynamic range, 62 dB. Shear wave imaging was performed before immunotherapy cocktail treatment on day 19, and on days 23 and 26.

[0174] Dynamic contrast-enhanced ultrasound (DCEUS). Tumor-associated vascular perfusion was evaluated by DCEUS after bolus injection of contrast agent (8 μl of sulfur hexafluoride microbubbles encapsulated in phospholipid shells with an average diameter of 2.5 μm, administered posteriorly orbitally). Ultrasound scanning of the tumor was performed using an L12-5 transducer. The first contrast harmonic signal was received at 8 MHz, and the mechanical index was 0.06. For all subjects, the depth was set to 3 cm to allow measurement of all tumor depths. The gain was set to 90% for each recording. When the tumor area was located using B-mode imaging, the focus was optimized and standardized for each subject. Real-time output modulation imaging was initiated after flashing the imaging with a high mechanical index to destroy microbubbles in the tumor tissue to the highest contrast brightness, enabling visualization of bubble replenishment. Image analysis was performed offline using ultrasound quantification and analysis software (QLAB, Phillips Medical Systems). From the generated time-luminance curves, the inventors used the average transit time and the time required to reach maximum luminance (rise time) as measurements of perfusion. Prior to each ultrasound application, mice were anesthetized by an IP injection of aveltin (200 mg / kg), and ultrasound gel was applied to the imaging area to prevent any pressure from the transducer on the underlying tissue.

[0175] Results. The experimental protocol detailed above was used for the treatment of mice with 4T1 tumors with a bosentan and immune checkpoint block (ICB) cocktail, as shown in Figure 5A. The experimental protocol was also used for the treatment of mice with MCA205 (experimental protocol shown in Figure 12A) or E0771 (experimental protocol shown in Figure 12B) tumors with tranilast. Bosentan treatment was initiated on day 6 post-cell transplantation, and tranilast treatment was initiated on day 7 post-cell transplantation. Anti-PD-1 + CTLA-4 treatment reduced tumor size to 300 mm compared to bosentan-treated mice. 3Treatment was initiated when the average tumor size reached (Figure 5A). In mice with MCA205 tumors treated with tranilast, tranilast treatment was initiated when the average tumor volume reached approximately 150 mm³. 3 The treatment began on day 7, and anti-PD-L1 therapy was initiated when the average tumor volume was approximately 300 mm². 3 It was started on day 11. In mice with E0771 tumors treated with tranilast, tranilast treatment was initiated when the average tumor volume was approximately 150 mm². 3 The treatment began on day 13, when the average tumor volume was approximately 350 mm². 3 It started on the 17th day.

[0176] Tumor volume as a function of time shows that when tumors were treated with the immune cocktail alone, they did not respond to the treatment and their growth rate was the same as the control group (Figure 5B). However, when tumors were pre-treated with bosentan, immunotherapy could effectively halt further tumor growth (Figure 5B). Bosentan treatment reduced tumor stiffness by more than half of a value close to 20 kPa, which did not change significantly between administrations of immunotherapy alone (Figure 5C), indicating a correlation between bosentan treatment, tumor softening, and the effectiveness of immunotherapy.

[0177] From the quantification of DCEUS time-intensity curves, two measurements of perfusion—mean transit time and rise time (Figures 6A-6B)—were calculated. Both measurements were higher in bosentan-treated tumors compared to control tumors or tumors treated with immunotherapy cocktails alone. The results illustrate the relationship between tumor softening and increased tumor perfusion observed in Figure 5C.

[0178] As shown in Figure 13, anti-PD-L1 therapy alone was only mildly effective in reducing tumor volume compared to controls in mice with MCA205 tumors. However, with tranilast pretreatment of 100 mg / kg or 200 mg / kg, anti-PD-L1 therapy was significantly effective in reducing tumor volume. Similar results were demonstrated in mice with E0771 tumors (Figure 14).

[0179] To further investigate the correlation between hardness, perfusion, and the therapeutic efficacy of immunotherapy, as well as the relative change in tumor volume from the time checkpoint treatment was initiated to the last day of the experiment, a series of correlations were plotted for both tranilast and bosentan-treated mice (Figures 15A–15E). For both treatments, strong, equal-quality correlations were observed between the two measurements, tumor hardness and perfusion, and the relative change in tumor volume. Similarly, a strong correlation was also observed between the elastic modulus and tumor perfusion.

[0180] Further correlations between tranilast alone and tranilast in combination with anti-PD-L1 therapy are shown in Figures 16A-16E and 17 for mice with MCA205 tumors. Correlations between tranilast alone and tranilast in combination with anti-PD-L1 therapy are shown in Figures 18A-18E and 19 for mice with E0771 tumors. In both tumor models, 100 mg / kg or 200 mg / kg tranilast pretreatment significantly enhanced the tumors against immunotherapy. Figures 20A-20E show the correlations for both models (mice with MCA205 or E0771 tumors). Together, these examples demonstrate that agents that reduce tumor stiffness and increase tumor perfusion can enhance tumors against immunotherapy. (Example 6) Additional tumor modifiers

[0181] This example demonstrates the effect of another agent that decompresses blood vessels. Two mouse models of distinct sarcoma subtypes, fibrosarcoma (MCA205 cells) and osteosarcoma (K7M2wt cells), were used in this study.

[0182] MCA205 mouse fibrosarcoma cell line (SCC173, Millipore) was cultured in RPMI-1640 augmented medium containing 2 mM L-glutamine, 1 mM sodium pyruvate, 10% fetal bovine serum, 1 × non-essential amino acids (TMS-001-C, Sigma), 1% antibiotic (A5955, Sigma), and 1 × β-mercaptoethanol. K7M2wt mouse osteosarcoma cell line (CRL2836®, ATCC®) was cultured in DMEM augmented medium supplemented with 10% FBS and 1% antibiotic. All cells were maintained at 37°C / 5% CO2.

[0183] The fibrosarcoma syngeneic tumor model consists of 2.5 × 10⁶ MCA205 cells in 50 μL of serum-free medium. 5 The individual tumors were created by subcutaneously transplanting them into the flanks of 6-week-old C57BL / 6 female mice. The osteosarcoma syngeneic tumor model was created by transplanting K7M2wt tumor fragments into the fat bodies of 6-week-old BALB / c female mice.

[0184] As shown in Figure 7A (MCA205 tumor) and Figure 7B (K7M2wt tumor), ketotifen alone did not show an antitumor effect in either mouse sarcoma model.

[0185] Interstitial fluid pressure (IFP) was measured in vivo in mice using the "wick-in-needle" technique after anesthesia with aveltin ip injection and before tumor resection. Additional information regarding the wick-in-needle technique is described in Dong et al., Involvement of mast cell chymase in burn wound healing in hamsters 2013;5:643-7 and Shankaran et al. IFNγ and lymphocytes prevent primary tumor development and shape tumor immunogenicity 2001;410:1107-11, the entirety of which is incorporated herein by reference. All doses of ketotifen reduced IFP, with the 10 mg / kg dose showing the greatest effect (Figure 8).

[0186] The mechanical therapeutic effect of ketotifen in reducing substrate stiffness was evaluated non-invasively and primarily in the longitudinal direction using ultrasound elastography. Tumor elasticity was assessed via shear wave elastography using a Philips EPIQ Elite ultrasound scanner with an eL 18-4 linear array. A dose of 10 mg / kg of ketotifen most effectively reduced tissue stiffness in mice with MCA205 fibrosarcoma tumors, reaching a Young's modulus of 20 kPa, similar to the elasticity of healthy tissue (Figure 9).

[0187] Vascular and functional perfusion were simultaneously measured using contrast-enhanced ultrasound during the course of ketotifen treatment in mice with MCA205 and mice with K7M2wt tumors. As shown in Figures 10A–10D, ketotifen induced a significant increase in vascular and functional perfusion in both sarcoma subtypes.

[0188] Given the effects of ketotifen demonstrated in a mouse tumor model, we evaluated the effects of ketotifen on the antitumor immune response of chemotherapeutic agents and anti-PD-L1 checkpoint inhibitors.

[0189] Mice with sarcoma tumors were pre-treated with daily administration of 10 mg / kg ketotifen, followed by treatment with three or four doses of doxorubicin and / or an immune checkpoint inhibitor, an anti-PD-L1 antibody. Mouse monoclonal anti-PD-L1 (B7-H1, clone 10F.9G2, BioXCell) was used. Doxorubicin hydrochloride was prepared as a pre-made solution at 2 mg / ml. The anti-PD-L1 antibody was administered at a final dose of 10 mg / kg, followed by doxorubicin at 5 mg / kg.

[0190] Mice with MCA205 tumors, with an average tumor size of 40 mm. 3 After reaching a certain stage, pre-treatment was performed daily with 10 mg / kg ketotifen or an equivalent volume of diluent (control group) before neoadjuvant chemotherapy. 3When the average size was reached (day 7), doxorubicin and anti-PD-L1 combination therapy was initiated, administered as intravenous injection in three doses every three days (days 7, 10, and 13). Daily administration of ketotifen continued until the completion of doxorubicin-anti-PD-L1 combination therapy.

[0191] The primary tumor was 700 mm on day 16. 3 Once the average size reached 80 mm, the primary tumor was excised and preserved, and the mouse was monitored for a re-challenge experiment. Similarly, K7M2wt tumors were monitored until the average tumor size reached 80 mm. 3 If the tumor size reached 150 mm (day 18), pretreatment was performed with daily administration of 10 mg / kg ketotifen or an equivalent amount of diluent (control group), and this was continued until the completion of preoperative chemotherapy. 3 When the average size reached 550 mm (day 22), doxorubicin and anti-PD-L1 combination therapy was initiated and repeated on days 25, 28, and 31. 3 The study was terminated when the average volume (day 33) was reached. Mice were sacrificed and tumors were collected for ex vivo analysis.

[0192] Neither anti-PD-L1 nor doxorubicin monotherapy had a significant effect on tumor growth in MCA205 or K7M2wt tumors, but when combined with ketotifen, they induced a significant antitumor response (Figure 11A for MCA205 tumors; Figure 11B for K7M2wt tumors).

Claims

1. A pharmaceutical composition for use in the treatment of solid tumors in subjects requiring treatment of solid tumors, comprising bosentan or a pharmaceutically acceptable salt thereof, wherein the treatment comprises administering the pharmaceutical composition to the subject in combination with a checkpoint inhibitor.

2. A pharmaceutical composition for use in the treatment of solid tumors in subjects requiring treatment of solid tumors, comprising a checkpoint inhibitor, wherein the treatment comprises administering the pharmaceutical composition to the subject in combination with bosentan or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition for the treatment of solid tumors in subjects requiring treatment of solid tumors, comprising a combination of bosentan or a pharmaceutically acceptable salt thereof and a checkpoint inhibitor.

4. A pharmaceutical composition for increasing blood flow to a solid tumor in a subject, comprising bosentan or a pharmaceutically acceptable salt thereof, wherein increasing blood flow to the solid tumor in the subject comprises administering the pharmaceutical composition to the subject in combination with a checkpoint inhibitor.

5. The pharmaceutical composition according to claim 4, characterized in that blood flow is measured using ultrasound-based blood flow measurement or histological techniques for measuring hypoxia.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein administration of the pharmaceutical composition increases the number of antitumor T cells co-localized with the solid tumor.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein administration of the pharmaceutical composition reduces the tissue hardness of the solid tumor.

8. The pharmaceutical composition according to claim 7, characterized in that the tissue hardness of the solid tumor is measured using ultrasound elastography.

9. The pharmaceutical composition according to any one of claims 1 to 5, wherein administration of the pharmaceutical composition reduces the level of extracellular matrix proteins in the solid tumor.

10. The pharmaceutical composition according to claim 9, wherein the extracellular matrix protein is collagen I or hyaluronan-binding protein (HABP).

11. The pharmaceutical composition according to any one of claims 1 to 5, wherein administration of the pharmaceutical composition reduces the hypoxic state in the solid tumor.

12. The pharmaceutical composition according to any one of claims 1 to 5, wherein the checkpoint inhibitor inhibits a checkpoint protein or a combination thereof selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, BMA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands.

13. The pharmaceutical composition according to claim 12, wherein the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or a combination thereof.

14. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that the pharmaceutical composition is administered to the subject at least once a day.

15. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that the pharmaceutical composition is administered to the subject in a dose of about 0.01 mg / kg to about 5 mg / kg.

16. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that the pharmaceutical composition is administered to the subject in a dose of about 100 mg to about 1200 mg.

17. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that the bosentan or a pharmaceutically acceptable salt thereof is administered to the subject before the checkpoint inhibitor is administered to the subject.

18. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that the administration of the bosentan or a pharmaceutically acceptable salt thereof to the subject is maintained for at least a portion of the period during which the checkpoint inhibitor is administered to the subject.

19. The pharmaceutical composition according to any one of claims 1 to 5, wherein one or more therapeutic effects in the subject are improved after administration of the pharmaceutical composition, wherein the one or more therapeutic effects are selected from the group consisting of the size of the solid tumor or tumor derived from the solid tumor, objective response rate, duration of response, time to response, progression-free survival, and overall survival.

20. The pharmaceutical composition according to any one of claims 1 to 5, wherein the size of the solid tumor or a tumor derived from the solid tumor is reduced by at least about 10% compared to the size of the solid tumor or a tumor derived from the solid tumor before administration of the pharmaceutical composition and the checkpoint inhibitor.

21. The pharmaceutical composition according to any one of claims 1 to 5, wherein the objective response rate is at least about 20%.

22. The pharmaceutical composition according to any one of claims 1 to 5, wherein the subject exhibits a progression-free survival period of at least about one month after administration of the pharmaceutical composition and the checkpoint inhibitor.

23. The pharmaceutical composition according to any one of claims 1 to 5, wherein the subject exhibits an overall survival period of at least about one month after administration of the pharmaceutical composition and the checkpoint inhibitor.

24. The pharmaceutical composition according to any one of claims 1 to 5, wherein the response period is at least about one month after administration of the pharmaceutical composition and the checkpoint inhibitor.

25. The pharmaceutical composition according to any one of claims 1 to 5, wherein the solid tumor is selected from the group consisting of breast cancer, lung metastasis of breast cancer, sarcoma, pancreatic cancer, ovarian cancer, liver metastasis, prostate cancer, brain cancer, melanoma, mesothelioma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, lung cancer, head and neck squamous cell carcinoma, urothelial carcinoma, esophageal squamous cell carcinoma, gastric cancer, esophageal cancer, cervical cancer, Merkel cell carcinoma, endometrial cancer, and cutaneous squamous cell carcinoma.

26. The pharmaceutical composition according to any one of claims 1 to 5, wherein the subject is a human.

27. (a) an effective amount of bosentan or a pharmaceutically acceptable salt thereof; (b) an effective dose of a checkpoint inhibitor; and (c) Instructions for use of the bosentan or a pharmaceutically acceptable salt thereof and the checkpoint inhibitor in accordance with any one of claims 1 to 5. A kit that includes this.

28. The pharmaceutical composition according to any one of claims 1 to 5, wherein the subject is selected for administration with the checkpoint inhibitor based on increased blood flow and / or decreased hardness of the solid tumor in response to bosentan or a pharmaceutically acceptable salt thereof.

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