Cancer treatment using a low volumetric flow rate of ultra-high concentration gaseous nitric oxide

Abstract

Embodiments of the present invention provide for methods of treating tumors in a subject in need thereof, the methods comprising locally administering to the tumor a therapeutically effective amount of ultra-high concentration gaseous nitric oxide (UNO), wherein the UNO is administered at a volumetric flow rate of less than 20 mL / min (mLPM).

Description

[0001] CANCER TREATMENT USING A LOW VOLUMETRIC FLOW RATE OF ULTRA- HIGH CONCENTRATION GASEOUS NITRIC OXIDE

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 573,933 filed April 3, 2024 and U.S. Provisional Application No. 63 / 716,164 filed November 4, 2024. The entire contents of the above-referenced applications are incorporated by reference herein.

[0004] FIELD OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to cancer treatment using a low volumetric flow rate of ultra-high concentration gaseous nitric oxide.

[0006] BACKGROUND OF THE INVENTION

[0007] Nitric oxide (NO) is a short-lived, endogenously produced gas that acts as a signaling molecule in the body. Increasing evidence highlights its wide spectrum of action in different pathologic conditions, including cancer and involvement in immune cell signaling against pathogens.

[0008] Preclinical studies testing the effect of exogenously administered nitric oxide (NO) demonstrated its anti-cancer properties and suggested that NO may serve as a potent tumoricidal ablation agent. While NO at low doses may possess pro-oncogenic properties; at high doses, NO may have a role in cancer therapy either as a single agent or in combination with other antineoplastic compounds. More specifically, high doses of NO were shown to promote oxidative / nitrosative stress and DNA damage. The generation of reactive nitric oxide species, including peroxynitrite can oxidize the DNA and induce single strand breaks.

[0009] Tumors can be treated with high doses of NO gas by administering relatively large volumes of the NO gas directly into the tumor. However, this has been known to increase swelling in and around the tumor treatment area. To reduce or prevent such swelling, the formation of an exit hole in the treatment area and / or tumor segregation may be required.

[0010] Accordingly, there is a need for delivery methods of high-dose NO gas that can avoid swelling in and around the treatment area. SUMMARY OF THE INVENTION

[0011] According to an aspect of some embodiments of the present invention there is provided a method of treating a tumor in a subject in need thereof, the method comprising: locally administering to the tumor a therapeutically effective amount of ultra-high concentration gaseous nitric oxide (UNO), wherein the UNO is administered at a volumetric flow rate of less than 20 mL / min (mLPM).

[0012] According to an aspect of some embodiments of the present invention, the UNO is administered at a volumetric flow rate between .01 mLPM and 20 mLPM. Preferably between .1 mLPM and 10 mLPM. Preferably between 1 mLPM and 5 mLPM. Preferably, 2 mLPM.

[0013] According to an aspect of some embodiments of the present invention, the UNO is administered at a volumetric flow rate between .01 mLPM and 10 mLPM. Preferably between .1 mLPM and 10 mLPM. Preferably between 1 mLPM and 5 mLPM. Preferably, 2 mLPM.

[0014] According to an aspect of some embodiments of the present invention, the UNO is administered for a time period of about 30 seconds to about 30 minutes. Preferably between 1 minute and 15 minutes. Preferably between 5 minutes and 10 minutes. Preferably, 8 minutes.

[0015] According to some embodiments of the invention, the UNO is administered at a dose between 1,000 ppm and 1,000,000 ppm. Preferably between 10,000 ppm and 500,000 ppm. Preferably between 20,000 ppm and 100,000 ppm.

[0016] According to an aspect of some embodiments of the present invention, locally administering the UNO is by intra-tumoral injection.

[0017] According to an aspect of some embodiments of the present invention, the method can further comprise monitoring a pressure parameter associated with the tumor; and controlling the volumetric flow rate of the locally administered UNO such that the monitored pressure parameter remains below a predetermined value.

[0018] According to an aspect of some embodiments of the present invention, the pressure parameter is the intra-tumoral pressure of the tumor.

[0019] According to an aspect of some embodiments of the present invention, the predetermined value is an absolute pressure between 1 atmosphere (ATM) and 2 ATM.

[0020] According to an aspect of some embodiments of the present invention, upon determining that the monitored pressure parameter has reached the predetermined value, ceasing or slowing the volumetric flow rate of the UNO.

[0021] According to an aspect of some embodiments of the present invention, the volumetric flow rate is controlled until (i) a total UNO administration time has elapsed, (ii) a total operation time has elapsed, or (iii) a total volume of UNO has been administered. According to an aspect of some embodiments of the present invention, UNO is administered either continuously or intermittently during the total operation time.

[0022] According to an aspect of some embodiments of the present invention, the UNO can be administered in combination with one or more checkpoint inhibitors.

[0023] According to an aspect of some embodiments of the present invention, the one or more checkpoint inhibitors are selected from the group consisting of an anti-PD-1 antibody, an anti- PD-L1 antibody, an anti-CTLA-4 antibody, and an anti-LAG-3 antibody.

[0024] According to an aspect of some embodiments of the present invention, the anti-PD-1 antibody is one of pembrolizumab, nivolumab, cemiplimab, spartalizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, JTX-4014, INCMGA00012, AMP -224, and AMP-514.

[0025] According to an aspect of some embodiments of the present invention, the anti-PD-Ll antibody is one of atezolizumab, durvalumab, avelumab, KN035, CK-301, AUNP12, CA-170 and BMS-986189.

[0026] According to an aspect of some embodiments of the present invention, the anti-CTLA- 4 antibody is one of ipilimumab and tremelimumab.

[0027] According to an aspect of some embodiments of the present invention, the anti-LAG-3 antibody is relatlimab.

[0028] According to some embodiments of the invention, the one or more checkpoint inhibitors are administered prior to the UNO.

[0029] According to some embodiments of the invention, the one or more checkpoint inhibitors are administered after the UNO.

[0030] According to some embodiments of the invention, the one or more checkpoint inhibitors are administered every 2-7 days.

[0031] According to some embodiments of the invention, the one or more checkpoint inhibitors are administered at least twice.

[0032] According to some embodiments of the invention, the tumor is refractory to treatment with a checkpoint inhibitor.

[0033] According to some embodiments of the invention, the tumor relapsed after previous treatment with the checkpoint inhibitor.

[0034] According to some embodiments of the invention, the subject achieved a prolonged stable disease state after previous treatment with the checkpoint inhibitor.

[0035] According to some embodiments of the invention, the tumor relapsed after previous treatment with another checkpoint inhibitor. According to some embodiments of the invention, the subject achieved a prolonged stable disease state after previous treatment with another checkpoint inhibitor.

[0036] According to some embodiments of the invention, the combination of UNO and the one or more checkpoint inhibitors results in a reduction of at least one of tumor size and volume.

[0037] According to some embodiments of the invention, the tumor is associated with a cancer that is negative for the microsatellite instability (MSI) and / or the mismatch repair deficient (dMMR) marker.

[0038] According to some embodiments of the invention, the tumor is associated with a cancer that is selected from the group consisting of colon, breast, melanoma (e.g., BRAF positive melanoma), lung (e.g., Non-Small Cell Lung Cancer (NSCLC)), Head and Neck Squamous Cell Cancer (HNSCC), Classical Hodgkin Lymphoma (cHL), Primary Mediastinal Large B- Cell Lymphoma (PMBCL), Urothelial Carcinoma, Gastric Cancer, Esophageal Cancer, Cervical Cancer, Hepatocellular Carcinoma (HCC), Merkel Cell Carcinoma (MCC), Renal Cell Carcinoma (RCC), Endometrial Carcinoma, Tumor Mutational Burden-High (TMB-H) Cancer, Cutaneous Squamous Cell Carcinoma (cSCC), Basal Cell Carcinoma (BCC), TripleNegative Breast Cancer (TNBC), Microsatellite Instability-Low, Microsatellite Instability- High, or Mismatch Repair Deficient Cancer, and Microsatellite Instability-Low, Microsatellite Instability-High, or Mismatch Repair Deficient Colorectal Cancer (CRC).

[0039] In some embodiments, UNO can be administered to treat tumors at a dose of one of 10,000 ppm, 15,000 ppm, 20,000 ppm, 25,000 ppm, 50,000 ppm, or 100,000 ppm through local administration, such as intra-tumoral injection. In some embodiments, UNO can be administered for a duration of 8 minutes at a flow rate of 2 mLPM, for a total volume of 16 mL. In some embodiments, UNO can be administered for one or more cycles. In some embodiments, a cycle can correspond to a one-week, two-week, three-week, or four-week period, with UNO being administered on day 1 of the cycle. In other embodiments, UNO can be administered on day 1 and / or day 8 and / or day 15 and / or day 22 of the respective cycle. In some embodiments, UNO can be administered in combination with a checkpoint inhibitor, wherein the checkpoint inhibitor is one of a PD-1 inhibitor, PD-L1 inhibitor, a CTLA-4 inhibitor, and a LAG-3 inhibitor. In some embodiments, the checkpoint inhibitor can be administered intravenously subsequent to the administration of UNO.

[0040] In some embodiments, UNO can be administered to treat tumors having relap sed / refractory or prolonged stable disease (> 12 weeks) after previous treatment single agent PD-1 inhibitor. In some embodiments, UNO can be administered intratumorally at a dose of 25,000 ppm ± 10% or 50,000 ppm ± 10% or 100,000 ppm ± 10% for a duration of 8 minutes at a volumetric flow rate between .2 mLPM and 2 mLPM. In some embodiments, UNO can be administered as a single dose. In some embodiments, UNO can be administered with a checkpoint inhibitor, wherein the checkpoint inhibitor is a PD-1 inhibitor, such as pembrolizumab, nivolumab, or cemiplimab. In some embodiments, the PD-1 inhibitor is administered intravenously at a therapeutically effective dose. In some embodiments, the PD-1 inhibitor is administered at 2- or 3-week intervals.

[0041] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0044] In the drawings:

[0045] FIG. 1 shows the absorption of UNO by concentration and type of applicator for live mouse tumors;

[0046] FIG. 2A shows the resultant tumor volume after a low volumetric flow rate treatment of UNO in combination with a checkpoint inhibitor;

[0047] FIG. 2B shows the resultant tumor volume after a low volumetric flow rate treatment of nitrogen in combination with a checkpoint inhibitor;

[0048] FIG. 3 shows the percentage of mice that underwent complete primary tumor regression after low volumetric flow rate treatments of (1) nitrogen in combination with a checkpoint inhibitor and (2) UNO in combination with a checkpoint inhibitor;

[0049] FIG. 4 shows the resultant mouse survival rate after low volumetric flow rate treatments of (1) nitrogen in combination with a checkpoint inhibitor and (2) UNO in combination with a checkpoint inhibitor; FIG. 5 shows the resultant primary tumor regression after (1) high volumetric flow rate treatments of UNO in combination with a checkpoint inhibitor, (2) low volumetric flow rate treatments of UNO in combination with a checkpoint inhibitor, (3) treatment with a checkpoint inhibitor alone, and (4) no treatment; and

[0050] FIG. 6A-6D shows the distribution of UNO in CT26 tumors, via nitrotyrosine staining, after (1) no treatment (FIG. 6A), (2) treatment with nitrogen (FIG. 6B), (3) high volumetric flow rate treatment of UNO (FIG. 6C), and (4) low volumetric flow rate treatment of UNO (FIG. 6D).

[0051] FIG. 6E shows a summary of the immunohistochemistry results for nitrotyrosine staining using a semi-quantitative scoring system;

[0052] FIG. 7 shows the resultant tumor volume after (1) low volumetric flow rate treatment of UNO with a checkpoint inhibitor, (2) high volumetric flow rate treatment of UNO with a checkpoint inhibitor, and (3) treatment with a checkpoint inhibitor alone; and

[0053] FIG. 8 A shows the probability of survival for MAT B III tumor-bearing rats after (1) low volumetric flow rate treatment of UNO (either 25,000 ppm or 100,000 ppm) in combination with a checkpoint inhibitor and (2) treatment with a checkpoint inhibitor alone.

[0054] FIG. 8B shows the resultant tumor volume after (1) low volumetric flow rate treatment of UNO with a checkpoint inhibitor and (2) treatment with a checkpoint inhibitor alone.

[0055] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0056] The present invention, in some embodiments thereof, relates to cancer treatment using a low volumetric flow rate of ultra-high concentration gaseous nitric oxide (UNO). The treatment further includes the use of a checkpoint inhibitor.

[0057] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0058] While reducing specific embodiments of the present invention to practice, the present inventors have now discovered that only a small volume of locally administered UNO is actually being absorbed by tumor tissue (Example 1 of the Examples section which follows). In fact, the present inventors have also discovered that more UNO is absorbed by the tumor tissue when it is administered at a lower volumetric flow rate, e.g., less than 20 mLPM, thereby obviating the need for either (i) an exit hole in the treated tumor or (ii) segregation of the treated tumor, as is required for higher volume / higher volumetric flow rate administration. As a result, the present inventors have also discovered that only a small volume of UNO may be needed in combination with a checkpoint inhibitor in order to achieve an improved anti-cancer effect (Example 2-3 of the Examples section which follows).

[0059] Thus, according to an aspect of the invention there is provided a method of treating cancer in a subject in need thereof, the method comprising: locally administering to a tumor a therapeutically effective amount of ultra-high concentration gaseous nitric oxide (UNO), wherein the UNO is administered at a volumetric flow rate of less than 20 mLPM.

[0060] As used herein the term “treating” refers to curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease or disorder (e.g., cancer). Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology (e.g., a malignancy), as discussed below.

[0061] As used herein throughout, the terms "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman organisms, i.e., animals. The term "nonhuman animals" of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like, for medical and / or laboratory research purposes. Preferably, the subject is a human subject. More preferably, the subject is a human patient diagnosed with cancer (e.g., pre-malignant or malignant tumor).

[0062] As used herein throughout, the term and “tumor” describes a plurality of cells or a tissue composed of the plurality of cells that are characterized by abnormal cell growth and which serve no physiological function.

[0063] By “abnormal cell growth” it is meant uncontrolled, progressive proliferation of the cells, which is no longer under normal bodily control. The growth of a tumor tissue typically exceeds, and is uncoordinated with, that of the normal cells or tissues around it.

[0064] "Abnormal cell growth” also describes cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition), including, for example, abnormal growth of: (1) cancerous (or cancer) cells that proliferate by expressing a mutated tyrosine kinase or over-expression of a receptor tyrosine kinase; (2) benign and malignant cells of other proliferative diseases in which aberrant tyrosine kinase activation occurs; (3) any tumors that proliferate by receptor tyrosine kinases; (4) any tumors that proliferate by aberrant serine / threonine kinase activation; and (5) benign and malignant cells of other proliferative diseases in which aberrant serine / threonine kinase activation occurs. A tumor as described herein can be a primary tumor or a secondary tumor.

[0065] The term “malignant tumor” describes a tumor that is not self-limited in its growth, is capable of invading into adjacent tissues, and may be capable of spreading to distant tissues (metastasizing). The term “benign tumor” describes a tumor which is not malignant (i.e., does not grow in an unlimited, aggressive manner, does not invade surrounding tissues, and does not metastasize).

[0066] The term “primary tumor” describes a tumor that is at the original site where it first arose.

[0067] The term “secondary tumor” describes a tumor that has spread from its original (primary) site of growth to another site, close to or distant from the primary site, and is also referred to herein and in the art as metastasis, or as metastasizing tumor. The term “secondary tumor” as used herein also describes recurrent tumor, which can ne at the original site as the primary tumor and / or at another site, as a metastasizing tumor.

[0068] According to some of any of the embodiments described herein, the tumor is a malignant tumor, for example, a malignant cancerous tumor, and the tumor cells are cancer or cancerous cells.

[0069] According to these embodiments, the methods and uses as described herein in any of the respective embodiments are for treating cancer or a cancerous tumor is a subject in need thereof.

[0070] The methods and uses as described herein are in the context of subjects having a primary cancer tumor, a metastasizing cancer and / or a recurrent cancer, as described herein.

[0071] The term “cancer” encompasses malignant and benign tumors as well as disease conditions evolving from primary or secondary tumors, as described herein.

[0072] Examples of benign tumors include, without limitation, lipomas, chondromas, adenomas, pilomatricomas, teratomas, and hamartomas.

[0073] Cancers treatable according to embodiments of the invention include, but are not limited to, carcinomas, sarcomas, blastomas, and germ cell tumors. Carcinomas include, without limitation, adenocarcinomas (e.g., small cell lung cancer, kidney, uterus, prostate, bladder, ovary and / or colon adenocarcinoma) and epithelial carcinomas.

[0074] Examples of cancers treatable according to embodiments of the invention include, without limitation, adenocarcinoma, adrenal tumors (e.g., hereditary adrenocortical carcinoma), biliary tract tumors, bladder cancer, bone cancer, brain cancer, breast cancer (e.g., ductal breast cancer, invasive intraductal breast cancer, sporadic breast cancer, susceptibility to breast cancer, type 4 breast cancer, breast cancer-1, breast cancer-3, and / or breast-ovarian cancer), bronchogenic large cell carcinoma, cervical cancer (e.g., cervical carcinoma), carcinosarcoma, choriocarcinoma, cystadenocarcinoma, dermatofibrosarcoma protuberans, ductal carcinoma, Ehrlich-Lettre ascites, embryonal rhabdomyosarcoma, endocrine neoplasia, endometrial cancer (e.g., endometrial carcinoma), ependimoblastoma, epidermoid carcinoma, epithelial adult tumor, epithelioma, erythroleukemia (e.g., Friend and / or lymphoblast), extraskeletal myxoid chondrosarcoma, fibrosarcoma, gallbladder carcinoma, ganglioblastoma, gastrointestinal tract tumors (e.g., colon carcinoma, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6, hereditary nonpolyposis type 7, small and / or large bowel carcinoma, esophageal carcinoma, tylosis with esophageal cancer, stomach carcinoma, pancreatic carcinoma, and / or pancreatic endocrine tumors), germ cell tumor (male germ cell tumor, and / or testicular and / or ovarian dysgerminoma), giant cell tumor, glial tumor, glioma, glioblastoma (e.g., glioblastoma multiforme, astrocytoma), head & neck cancer, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B-cell), hypernephroma, insulinoma, islet tumor, keratoma, large cell carcinoma, leiomyoblastoma, leiomyosarcoma, leukemia (e.g., acute lymphatic leukemia, acute lymphoblastic leukemia, acute lymphoblastic pre-B cell leukemia, acute lymphoblastic T cell leukemia, acute megakaryoblastic leukemia, monocytic leukemia, acute myelogenous leukemia, acute myeloid leukemia, acute myeloid leukemia with eosinophilia, B- cell leukemia, basophilic leukemia, chronic myeloid leukemia, chronic B-cell leukemia, eosinophilic leukemia, Friend leukemia, granulocytic or myelocytic leukemia, hairy cell leukemia, lymphocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, monocytic leukemia, monocytic-macrophage leukemia, myeloblastic leukemia, myeloid leukemia, myelomonocytic leukemia, plasma cell leukemia, pre-B cell leukemia, promyelocytic leukemia, subacute leukemia, T-cell leukemia, lymphoid neoplasm, predisposition to myeloid malignancy, and / or acute nonlymphocytic leukemia), Li-Fraumeni syndrome, liposarcoma, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular cancer, and / or hepatoma), lung cancer (e.g., Lewis lung carcinoma, small cell carcinoma and / or non-small cell carcinoma) lymphoma (e.g., Hodgkin’s disease, non-Hodgkin’s lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, cutaneous T-cell lymphoma, histiocytic lymphoma, lymphoblastic lymphoma, T-cell lymphoma, and / or thymic lymphoma), lymphosarcoma, lynch cancer family syndrome II, mammary tumor, mastocytoma, medulloblastoma, medullary carcinoma, melanoma, mesothelioma, metastatic tumor, monocyte tumor, mucoepidermoid carcinoma, multiple glomus tumors, multiple meningioma, myelodysplastic syndrome, myeloma (e.g., multiple myeloma), nasopharyngeal cancer, nephroblastoma, nervous tissue glial tumor, nervous tissue neuronal tumor, neurinoma, neuroblastoma, neurogenic tumor, non-melanoma skin cancer, oat cell carcinoma, oligodendroglioma, osteochondroma, osteomyeloma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma, serous ovarian cancer, and / or ovarian sex cord tumors), papillary carcinoma, papilloma, paraganglioma (e.g., familial nonchromaffin), pheochromocytoma, pituitary tumor (invasive), placental site trophoblastic tumor, plasmacytoma, prostate cancer (e.g., prostate adenocarcinoma), renal cancer (e.g., Wilms’ tumor type 2 or type 1), retinoblastoma, rhabdoid tumors (e.g., rhabdoid predisposition syndrome), rhabdomyosarcoma, sacrococcygeal tumor, sarcoma (e.g., Ewing’s sarcoma, histiocytic cell sarcoma, Jensen sarcoma, myxosarcoma, osteosarcoma, reticulum cell sarcoma, soft tissue sarcoma and / or synovial sarcoma), schwannoma, small cell carcinoma, spindle cell carcinoma, spinocellular carcinoma, squamous cell carcinoma (e.g., in head and neck), basal cell carcinoma, subcutaneous tumor, teratocarcinoma (e.g., pluripotent), teratoma (e.g., immature teratoma of ovary), testicular cancer (e.g. testicular germ cell tumor), transitional cell carcinoma, Turcot syndrome with glioblastoma, thymoma, thyroid cancer (e.g., follicular, medullary and / or papillary thyroid cancer), trichoepithelioma, trophoblastic tumor, undifferentiated carcinoma, uterine cancer, uterine cervix carcinoma.

[0075] Methods and uses of the present embodiments can be used to treat one or more solid tumors.

[0076] As used herein, the term "solid tumor" refers to those conditions, such as cancer, that form an abnormal tumor mass, such as sarcomas, carcinomas, and lymphomas. For example, solid tumors can include, but are not limited to, ovarian tumors, prostate tumors, skin tumors, lung tumors, breast tumors, liver tumors, brain tumors, CNS tumors, kidney tumors, colon tumors, bladder tumors, intestinal tumors, melanomas, gliomas, ependymomas, oligodendrogliomas, oligoastrocytomas, astrocytomas, glioblastomas, and medulloblastomas. Suitable examples of solid tumor diseases include, but are not limited to, non-small cell lung cancer (NSCLC), neuroendocrine tumors, thyomas, fibrous tumors, metastatic colorectal cancer (mCRC), and the like. In certain embodiments, the solid tumor disease is an adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and the like.

[0077] According to some embodiments, the cancer is or comprises a solid tumor, and can be, for example, adenocarcinoma, adrenal tumors (e.g., hereditary adrenocortical carcinoma), biliary tract tumors, bladder cancer, bone cancer, brain cancer, breast cancer (e.g., ductal breast cancer, invasive intraductal breast cancer, sporadic breast cancer, susceptibility to breast cancer, type 4 breast cancer, breast cancer-1, breast cancer-3, and / or breast-ovarian cancer), bronchogenic large cell carcinoma, cervical cancer (e.g., cervical carcinoma), carcinosarcoma, choriocarcinoma, cystadenocarcinoma, dermatofibrosarcoma protuberans, ductal carcinoma, Ehrlich-Lettre ascites, embryonal rhabdomyosarcoma, endocrine neoplasia, endometrial cancer (e.g., endometrial carcinoma), ependimoblastoma, epidermoid carcinoma, epithelial adult tumor, epithelioma, extraskeletal myxoid chondrosarcoma, fibrosarcoma, gallbladder carcinoma, ganglioblastoma, gastrointestinal tract tumors (e.g., colon carcinoma, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6, hereditary nonpolyposis type 7, small and / or large bowel carcinoma, esophageal carcinoma, tylosis with esophageal cancer, stomach carcinoma, pancreatic carcinoma, and / or pancreatic endocrine tumors), germ cell tumor (male germ cell tumor, and / or testicular and / or ovarian dysgerminoma), giant cell tumor, glial tumor, glioma, glioblastoma (e.g., glioblastoma multiforme, astrocytoma), head & neck cancer, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B-cell), hypernephroma, insulinoma, islet tumor, keratoma, large cell carcinoma, leiomyoblastoma, liposarcoma, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular cancer, and / or hepatoma), lung cancer (e.g., Lewis lung carcinoma, small cell carcinoma and / or non-small cell carcinoma) lymphoma (e.g., Hodgkin’s disease, non-Hodgkin’s lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, cutaneous T-cell lymphoma, histiocytic lymphoma, lymphoblastic lymphoma, T-cell lymphoma, and / or thymic lymphoma), lymphosarcoma, lynch cancer family syndrome II, mammary tumor, mastocytoma, medulloblastoma, medullary carcinoma, melanoma, mesothelioma, metastatic tumor, monocyte tumor, mucoepidermoid carcinoma, multiple glomus tumors, multiple meningioma, myelodysplastic syndrome, myeloma (e.g., multiple myeloma), nasopharyngeal cancer, nephroblastoma, nervous tissue glial tumor, nervous tissue neuronal tumor, neurinoma, neuroblastoma, neurogenic tumor, non-melanoma skin cancer, oat cell carcinoma, oligodendroglioma, osteochondroma, osteomyeloma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma, serous ovarian cancer, and / or ovarian sex cord tumors), papillary carcinoma, papilloma, paraganglioma (e.g., familial nonchromaffin), pheochromocytoma, pituitary tumor (invasive), placental site trophoblastic tumor, plasmacytoma, prostate cancer (e.g., prostate adenocarcinoma), renal cancer (e.g., Wilms’ tumor type 2 or type 1), retinoblastoma, rhabdoid tumors (e.g., rhabdoid predisposition syndrome), rhabdomyosarcoma, sacrococcygeal tumor, sarcoma (e.g., Ewing’s sarcoma, histiocytic cell sarcoma, Jensen sarcoma, myxosarcoma, osteosarcoma, reticulum cell sarcoma, soft tissue sarcoma and / or synovial sarcoma), schwannoma, small cell carcinoma, spindle cell carcinoma, spinocellular carcinoma, squamous cell carcinoma (e.g., in head and neck), basal cell carcinoma, subcutaneous tumor, teratocarcinoma (e.g., pluripotent), teratoma (e.g., immature teratoma of ovary), testicular cancer (e.g. testicular germ cell tumor), transitional cell carcinoma, Turcot syndrome with glioblastoma, thymoma, thyroid cancer (e.g., follicular, medullary and / or papillary thyroid cancer), trichoepithelioma, trophoblastic tumor, undifferentiated carcinoma, uterine cancer, uterine cervix carcinoma.

[0078] According to a specific embodiment, the cancer is selected from the group consisting of colon, breast, melanoma, lung, Head and Neck Squamous Cell Cancer (HNSCC), Classical Hodgkin Lymphoma (cHL), Primary Mediastinal Large B-Cell Lymphoma (PMBCL), Urothelial Carcinoma, Gastric Cancer, Esophageal Cancer, Cervical Cancer, Hepatocellular Carcinoma (HCC), Merkel Cell Carcinoma (MCC), Renal Cell Carcinoma (RCC), Endometrial Carcinoma, Tumor Mutational Burden-High (TMB-H) Cancer, Cutaneous Squamous Cell Carcinoma (cSCC), Basal Cell Carcinoma (BCC), Triple-Negative Breast Cancer (TNBC), Microsatellite Instability-High or Mismatch Repair Deficient Cancer and Microsatellite Instability-High or Mismatch Repair Deficient Colorectal Cancer (CRC).

[0079] According to a specific embodiment, the cancer is colon cancer.

[0080] According to a specific embodiment, the cancer is colon carcinoma.

[0081] According to a specific embodiment, the cancer is breast cancer.

[0082] According to a specific embodiment, the cancer is adenocarcinoma.

[0083] According to specific embodiments, the cancer is a respiratory tract tumor.

[0084] According to other specific embodiments, the cancer does not comprise a respiratory tract tumor.

[0085] According to specific embodiments, the cancer is refractory to treatment with a checkpoint inhibitor. This may present as innate resistance (also known as primary resistance, meaning that the patient does not respond at all to treatment) or an acquired resistance (also known as secondary resistance, meaning that patient initially responds to treatment but develops resistance later on).

[0086] According to specific embodiments, the cancer is in a relapsed state after a previous treatment with a checkpoint inhibitor.

[0087] According to specific embodiments, the cancer is in a prolonged stable disease state after a previous treatment with a checkpoint inhibitor. As used herein the term “checkpoint inhibitor” refers to a molecule that inhibits the activity of one or more immune checkpoint proteins, resulting in activation of an immune cell.

[0088] As used herein the term “immune checkpoint protein” refers to an antigen independent protein that regulates an immune cell activation or function in response to an antigen. Immune checkpoint proteins can be either co-stimulatory proteins (i.e., transmitting a stimulatory signal resulting in activation of an immune cell) or inhibitory' proteins (i.e., transmitting an inhibitory signal resulting in suppressing activity of an immune cell). According to some embodiments, the immune check-point protein regulates activation or function of a T cell. Numerous checkpoint proteins are known in the art and include, but not limited to, PD-1, PD-L1, A2aR, B7-H2, B7-H3, B7-H4, CTLA-4, CD73, CD80, CD86, LAG-3, TIM-3, NKG2A, PVRIG / PVRL2, CEACAM1, CEACAM 5 / 6, FAK, CCL2 / CCR2, LIF, KIR, IDO, CD 19, 0X40, 4-1BB (CD137), CD27, CD47 / SIRPa, CD70, CD40, CSF-1, GITR, CD28, IL-1, IL- IR3, IL-8, SEMA4D, Ang-2, CLEVER- 1, Axl, phosphatidylserine, and ICOS (CD278).

[0089] According to specific embodiments, the immune checkpoint protein is presented on an immune cell e.g., T cell or antigen-presenting cells.

[0090] According to specific embodiments, the immune checkpoint protein is presented on a cancerous cell.

[0091] According to specific embodiments, cells of the cancer present said immune checkpoint protein or a binding pair thereof (i.e., receptor or ligand) following administration of said UNO.

[0092] Thus, according to specific embodiments, the method comprises determining presentation of an immune checkpoint protein or a binding pair thereof in a biological sample obtained from the subject. Methods of determining expression and / or presentation are well known in the art and include PCR, Western blot, immunostaining, flow cytometry, and the like.

[0093] According to specific embodiments, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, CTLA-4, and LAG-3. According to specific embodiments, the immune checkpoint protein is PD-1. According to specific embodiments, the immune checkpoint protein is PD-L1. According to specific embodiments, the immune checkpoint protein is CTLA-4. According to specific embodiments, the immune checkpoint protein is LAG-3.

[0094] According to specific embodiments, the checkpoint inhibitor comprises an antibody.

[0095] Non-limiting examples of PD-L1 inhibitors include, without limitation, atezolizumab, durvalumab, avelumab, KN035, CK-301, AUNP12, CA-170 and BMS-986189; non-limiting examples of PD-1 inhibitors include, without limitation, pembrolizumab, nivolumab, cemiplimab, spartalizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, JTX-4014, INCMGA00012, AMP -224, and AMP-514; non-limiting examples of CTLA-4 inhibitors include, without limitation, ipilimumab, tremelimumab; non-limiting examples of LAG-3 inhibitors include, without limitation, relatlimab.

[0096] According to specific embodiments, the cancer is refractory to treatment with a PD-1 inhibitor.

[0097] According to specific embodiments, the cancer is refractory to treatment with a PD-L1 inhibitor.

[0098] According to specific embodiments, the cancer is refractory to treatment with a CTLA- 4 inhibitor.

[0099] According to specific embodiments, the cancer is refractory to treatment with a LAG-3 inhibitor.

[0100] According to specific embodiments, the cancer is in a relapsed state after previous treatment with a PD-1 inhibitor.

[0101] According to specific embodiments, the cancer is in a relapsed state after previous treatment with a PD-Ll inhibitor.

[0102] According to specific embodiments, the cancer is in a relapsed state after previous treatment with a CTLA-4 inhibitor.

[0103] According to specific embodiments, the cancer is in a relapsed state after previous treatment with a LAG-3 inhibitor.

[0104] According to specific embodiments, the cancer is in a prolonged stable disease state after a previous treatment with a PD-1 inhibitor.

[0105] According to specific embodiments, the cancer is in a prolonged stable disease state after a previous treatment with a PD-L1 inhibitor.

[0106] According to specific embodiments, the cancer is in a prolonged stable disease state after a previous treatment with a CTLA-4 inhibitor.

[0107] According to specific embodiments, the cancer is in a prolonged stable disease state after a previous treatment with a LAG-3 inhibitor.

[0108] According to specific embodiments, the cancer is positive for the microsatellite instability (MSI) [e.g., high microsatellite instability (MSI-H)] and / or the mismatch repair deficient (dMMR) marker.

[0109] According to other specific embodiments, the cancer is negative for the microsatellite instability (MSI) and / or the mismatch repair deficient (dMMR) marker. Methods of determining MSI / dMMR are well known in the art and include Nextgeneration sequencing (NGS), Fluorescent multiplex PCR and C, immunohistochemistry, single-molecule molecular inversion probes (smMIPs).

[0110] One of skill in the art will appreciate that the methods and uses provided herein for inhibiting abnormal growth of tumor cells or tissue and are for treating cancer may be generally applicable to all known or to-be-discovered cancerous cell phenotypes and cancerous growths.

[0111] According to specific embodiments, cells of the cancer present PD-L1 and / or CTLA- 4 and / or LAG-3 on their cell membrane.

[0112] Thus, according to specific embodiments, the method comprises determining presentation of one of PD-L1, CTLA-4, and / or LAG-3 in a biological sample obtained from the subject. Methods of determining expression and / or presentation are well known in the art and include PCR, Western blot, immunostaining, flow cytometry and the like.

[0113] The present embodiments relate to any size and shape of tumors, including large, spread and amorphic cancerous outgrowths.

[0114] The methods and uses provided herein may be especially useful for the treatment, control, and / or prevention of tumors (e.g., cancerous tumors) at localized sites, including inoperable tumors, tumors where localized treatment would be beneficial, and solid tumors.

[0115] According to some of any of the embodiments described herein, the methods and uses of the present embodiments are for inhibiting growth of cells of a primary tumor.

[0116] According to a specific embodiment, an efficacious treatment with UNO is considered when between about 10 % and about 100 % (e.g., 10-100 %, 15-100 %, 20-100 %, 25-100 %, 30-100 %, 35-100 %, 40-100 %, 45-100 %, 50-100 %, 55-100 %, 60-100 %, 65-100 %, 70- 100 %, 75-100 %, 80-100 %, 85-100 %, 90-100 %) of the cancerous cells may be killed by the gaseous nitric oxide over the course of one or more administrations, as described herein in any of the respective embodiments. UNO is defined as the delivery of gaseous nitric oxide in a preferably inert gas such as N2 at a concentration of between about 10,000 and 1,000,000 ppm, as is described in further detail hereinafter.

[0117] The methods of delivering UNO may include administration of UNO in a continuous or pulsed manner.

[0118] According to some embodiments of the present invention, the methods are affected by locally administering the UNO.

[0119] By “locally administering of UNO” it is meant directly contacting the tumor cells or tissue with UNO, such that UNO is applied directly to the tumor and / or its close vicinity. In some embodiments, local administration of UNO is affected intratumorally, by applying UNO directly into the tumor cells or tissue. In some embodiments, local administration (intra- tumoral) of UNO is affected by applying UNO to the surface of the tumor tissue, for example, by contacting the surface of the tumor with UNO. In some embodiments, local administration is affected by bringing UNO in close vicinity to the tumor cells or tissue, for example, directly or up to 2 cm, or up to 1 cm, from at least one and preferably all of the tumor surfaces.

[0120] According to some of any of the embodiments described herein, the UNO is locally administered to the primary tumor and / or to a metastasizing tumor.

[0121] Exemplary organs to which UNO can be locally administered according to some of any of the respective embodiments as described herein include, but are not limited to, the adrenal gland, bladder, bones, brain, breast, cervix, colon, colorectum, esophagus, gastrointestinal tract, heart, kidney, liver, large intestine, lungs, mouth, ovaries, pancreas, parathyroid, pituitary gland, prostate, salivary gland, skin, small intestine, spleen, stomach, thymus, thyroid, testicles, urinary tract, uterus, or vagina. According to some embodiments, the UNO is locally administered to the liver, in case of a primary liver cancer or of liver metastases.

[0122] According to some embodiments, the UNO is locally administered to a lymph node.

[0123] According to some embodiments, the UNO is locally administered to the colon, e.g., in case of a primary colon cancer or of colon metastases.

[0124] According to some embodiments, the UNO is locally administered to the breast, e.g., in case of a primary breast cancer or of breast metastases.

[0125] In some of any of the embodiments described herein, local administration is affected intratumorally, such that UNO is injected or otherwise delivered into the tumor.

[0126] In some of any of the embodiments described herein, local administration is affected by delivering UNO to a physiological space or cavity which contains at least a portion of the tumor tissue, such that the tumor is contacted with the UNO, and the UNO enters the tumor via e.g., diffusion.

[0127] According to some of any of the embodiments described herein for local administration of UNO is administered locally, as described herein.

[0128] In some of any of the embodiments described herein, the high dose of UNO is reflected by its relatively high concentration in the total amount of gas that is locally administered to the tumor and is presented by ppm (part per million) units.

[0129] In some of any of the embodiments described herein, the high dose of UNO is presented as its fraction, in ppm units, in the gas carrier. The gas carrier can be an inert gas such as N2 or argon (Ar), preferably N2. In some of any of the embodiments described herein, the high dose of UNO is reflected by the mass of UNO that is locally administered to the tumor, per a volumetric unit of the tumor.

[0130] According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, at a concentration of from about 10,000 ppm to about 1,000,000 ppm (1% to 100%), including any intermediate values and subranges therebetween, for example, from about 10,000 ppm to about 200,000 ppm, or from about 10,000 ppm to about 100,000 ppm, or from about 15,000 ppm to about 100,000 ppm, or from about 20,000 ppm to about 100,000 ppm, or from about 25,000 ppm to about 100,000 ppm, or from about 25,000 ppm to about 75,000 ppm, or from about 10,000 ppm to about 50,000 ppm, or from about 50,000 ppm to about 100,000 ppm, including any intermediate values and subranges between any of the foregoing, or is about 50,000 ppm.

[0131] In some of any of the embodiments described herein in the context of high dose of UNO, the high dose can be obtained by locally administering UNO at a dose of at least 10,000 ppm, or at least 20,000 ppm, or at least 50,000 ppm, and optionally up to about 1,000,000 ppm.

[0132] In exemplary embodiments, the UNO is administered, as described herein in any of the respective embodiments, at a concentration of about 20,000 to 200,000 ppm.

[0133] In exemplary embodiments, the UNO is administered, as described herein in any of the respective embodiments, at a concentration of about 20,000 to 100,000 ppm. In exemplary embodiments, the UNO is administered, as described herein in any of the respective embodiments, in a concentration of about 50,000 ppm.

[0134] In exemplary embodiments, the UNO is administered, as described herein in any of the respective embodiments, at a concentration of about 25,000 ppm.

[0135] In exemplary embodiments, the UNO is administered, as described herein in any of the respective embodiments, at a concentration of about 20,000 ppm.

[0136] In exemplary embodiments, the UNO is administered, as described herein in any of the respective embodiments, at a concentration of about 10,000 ppm.

[0137] In exemplary embodiments, the UNO is administered, as described herein in any of the respective embodiments, at a concentration of about 100,000 ppm.

[0138] In exemplary embodiments, the UNO is administered, as described herein in any of the respective embodiments, at a concentration of from about 100,000 ppm to about 200,000 ppm, or from about 200,000 ppm to about 500,000 ppm, or from about 500,000 ppm to about 1,000,000 ppm or from about 50,000 ppm or about 100,000 ppm or about 200,000 ppm or about 500,000 ppm or about 1,000,000 ppm. According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, at a volumetric flow rate of from about .01 mLPM to 20 mLPM. Preferably between .1 mLPM and 10 mLPM. Preferably between 1 mLPM and 5 mLPM. Preferably, 2 mLPM.

[0139] According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, at a volumetric flow rate of from about .01 mLPM to 10 mLPM. Preferably between .1 mLPM and 10 mLPM. Preferably between 1 mLPM and 5 mLPM. Preferably, 2 mLPM.

[0140] According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, at a volumetric flow rate of about 0.5 mLPM. According to some of any of the embodiments described herein, the UNO is administered for a time period that ranges from about 0.1 seconds to about 10 hours, per administration, including any intermediate values and subranges therebetween. For example, the time period can be from about 0.1 second to about 1 hour, or from about 1 second to about 10 minutes, or from about 1 minute to about 10 minutes, or from about 10 seconds to about 10 minutes, or from about 0. 1 second to about 10 minutes, or from about 30 seconds to about 3 minutes, or from about 1 minute to about 30 minutes, or from about 10 minutes to about 60 minutes, or from about 60 minutes to about 180 minutes, or from about 180 minutes to about 600 minutes, including any intermediate values and subranges between any of foregoing, or it can be about 30 seconds, about 10 minutes, about 30 minutes, or about 60 minutes.

[0141] According to some of any of the embodiments described herein, the UNO is administered for a time period that ranges from about 30 seconds to about 30 minutes.

[0142] According to some of any of the embodiments described herein, the UNO is administered for about 10 minutes.

[0143] According to some of any of the embodiments described herein, the UNO is administered intermittently, i.e., more than once per day; e.g., such that a time of administration per day according to any of the respective embodiments described herein represents a sum of two or more separate administration periods, which may be of the same length or of different lengths.

[0144] In some of any of the embodiments, UNO can be administered, as described herein in any of the respective embodiments, at a dose of at least 10,000 ppm, and optionally up to about 1,000,000, or up to about 500,000 ppm, or up to about 200,000 ppm, or up to about 100,000 ppm, for a time period of from about 1 second to about 60 minutes at a volumetric flow rate (flow volume) of from about .01 mLPM to 20 mLPM, including any intermediate values and subranges between any of the foregoing. Preferably between .1 mLPM and 10 mLPM. Preferably between 1 mLPM and 5 mLPM. Preferably, 2 mLPM.

[0145] In some of any of the embodiments, UNO can be administered, as described herein in any of the respective embodiments, at a dose of at least 10,000 ppm, and optionally up to about 1,000,000, or up to about 500,000 ppm, or up to about 200,000 ppm, or up to about 100,000 ppm, for a time period of from about 1 second to about 60 minutes at a volumetric flow rate (flow volume) of from about .01 mLPM to 10 mLPM, including any intermediate values and subranges between any of the foregoing. Preferably between .1 mLPM and 10 mLPM. Preferably between 1 mLPM and 5 mLPM. Preferably, 2 mLPM.

[0146] In some of any of the embodiments, UNO can be administered at a dose of at least 10,000 ppm, or at least 20,000 ppm, or at least 50,000 ppm, and optionally up to about 1,000,000 ppm, for a time period of at least 1 second, or at least 10 seconds, or at least 30 seconds, or at least 1 minute, and optionally up to about 60 minutes, at a volumetric flow rate (flow volume) of at least .01 mLPM and optionally up to about 20 mLPM, including any intermediate values and subranges between any of the foregoing. Preferably between .1 mLPM and 10 mLPM. Preferably between 1 mLPM and 5 mLPM. Preferably, 2 mLPM.

[0147] In some of any of the embodiments, UNO can be administered at a dose of at least 10,000 ppm, or at least 20,000 ppm, or at least 50,000 ppm, and optionally up to about 1,000,000 ppm, for a time period of at least 1 second, or at least 10 seconds, or at least 30 seconds, or at least 1 minute, and optionally up to about 60 minutes, at a volumetric flow rate (flow volume) of at least .01 mLPM and optionally up to about 10 mLPM, including any intermediate values and subranges between any of the foregoing. Preferably between .1 mLPM and 10 mLPM. Preferably between 1 mLPM and 5 mLPM. Preferably, 2 mLPM.

[0148] Alternatively, or in addition, the amount of UNO administered to the tumor ranges from about 0.1 mg to about 300 mg, per cm3tumor, per administration. The parameters of the UNO concentration (ppm), volumetric flow rate (LPM) and time to achieve a desired mass of UNO, and vice versa, the UNO mass achieved by administering UNO at a concentration, volumetric flow rate and time, can be calculated using the known ideal gas equation, PV=nRT, wherein P is the pressure, V is the volume, n is the number of moles, R is the gas constant and T is the temperature.

[0149] More specifically, these relations can be calculated or converted one to the other using the following equations: 1) X = y x 10“6

[0150] 2) V = V x t

[0151] V x 10“3x 101325

[0152] 3) n = -

[0153] 8.314 X 298

[0154] 4) m = n x X x 30.01 x 103

[0155] ( x t) x 10“3x 101325

[0156] 5) m = ( ) X (y X 10“6) x 30.01 x 10

[0157] 8.314 X 298 y is the concentration in ppm units;

[0158] X is the concentration in molar fraction units, and equation 1 presents the relation between molar fraction and ppm (y);

[0159] V is the volume, V is the volumetric flow rate in LPM and t is the time in minutes, and equation 2 presents the relation between Volume, volumetric flow rate and time;

[0160] Equation 3 is the Ideal gas equation, and the 10'3factor is added for transformation from Liters to m3;

[0161] Equation 4 presents the relation between the mass (m) the mole number (n), the molar fraction X and Nitric Oxide molar mass (30.01 grams / mol); and

[0162] Equation 5 is a combination of equations 1-4 into a single equation, reflecting the relation between the mass of UNO and the molar fraction, volumetric flow rate and time. The 103is for obtaining the mass m on a milligram (mg) scale.

[0163] Thus, for example, at a volumetric flow rate of 0.5 mLPM, time of 1 minute and concentration of 50,000 ppm, using equation 5 above, about .03 mg UNO is administered.

[0164] According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, at a concentration of from about 10,000 ppm to about 1,000,000 ppm (1% to 100%), preferably from about 10,000 ppm to about 500,000 ppm, or from about 10,000 ppm to about 100,000 ppm, or at about 50,000 ppm; at a volumetric flow rate of from about 0.01 mLPM to about 20 mLPM. Preferably between .1 mLPM and 10 mLPM. Preferably between 1 mLPM and 5 mLPM. Preferably, 2 mLPM.

[0165] According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, at a concentration of from about 10,000 ppm to about 1,000,000 ppm (1% to 100%), preferably from about 10,000 ppm to about 500,000 ppm, or from about 10,000 ppm to about 100,000 ppm, or at about 50,000 ppm; at a volumetric flow rate of from about 0.01 mLPM to about 10 mLPM. Preferably between .1 mLPM and 10 mLPM. Preferably between 1 mLPM and 5 mLPM. Preferably, 2 mLPM. According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, at a concentration (dose) of from about 20,000 ppm to about 100,000 ppm or from about 20,000 ppm to about 50,000 ppm; for a time period that ranges from about 30 seconds to about 30 minutes; at a volumetric flow rate (flow volume) of from about 0.1 mLPM to about 20 mLPM, per administration. Preferably between .1 mLPM and 10 mLPM. Preferably between 1 mLPM and 5 mLPM. Preferably, 2 mLPM.

[0166] According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, at a concentration (dose) of from about 20,000 ppm to about 100,000 ppm or from about 20,000 ppm to about 50,000 ppm; for a time period that ranges from about 30 seconds to about 30 minutes; at a volumetric flow rate (flow volume) of from about 0.1 mLPM to about 10 mLPM, per administration. Preferably between .1 mLPM and 10 mLPM. Preferably between 1 mLPM and 5 mLPM. Preferably, 2 mLPM.

[0167] According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, in an amount of no more 1 mg UNO per 100 mm3tumor volume, per administration, so as to avoid possible damage to healthy tissues adjacent to, or surrounding, the treated tumor.

[0168] According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, in an amount of about 250 mg per cm3tumor, per administration.

[0169] According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, in an amount of from about 0.01 mg to about 100 mg, or from about 0.1 to about 10 mg per a tumor of 20 mm3or less, including any intermediate values and subranges therebetween.

[0170] According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, in an amount of from about 0.1 to about 300 mg including any intermediate values and subranges therebetween, per 1 cm3tumor, per administration. For example, the UNO is administered in an amount of from about 0.1 mg to about 250 mg, or from 0. 1 mg to about 100 mg, or from 1 mg to about 50 mg, or from about 1 mg to about 100 mg, or from about 1 mg to about 300 mg, of from about 50 mg to about 100 mg, or from about 50 mg to about 300 mg, or from about 100 mg to about 150 mg, or from about 100 mg to about 300 mg, or from about 10 mg to about 100 mg, or of from about 10 mg to about 250 mg, or from about 0.1 mg to about 10 mg, or from about 10 mg to 200 mg, including any intermediate values and subranges of any of the foregoing, per 1 cm3tumor, per administration.

[0171] According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, to a tumor having a volume of up to 20 mm3, and an amount of UNO that is administered as described herein in any of the respective embodiments is from about 0.001 mg to about 10 mg, or from about 0.01 mg to about 20 mg, or from about 0.01 mg to about 2 mg, or from about 0. 1 mg to about 1.0 mg, or from about 0.2 mg to about 0.8 mg, including any intermediate values and subranges between any of the foregoing, per administration. In other embodiments, the tumor has a volume greater than 20 mm3.

[0172] Without being bound by any particular theory, it is assumed that a high dose (concentration or amount) as described herein in any of the respective embodiments, inhibits the growth of tumor cells, reduces tumor volume and / or stimulates an anti-tumor immune response, as described herein in any of the respective embodiments, without causing a harmful effect to healthy tissues in the vicinity of the tumor.

[0173] For any of the embodiments described herein for administration of UNO, the administration can be either continuous or pulsed, such that for each administration, the indicated dose of UNO is administered either continuously or in a pulsed manner. When the dose is referred to in ppm units, each pulse is at the indicated dose concentration, as described herein in any of the respective embodiments. When the dose is referred to as the total mass per administration, the indicated dose is divided into pulses.

[0174] According to some of any of the embodiments described herein, UNO is pulsed from about 2 to about 50 times, or from about 2 to about 30 times, or from about 2 to about 20 times, or from about 2 to about 15 times, or from about 5 to about 15 times, including any intermediate values and subranges therebetween, or about 10 times, per administration.

[0175] According to some of any of the embodiments described herein, each pulse is between 10,000 ppm and about 1,000,000 ppm of UNO, at a volumetric flow rate (flow volume) of from about 0.1 mLPM to about 20 mLPM, wherein each pulse is, independently, between about 0.1 second and about 30 minutes per pulse with a break of from about 0.1 second to about 30 minutes between pulses.

[0176] According to some of any of the embodiments described herein, each pulse of UNO is, independently, from about 10 seconds per pulse to about 45 seconds per pulse, including any intermediate values and subranges therebetween. According to some of any of the embodiments described herein, each pulse of UNO is about 30 seconds per pulse.

[0177] According to some of any of the embodiments described herein, UNO is not administered between pulses and the time between each two pulses is, independently, from about 1 second to about 300 seconds, or from about 1 second to about 200 seconds, or from about 1 second to about 100 seconds, or from about 1 second to about 50 seconds, or from about 10 seconds to about 50 seconds, including any intermediate values and subranges therebetween, or is about 20 seconds. According to some of any of these embodiments, the ratio between the time of UNO pulsed administration and the resting time between pulses ranges from 1 :2 to 1 :5. For example, for each pulse of UNO administration for 5 seconds, a following resting time is independently from 10 to 50 seconds. Preferably, the UNO is pulsed such that about 33 % of the time UNO is delivered and 66 % of the time is resting or waiting time between pulses.

[0178] According to some of any of the embodiments described herein, the UNO is administered, as described herein in any of the respective embodiments, at two or more administration sites in or on the tumor (depending in the administration mode). In some of these embodiments, the distance between the two administration sites is, independently, from about 2.5 mm to about 1 cm, or from about 0.25 cm to about 0.5 cm, including any intermediate values and subranges therebetween.

[0179] When UNO is administered to two or more tumor sites, each administration is at the ppm dose or mass amount indicated herein in any of the respective embodiments, or the total mass (amount) administered to all tumor sites is as indicated herein in any of the respective embodiments.

[0180] According to some of any of the embodiments described herein, the method further comprises scavenging excess UNO from the one or more administration sites, as described in further detail hereinunder. In exemplary embodiments, the scavenging comprises applying a reduced pressure (vacuum) around the administration site(s).

[0181] According to some of any of the embodiments described herein for high dose administration of UNO, the administration is performed one or more times per a treatment session.

[0182] In some embodiments, it is performed once during a treatment session. In some embodiments, it is performed twice, thrice or more times during a treatment session. In some of these embodiments, the administration is performed once daily during the treatment session. Preferably, the administration is performed such that a time interval between the two administrations is at least one day, or at least two days, or at least three days, or at least four days, or at least five days, or at least six days, or at least one week, during a treatment session.

[0183] The duration of a treatment session can be determined by skilled persons such as physicians, in accordance with the subject’s response to the treatment, that is, in accordance with the effect of the treatment on the growth of the cells of the primary and / or secondary tumor, as described herein. According to some of any of the embodiments described herein for administration of UNO, the administration is performed once a day, although two or more times a day are also contemplated.

[0184] According to some embodiments, the local administration of UNO comprises injecting, or exposing the outer layer of a tumor to, or filling a space or cavity containing a tumor with, a high dose UNO, and in some embodiments, the UNO is at a concentration ranging from about 10,000 ppm to about 1,000,000 ppm, preferably 25,000, or 50,000 ppm, or 100,000 ppm, as described herein in any of the respective embodiments. The local administration to the tumor is for a period of time from about 1 second to about 3 hours, depending on the size and location of the tumor, with a very low volume in the order of up to 0.1 LPM and preferably 0.01 LPM.

[0185] According to some of any of the embodiments described herein, in cases where the tumor is covered by skin, peritoneum, crust or any other thick layer, this layer can be removed prior to or during exposure of the tumor to the UNO local administration.

[0186] In any of the embodiments described herein in the context of UNO administration, the UNO may be provided by an external source, for example, a reservoir of UNO or a chemical generator of UNO. In some embodiments, UNO is provided by a reservoir of UNO, preferably of a small volume of, for example, a single administration dosage (that is, the dose of UNO used per a single administration, as described herein in any of the respective embodiments). Such reservoirs are described in further detail hereinunder.

[0187] The UNO is preferably of medical purity, that is, preferably at least about 95 %, more preferably at least about 99 %, and even more preferably at least about 99.5 % pure UNO. The UNO is preferably provided as a mixture of UNO and other gases, preferably an inert gas such as, for example, N2, and its ppm concentration is within the gas it is mixed with.

[0188] Embodiments of the present invention further relate to a system, which is also referred to herein interchangeably as “device”, which is configured for locally administering UNO to a tumor as described herein in any of the respective embodiments. Such a system is also referred to herein as a delivery system. Such a system is also described in WO 2021 / 105900 and, alternatively, WO 2024 / 243017, which are fully incorporated herein by reference. Generally, but not obligatory, a system for locally administering UNO can include a pressure regulator, a flow meter, optionally an exposure box or container, one or more delivery lines, which are optionally terminated by or connected to a delivery device or configuration through which the UNO is administered and further optionally, a NO and / or NOx (as defined hereinunder) detector. Purging the UNO delivery system with an inert gas, such as N2, may be desired.

[0189] The volume and / or flow rate of the administered (delivered) gas can be regulated by a mass flow controller (digital or analog), designed to deliver low volumes or flow rates of gas, from 0.01 mLPM to 20 mLPM per cm3of tissue, in accordance with any of the respective embodiments as described herein. Purging of the UNO delivery system, including purging the pressure regulator, flow meter and delivery lines can be performed before and / or after UNO local administration. The gas purge can preferably last at least 1 minute or until the NO and NOx (as described below) detectors read no signal. In exemplary embodiments, N2 is used as the purging gas at a flow rate of at least 0.1 mLPM.

[0190] According to some of any of the embodiments described herein, the delivery device is inserted into a body and advanced adjacent to an administration site, on or near tumor cells or tissue. When a delivery device or configuration is appropriately positioned, UNO is supplied and exits the device into the tumor or on or above a surface of the tumor, depending on the nature of the delivery device and the local administration mode of choice. In some embodiments, the delivery device extends against a tumor in order to form a seal, thus further reducing damage that may be caused by the UNO to adjacent normal cells that are outside of the area sealed off by the device.

[0191] The delivery device as described herein is meant to describe a component or configuration of the delivery system through which the gas exits the delivery' system and contacts the administration site (e g., the tumor or its close vicinity).

[0192] According to some of any of the embodiments described herein, administering UNO to a tumor as described herein in any of the respective embodiments can be accomplished by delivery device means such as one or more needles, including, for example, perforated needles, perforated spray needles, non-perforated and non-spray needles, umbrella needles, closed-tip needles, or other needles. The needles can optionally be nano-sized, micron-sized or macrosized needles (having a diameter of 1 mm or higher). Other delivery devices are described hereinunder. Embodiments in which needles are used are typically used when the UNO is locally administered intratumorally, e.g., by intra-tumoral injection. In this regard, the needle is inserted into the tumor. In certain embodiments, the needle can be inserted all the way through the tumor such that an exit hole is created and then retracted back to position the tip of the needle somewhere within the tumor. In other embodiments, the needle is inserted only to a position within the tumor, without creation of any exit hole.

[0193] According to some of any of the embodiments described herein, the administered UNO can be absorbed at least partially by the injected tumor. In some embodiments, the volumetric flow rate of the administered UNO can affect the eventual absorption by the injected tumor. In this regard, slower volumetric flow rates, e.g., less than 20 mLPM, can result in better absorption. According to an embodiment, the absorption of UNO can be determined using staining mechanisms that detect reactive nitrogen species, e.g., nitrotyrosine staining. According to another embodiment, the absorption can also be determined using nitrites, ascorbic acid, hydrogen peroxide, catecolamines, etc. Further, NO absorption can also be detected electrically, e.g., via NO sensor probes.

[0194] In some of any of the embodiments described herein in the context of a delivery system, the opening from which the UNO is delivered can be adjusted to the size of the tumor, so as to prevent damage to the area surrounding the cancer. In some embodiments, the opening does not exceed the size of the tumor. The methods of locally administering UNO to a tumor can include contacting at least a portion of the tumor with the gaseous nitric oxide. The methods of locally administering UNO to a tumor can further include subsequently removing gaseous nitric oxide and NOx gas molecules from the treated site during or after the administration step. NOx encompasses NO, when x is 1, and oxidized forms of NO, which can be formed when UNO is in contact with a physiological environment and / or the subj ect’ s environment, whereby x can be, for example, 2. Vacuuming the gas can be done in a pulsed or continuous manner, preferably synchronized with the UNO mode of administration.

[0195] A delivery system can include a full-body, or a differently sized chemical hood designed to evacuate excessive UNO or NOx during treatment, in a pulsed or continuous manner, preferably synchronized with the UNO mode of administration.

[0196] The delivery system can include an evacuation cylinder, which is connected directly to a regulator of the UNO tank. To purge the regulator safely, the evacuation cylinder can be filled with a gas accumulated in the regulator.

[0197] Another embodiment of controlling UNO includes the use of a one-way valve where disconnecting the regulator or flow meter from the cylinder locks the valve, thereby preventing gas release from a gas tank.

[0198] According to some of any of the embodiments described herein, the methods, uses and delivery systems as described herein utilize a UNO cylinder, optionally equipped with a gas regulator, and one or more valves, and further optionally, the cylinder further comprises a delivery device for executing the local administration. The delivery device is in fluid communication with the cylinder, preferably via the valves and gas regulator (e.g., flow controller). Alternatively, the cylinder comprises means to connect the delivery device to the cylinder, to obtain fluid communication therebetween. The delivery device can be, for example, a scope with an annular-shape or a needle or a device configured for spraying the tumor, or else, as described in further detail hereinunder.

[0199] According to some of any of the embodiments described herein, the UNO cylinder is a miniature or at least portable cylinder.

[0200] According to some embodiments, the cylinder is of a volume of less than 3 liters, or less than 1 liter, or less than 0.8 liter, or less than 0.75 liter, or less than 0.5 liter, or less than 0.3 liter.

[0201] According to some of any of the embodiments described herein, the methods and uses can involve scavenging UNO and optionally other gases, and the scavenging can be performed by applying vacuum so as to remove UNO and other gases from the administration site. A delivery system as described herein is configured, according to some embodiments, as being capable of scavenging UNO from an administration site.

[0202] A delivery system according to some of the present embodiments can additionally or alternatively comprise one or more, preferably two, vacuum devices for scavenging gaseous nitric oxide and other gases that may form during the local administration. The vacuum devices can be placed or held above, or distal to, the tumor, or to the delivering device, during administration, for example, about 15 cm away. The vacuum devices can vacuum all the gases from the area at a rate of at least about 50 LPM. The vacuuming of the gas can be done in a pulsed or continuous manner, preferably synchronized with UNO administration. Purging the UNO delivery system, for example, with N2, before and / or after can also be performed. Purging can also be performed intermittently during the procedure.

[0203] According to some embodiments, a chemical hood is placed above the tumor or tumor mass and used to apply vacuum and scavenge UNO. The hood can be placed about 15 cm above the tumor. The hood can vacuum gas at a flow rate of at least about 50 LPM.

[0204] A whole-body chemical hood that can accommodate the patient’s body can also be used. The patient’s head can be placed outside the hood to minimize the risk of breathing NOx molecules. UNO can be delivered as described herein. The vacuum system can exchange the gas at a flow rate of at least 50 LPM. In each of the embodiments that relate to a vacuum application, a NO and NOx filtering pump can be placed in the discharge line. A soda lime filter, such as a Sofnolime filter, or a similar filter that can absorb NOx molecule, can be used. In another embodiment, the NOx filter can be potassium permanganate, sodium hydroxide solution, metal oxides, activated charcoal, or alkaline activated carbon filter. In each instance, vacuuming can be done in a pulsed or continuous manner, preferably synchronized with UNO administration or not.

[0205] According to some of any of the embodiments described herein, the delivery system is configured for delivering UNO to one or more administration sites by a positive pressure gradient, and scavenging UNO from the one or more administration sites by a negative pressure gradient. In this way, the delivery system may deliver UNO to one or more administration sites with reduced or nullified damage to collateral host cells.

[0206] It is to be noted that a certain level of damage to collateral cells may be tolerated, and that the conditions under which the UNO gas is administered may be optimized to decrease damage to collateral cells while also providing the therapeutic effects described herein.

[0207] According to some of any of the embodiments described herein, the delivery system comprises a gas supply passage in fluid communication with gas supply openings. The delivery system can further comprise an exhaust passage in fluid communication with an exhaust opening. The delivery system can comprise one or more gas supply openings and / or one or more exhaust openings, and / or one or more gas supply passages and / or one or more exhaust passages.

[0208] An exemplary system for the local administration and scavenging of gaseous nitric oxide comprises a container or box, as illustrated, for example in FIG. 22 of W02021 / 105901 and described in further detail hereinunder, that can be filled with UNO at a volume of at least 0.5 LPM supplied from a tank (a gas reservoir). The container can have 2 or more holes, or ports. A first hole or port can be an input hole, sized to allow insertion of at least a portion of the tumor or of a bodily organ containing the tumor. An output hole can be connected to a discharge conduit or pipe that can remove or evacuate excessive NOx gases out from the box to the outside air, avoiding or minimizing the risk of contaminating the room and overexposing staff and the treated subjects (patients). Applying a vacuum can be in a pulsed or continuous manner, preferably synchronized with UNO administration.

[0209] An exemplary delivery system can comprise a small-bore inner cannula that delivers an adequate dose of UNO to the target site, for example, a target site of from about 1 mm2to about 2 cm2in size. The delivery system can further comprise an outer lumen through which a vacuum may be applied to scavenge excess UNO away from tumor cells and tissue that surround and border the tumor site. Such a configuration allows locally administering UNO to the target site (a tumor), without excessive damage to healthy host tissues.

[0210] In an exemplary method, a tumor or a portion thereof is inserted into a hole that substantially matches the tumor’s diameters. An additional output pore in the box enables lowering the pressure. The excessive gas is cleared up from the box through this output pore as described above. For example, the gas can flow into the box containing the tumor for 2 seconds followed by a suction of the gas for 2 seconds.

[0211] An exemplary delivery system comprises an outer lumen or cannula, trocar, tube, etc. An inner lumen or cannula, tube, etc. can be disposed coaxially inside of the outer lumen. The inner lumen is disposed approximately centrally in the outer lumen, although other configurations are also contemplated.

[0212] In some embodiments of such an exemplary delivery system, a space, preferably an exhaust space, is between the outer lumen and inner lumen. The exhaust space can be annular or may take any other configuration and / or geometry. A tip can be attached to the inner lumen at the distal end and is in fluid communication with the inner lumen. In some embodiments, the tip comprises a wire mesh or screen that accesses the space inside of the inner lumen. The delivery device may be advanced to an administration site in the retracted configuration. In some embodiments, the tip is rounded and seals the outer lumen when it is in the retracted position, thus easing insertion of the tip into the body where it is brought adjacent to an administration site.

[0213] When the tip of the system is brought adjacent to an administration site, the system is adjusted to its extended configuration in order to affect the administration of the UNO. In the extended configuration, an exhaust path is opened between the distal end of the outer lumen and the tip. UNO is delivered through the inner lumen. The UNO exits the inner lumen at the tip that is in fluid communication with the inner lumen. A wire mesh or screen at the distal end of the tip may assist in diffusing the UNO gas as it exits the device. The exhausted UNO gas returns to the device at the exhaust path. In embodiments, a vacuum is applied to the exhaust space between the outer lumen and the inner lumen in order to attract the exhausted UNO. The exhausted UNO is then brought through the exhaust space to exit the body and be disposed of appropriately. In this way, the device is capable of scavenging UNO from an administration site.

[0214] In an alternative configuration, the flow of UNO could be reversed such that UNO is delivered through the space between the outer lumen and inner lumen and removed from the administration site through the inner lumen. In this alternative configuration, a vacuum can be applied to the inner lumen and a positive pressure of UNO is applied to the space defined by the inner and outer lumens. In another alternative, the tip is permanently secured to the outer lumen as well as the inner lumen, such that the tip does not have retracted and extended configurations. In this alternative, permanent passages are provided in the outer lumen for UNO to be expelled from the device or sucked into the device by vacuum. For example, the permanent passages could be small holes or slits radially disposed around the outer lumen, preferably near the distal end of the outer lumen so as to be near the tip of the device.

[0215] According to some of any of the embodiments described herein, a delivery device is attached to the end of an endoscope or bronchoscope (e.g., a blue-fluorescence endoscope or bronchoscope) so that the insertion of the delivery device into the body, advancement towards the administration site, and retraction from the body can be visually observed by or otherwise made known to the operator or someone working in concert with the operator. Alternatively, the delivery device can be attached to a guidewire to insert, advance, and retract the device more effectively. Additionally, the device can be coated with a fluoroscopic material or have one or more fluoroscopic tags attached to it so that its insertion, advancement, and retraction could be fluoroscopically observed.

[0216] In some embodiments, a system for delivering UNO to and scavenging NOx from one or more administration sites is intended to fit over the distal end or tip of an endoscope or bronchoscope. The device can be, for example, annular-shaped, having a hole in or about its center and is approximately circular in shape. The hole is preferably sized to accommodate the distal end of an endoscope or bronchoscope. For example, the hole is from about 0.5 cm to about 10 cm in diameter. The hole can be sized so as to fit snugly over the distal end of an endoscope or bronchoscope.

[0217] According to some of any of the embodiments described herein, the delivery system comprises a double-needle system in which a suction needle is located adjacent to or proximal to the gas delivery needle (e.g., a distance of between 3 mm and 1 cm). The suction needle can decrease or maintain intra-tumoral pressure. Further, two or more needles can be spaced at least about 2 mm apart. UNO can be delivered at a flow rate of at least about 0.01 mLPM as described herein. The needles can be designed to have holes along the length of the needle. In example, the diameter of the holes is about 1 mm and disposed every 2 mm. The needles can be disposed within a lumen, placed outside of the tumor mass, while the shaft of the needle can be placed inside the tissue. The length of the needles can be selected to be at least half the tumor’s longest dimension. Vacuuming gas through one or more suction needles or holes can be done in a pulsed or continuous manner, preferably synchronized with UNO administration. For example, the UNO can flow into the tumor for 2 seconds followed by applying a vacuum or suction for 2 seconds. One or more, such as a plurality, of needles or an array of needles, such as nano-sized or micron-sized needles can be used. In some embodiments, the gas is injected into the tumor by means of an array of needles with a spacing of about 0.5 cm. The ratio of suction needles and delivery needles can be 1 : 10 to 10: 1, preferably 1 : 1. The suction needles can be designed to remove less than about 1 LPM per cm gas or fluid.

[0218] According to some of the any of the embodiments described herein, UNO is applied to a targeted tumor through one or more intra-tumoral channels. For example, a channel 2 mm in width respective to every 4 mm of tissue can be formed through which gas can be delivered, for example at a flow rate higher than 0.01 mLPM, directly to the tumor mass by a needle that is placed at the center of each channel, while the gas is cleared up from a scavenging channel. The flow rate in the scavenging channel can be lower than the delivery channel, such as at least 0.001 mLPM less than the UNO delivery flow rate. The vacuuming can be accomplished through suction needles or hoses and can be pulsed or continuous, preferably synchronized with UNO administration. For example, the UNO can flow into the tumor for 2 seconds followed by a suction of the gas for 2 seconds.

[0219] According to some of the any of the embodiments described herein, UNO is sprayed onto a targeted tumor, optionally when a method as described herein is used in combination with surgical treatment (e.g., tumor resection) and / or in combination with UNO intra-tumoral injection and / or when the tumor is inoperable, flat or amorphous.

[0220] According to some of any of the embodiments described herein, exemplary methods of treatment with UNO can also include: (i) monitoring a pressure parameter associated with the tumor; and (ii) controlling the volumetric flow rate of the locally administered UNO such that the monitored pressure parameter remains below a predetermined value. The pressure parameter is the intra-tumoral pressure of the tumor. According to an embodiment, the pressure parameter can be monitored using a flow control module as illustrated in FIGS. 1 A-1C of WO 2024 / 243017. In this regard, the flow control module is a mass flow controller (digital or analog), which monitors the intra-tumoral pressure via the back pressure. In some embodiments, the predetermined value is an absolute pressure between 1 and 2 ATM, i.e., approximately 14.7 pounds per square inch (PSI) to approximately 29.4 PSI. For example, the predetermined value can be 16.7 PSI. Upon determining that the predetermined value has been reached, UNO administration is ceased until the monitored pressure parameter falls, for example, to 1 ATM (about 14.7 PSI), at which point the local administration is restarted. In another embodiment, instead of ceasing UNO administration, the volumetric flow rate of the locally administered UNO can be slowed down / reduced upon determining that the predetermined value has been reached. In this regard, the volumetric flow rate can be decreased (gradually or immediately) to a lower volumetric flow rate until the pressure parameter falls, for example to about an absolute value of 1 ATM is reached, at which point the volumetric flow rate is returned to the original value. For example, if the original volumetric flow rate is 2 mLPM, then the volumetric flow rate can either reduced (gradually or immediately) to a lower volumetric flow rate, e.g., .2 mLPM, until 1 ATM is reached, after which the volumetric flow rate may return (gradually or immediately) to the original value.

[0221] Further, according to another embodiment, after the volumetric flow rate of the locally administered UNO is slowed down / reduced, the absolute pressure can be monitored and based on the monitored pressure, the volumetric flow rate can be maintained or ceased. For example, the volumetric flow rate can be maintained if the monitored pressure plateaus, and ceased if the monitored pressure increases.

[0222] Further, according to an embodiment, the volumetric flow rate of the UNO can also be controlled until (i) a total UNO administration time has elapsed, (ii) a total operation time has elapsed, or (iii) a total volume of UNO has been administered, after which UNO administration ceases. In this regard, UNO can be administered either continuously or intermittently during the total operation time. For example, the total UNO administration can be 10 minutes, the total operation time can be 60 minutes, and the total volume can be 20 mL. As such, if either the total UNO administration time, e.g., 10 minutes, or total volume, e.g., 20 mL, is reached before the end of the total operation time, e.g., 60 minutes, then the UNO administration will cease. Otherwise, UNO administration will cease at the end of the total operation time.

[0223] The present invention provides methods to treat cancer by a combined treatment comprising one or more checkpoint inhibitors.

[0224] According to specific embodiments, treatment with UNO and a checkpoint inhibitor has a combined improved anti-cancer or anti-tumor activity. As used herein the phrase "combined improved anti-cancer or anti-tumor activity" refers to at least additive but also synergistically improved anti-cancer or anti-tumor activity as compared to treatment with each of the agents when administered as a single agent, which may be determined by the effect on e.g., tumor size, tumor regression, symptoms of the disorder or subject’s survival.

[0225] According to some embodiments, the combined improved anti-cancer or anti-tumor activity can be improved by increasing the absorption of UNO in the treated tumor. The checkpoint inhibitor of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

[0226] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0227] Herein the term "active ingredient" refers to the checkpoint inhibitor accountable for the biological effect.

[0228] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

[0229] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0230] Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0231] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0232] According to specific embodiments, the pharmaceutical composition is administered intravenously.

[0233] According to specific embodiments, pharmaceutical composition is administered as an intravenous infusion over 20-60 minutes e.g., 30 minutes after dilution.

[0234] Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.

[0235] Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

[0236] According to specific embodiments, the pharmaceutical composition is administered intratumorally or in close vicinity to the tumor.

[0237] Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0238] Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0239] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0240] For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0241] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0242] Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0243] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0244] For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0245] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0246] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0247] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

[0248] The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0249] Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

[0250] In some of any of the embodiments described herein, co-administering of UNO and a checkpoint inhibitor allows the administration of the UNO and / or the checkpoint inhibitor at a sub-therapeutic dosage; which may, for example, reduce the adverse effects of the treatment.

[0251] Herein, the term “sub-therapeutic dosage” or “sub-therapeutic dose” refers to a dosage of an agent which is lower than a dosage of the agent effective (when administered alone) to prevent, alleviate or ameliorate symptoms of a disorder (e.g. cancer) or prolong the survival of the subject being treated; for example, a dosage lower than a dosage of the agent recognized in the art to be effective (when administered alone) for such a purpose, or a dosage that was shown to be effective (when administered alone) to prevent, alleviate or ameliorate symptoms of a disorder (e.g., a tumor) or prolong the survival of a specific subject. In other words, the term “sub-therapeutic dosage” or “sub-therapeutic dose” refers to a dosage of an agent which is lower than a therapeutically effective amount of the agent (when administered alone) to the subject, as defined herein.

[0252] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0253] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0254] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

[0255] Dosage amount and interval may be adjusted individually to provide levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0256] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0257] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0258] According to specific embodiments, the checkpoint inhibitor is administered in a dose of 1 - 10 mg / kg.

[0259] According to specific embodiments, the checkpoint inhibitor is administered in a dose of 100 - 500 mg (e.g., 200 mg or 400 mg).

[0260] Below are non-limiting exemplary known protocols for administration of a checkpoint inhibitor, e.g., pembrolizumab, that can be used with specific embodiments of the invention: Melanoma: 200 mg every 3 weeks or 400 mg every 6 weeks; 2 mg / kg (up to 200 mg) every 3 weeks for pediatrics; NSCLC: 200 mg every 3 weeks or 400 mg every 6 weeks; HNSCC: 200 mg every 3 weeks or 400 mg every 6 weeks; cHL or PMBCL: 200 mg every 3 weeks or 400 mg every 6 weeks for adults; 2 mg / kg (up to 200 mg) every 3 weeks for pediatrics; Urothelial Carcinoma: 200 mg every 3 weeks or 400 mg every 6 weeks; MSI-H or dMMR Cancer: 200 mg every 3 weeks or 400 mg every 6 weeks for adults; 2 mg / kg (up to 200 mg) every 3 weeks for pediatrics; MSI-H or dMMR CRC: 200 mg every 3 weeks or 400 mg every 6 weeks; MSI-H or dMMR Endometrial Carcinoma: 200 mg every 3 weeks or 400 mg every 6 weeks; Gastric Cancer: 200 mg every 3 weeks or 400 mg every 6 weeks; Esophageal Cancer: 200 mg every 3 weeks or 400 mg every 6 weeks; Cervical Cancer: 200 mg every 3 weeks or 400 mg every 6 weeks; HCC: 200 mg every 3 weeks or 400 mg every 6 weeks; MCC: 200 mg every 3 weeks or 400 mg every 6 weeks for adults; 2 mg / kg (up to 200 mg) every 3 weeks for pediatrics; RCC: 200 mg every 3 weeks or 400 mg every 6 weeks as a single agent in the adjuvant setting, or in the advanced setting with either: oaxitinib 5 mg orally twice daily or o lenvatinib 20 mg orally once daily; Endometrial Carcinoma: 200 mg every 3 weeks or 400 mg every 6 weeks with lenvatinib 20 mg orally once daily; TMB-H Cancer: 200 mg every 3 weeks or 400 mg every 6 weeks for adults; 2 mg / kg (up to 200 mg) every 3 weeks for pediatrics; cSCC: 200 mg every 3 weeks or 400 mg every 6 weeks; TNBC: 200 mg every 3 weeks or 400 mg every 6 weeks.

[0261] In some of any of the embodiments described herein, administration of the UNO may optionally be performed prior to, subsequent to, and / or concomitant with administering the checkpoint inhibitor.

[0262] According to specific embodiments, the checkpoint inhibitor is administered prior to the UNO.

[0263] According to specific embodiments, the checkpoint inhibitor is administered at least once prior to the UNO followed by administration concomitantly and / or subsequently to the UNO.

[0264] According to specific embodiments, the checkpoint inhibitor is administered at least two, at least three, at least 4 or at least 5 times.

[0265] According to specific embodiments, the checkpoint inhibitor is administered every 1-50 days, every 1-30, every 1-21 days, every 2-10 days or every 2-5 days.

[0266] According to specific embodiments, the checkpoint inhibitor is administered every day or every two days. According to specific embodiments, the checkpoint inhibitor is administered every two days.

[0267] According to specific embodiments, the checkpoint inhibitor is administered every 1 - 10 weeks, every 2-8 weeks or every 3-6 weeks.

[0268] According to specific embodiments, the checkpoint inhibitor is administered every week, every two weeks, every three weeks, every four weeks, every 5 weeks or every 6 weeks.

[0269] Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

[0270] The term throughout "about" as used herein when referring to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass variations of + / - 20% or + / - 10% from the specified amount, as such variations are appropriate to perform the disclosed method. In embodiments, the term “about” is meant to encompass variations of + / - 5%. In embodiments, the term “about” is meant to encompass variations of + / - 1%. In embodiments, the term “about” is meant to encompass variations of + / - 0.1%.

[0271] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0272] The term “consisting of’ means “including and limited to”.

[0273] The term "consisting essentially of' means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0274] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0275] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0276] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0277] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0278] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0279] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques.

[0280] EXAMPLE 1

[0281] AMOUNT OF UNO ABSORBED BY TUMOR TISSUE

[0282] The amount of UNO being absorbed by tumor tissue was evaluated. Specifically, the absorption of UNO by concentration (25,000 ppm v. 50,000 ppm) and type of applicator (23 gauge v. fenestrated needle) for live mouse tumors were evaluated. The amount of UNO absorbed was determined using a dilution system which collected and diluted the nitric oxide down (e.g., by 20-100x) to the level current nitric oxide sensors (e.g., electrochemical) can measure (e.g., <800 ppm, normally calibrated at 20-200 ppm) without becoming saturated. The resultant data is depicted in FIG. 1. Surprisingly, only a small percentage of UNO administered is being absorbed by the tumor tissue.

[0283] EXAMPLE 2

[0284] EFFICACY OF LOW VOLUMETRIC FLOW RATE OF UNO IN MICE

[0285] The efficacy and safety of 2.5 minutes of low volumetric flow rate administration of 100,000 ppm of UNO at 0.5 mLPM administration on the primary CT26 tumor when combined with anti-mPD-1 mAb at 5 mg / kg was evaluated.

[0286] Methods: Mice were inoculated with CT26 cells to the right flank (primary inoculation). Ten days following primary tumor inoculation, CT26 tumor-bearing mice were treated in situ on the primary tumor with UNO 100,000 ppm delivery at 0.5 mLPM for 2.5 minutes. A control group contained mice treated for 2.5 minutes with 0.5 mLPM of nitrogen. All mice were administered with six doses of murine anti-PD-1 (anti-mPD-1) monoclonal (mAb) at 5 mg / kg.

[0287] Test Items: UNO (10%) stabilized in nitrogen (90%).

[0288] Test Animals: Ten BALB / c mice, female, 10 weeks of age. Immune Checkpoint Inhibitor: anti-PD-1 mAb.

[0289] Mice were administered anti-mPD-1 mAb 6 times, with anti-PD-1 mAb at concentration of 5 mg / kg, intraperitoneally using a 1 ml syringe with a 27G needle. The injection was administered intraperitoneally to approximately the same location each time.

[0290] Results: Primary tumors of CT26 tumor-bearing mice were treated with intra-tumoral delivery of 100,000 ppm of UNO or nitrogen at a flow of 0.5 mLPM, for 2.5 minutes, ten days following primary tumor inoculation. Flow of 0.5 mLPM (with either 100,000 ppm of UNO or nitrogen) was well tolerated by all mice. Both groups were administered 6 injections of 5 mg / kg anti-mPD-1 mAb, beginning one day after UNO / nitrogen treatment, and continuing every three days until the 6thinjection. The volume of the treated tumors was monitored by measuring the length and width of the tumor three times per week using a caliper. Tumor-bearing mice treated with 100,000 ppm of UNO, combined with anti-mPD-1 mAb injections, displayed a higher percentage of complete primary tumor regression, compared to the nitrogen and anti-mPD-1 mAb group: 2 out of 5 mice (40%) and 0 out of 5 mice (0%), respectively (FIGS. 2A and 2B). Further, monitoring of mice survival for 50 days, based on clinical signs and tumor volume, shows improved survival of mice treated with UNO and anti-mPD-1 mAb compared to nitrogen and anti-mPDl mAb, 2 / 5 compared to 0 / 5 (FIG. 4).

[0291] In sum, treatment of 0.5 mLPM of 100,000 ppm of UNO or nitrogen was well tolerated by all mice. Further, combination treatment of .5 mLPM of UNO at 100,000 ppm for 2.5 minutes with 6 x 5 mg / kg anti-mPD-1 mAb injections showed an improved effect on the treated tumor volume (FIGS. 2A and 2B) and complete tumor regression (FIG. 3) compared to the control nitrogen and anti-mPD-1 mAb group. Mouse survival was also improved in the UNO arm compared to the nitrogen arm (FIG. 4).

[0292] EXAMPLE 3 COMPARING LOW VOLUMETRIC FLOW RATE TO HIGH VOLUMETRIC FLOW RATE DELIVERY OF UNO

[0293] Using the CT26 mouse model, the safety and efficacy of 2.5- and 5-minute administration of 100,000 ppm UNO treatment at 0.5 mLPM (low volumetric flow rate or LF), when combined with anti-mPD-1 mAb administration, relative to 100,000 ppm UNO treatment at 200 mLPM (high volumetric flow rate or HF) administered for 2.5 and 5 minutes, in combination with anti-mPD-1 mAb, was evaluated. Methods: Mice were inoculated with CT26 cells in the right flank (primary tumor) and two days later, CT26 cells were inoculated in the left flank (secondary tumor). Thirteen days following primary tumor inoculation, CT26 tumor-bearing mice were treated in situ on the primary tumor with UNO 100,000 ppm using one of two methods: (1) UNO delivery at 0.5 mLPM for 2.5 minutes or 5 minutes and, (2) UNO delivery at 200 mLPM for 2.5 minutes or 5 minutes.

[0294] Mice treated with LF did not have the tumor segregated, while those treated with HF had the tumor segregated with a hemostat to reduce chances of swelling owing to the high volume of administered gas. In addition, an exit hole was not formed in the mice treated with LF UNO as opposed to the mice treated with HF UNO.

[0295] Mice treated with either HF or LF UNO were dosed with anti-mPD-1 mAh.

[0296] Test Items: UNO (10%) stabilized in nitrogen (90%).

[0297] Test Animals: Seventy female (70) BALB / c mice, 8 weeks of age.

[0298] Immune Checkpoint Inhibitor: anti-PD-1 mAb.

[0299] Mice were administered anti-mPD-1 mAb 5-6 times intraperitoneally using a 1 ml syringe with a 27G needle. The injection was administered to approximately the same location each time.

[0300] Safety Results: LF UNO was well tolerated for approximately 94% of the mice. Most of the animals showed normal behavior and recovered quickly after treatment.

[0301] Efficacy Data: Further, primary tumor regression was observed in both the LF and HF UNO groups as opposed to untreated mice or anti-mPD-1 mAb only arms, the latter two groups having primary tumor regression rates of 0% (FIG. 5). Regarding systemic effect, two mice from the LF arms and one mouse from each HF arms, did not have a secondary tumor on day 25 post UNO treatment, while two mice from anti-mPD-1 mAb arm and untreated arm did not have a secondary tumor that day as well. When looking at the combined sum of mice who were both primary and secondary tumor free, one mouse in the LF 2.5 min UNO group that had a complete response (CR) of the primary tumor developed a secondary tumor, meaning that only one mouse in the LF group was completely tumor free. This was a similar rate as to what was observed in the LF 5 min UNO + anti-mPD-1 mAb and the HF 2.5 min UNO + anti-mPD-1 mAb.

[0302] In sum, both HF and LF UNO treatments were well tolerated. Further, at day 25 post UNO treatment, combined treatment of UNO 100,000 ppm at 0.5 mLPM or 200 mLPM administered for 2.5 or 5 minutes with anti-mPD-1 mAb led to a total of 3 CRs from the LF groups and 1 CR from the HF group in the primary tumor while no CRs were seen in the primary tumors from the control or anti-mPD-1 mAb only groups. All treatment groups and the control group showed similar rates of response in the secondary tumors, presenting 1 or 2 secondary tumor CRs.

[0303] EXAMPLE 4

[0304] ABSORPTION DISTRIBUTION OF UNO IN TUMOR TISSUE

[0305] Using the CT26 mouse model, the absorption distribution of UNO in CT26 tumors was assessed by nitrotyrosine staining tumor slides.

[0306] Methods: Thirty Balb / c mice were inoculated with CT26 cells in the right flank (primary tumor). Ten days following primary tumor cell inoculation, 12 animals were selected for treatment and randomized into 4 experimental groups. Eight tumor-bearing mice were treated in situ on the primary tumor with UNO 100,000 ppm: 1) half of the mice were treated for two 2-minute cycles at a flow of 0.25 mLPM (LF) and 2) four mice were treated for 2.5 minutes as 200 mLPM (HF). Nitrogen and sham treatments served as the control arms of the study.

[0307] Immediately after treatment, mice were euthanized, their tumors were resected, stained with Indian ink and preserved in 4% paraformaldehyde for a further histology and immunohistochemical (IHC) analysis.

[0308] Results: The levels of 3-NT were highest in the LF-treated tumors (FIG. 6D), with the lowest being seen in the sham (FIG. 6 A) and the nitrogen control arm (FIG. 6B). Further, HF- treated tumors resulted in 33% increase in the 3-NT level compared to the nitrogen control (FIG. 6C and 6E). In other words, more UNO was absorbed by LF-treated tumors than HF-treated tumors.

[0309] Further, in terms of tumor volume, mice treated with LF UNO and an anti-mPD-1 mAb compared favorably to mice treated with HF UNO and an anti-mPD-1 mAb (FIG. 7). Thus, demonstrating that tumors with greater NO absorption interact better with checkpoint inhibitors such as anti-mPD-1 mAb to affect tumor volume.

[0310] EXAMPLE 5

[0311] EFFICACY OF LOW VOLUMETRIC FLOW RATE OF UNO IN RATS

[0312] The combination of low volumetric flow rate of UNO and anti-rPD-Ll was assessed in MAT B III tumor-bearing rats.

[0313] MAT B III tumor-bearing rats were treated with (1) UNO 25,000 ppm at 3 mLPM administered for 5 or 8 minutes with an anti-rPD-Ll mAb, (2) UNO 100,000 ppm at 2.5 mLPM administered for 2.5 minutes with an anti-rPD-Ll mAb, (3) 25,000 ppm at 2 mLPM administered for 8 minutes, and (4) an anti-rPD-Ll mAb alone.

[0314] Anti-rPD-Ll mAb in combination with either LF of UNO 25,000 ppm or 100,000 ppm resulted in prolonged survival as compared to the anti-rPD-Ll mAb alone (FIG. 8A). Specifically, the combination of anti-rPD-Ll mAb in combination with either LF of UNO 25,000 ppm or 100,000 ppm doubled survival by day 37 versus anti-rPD-Ll mAb alone. Moreover, LF of UNO 25,000 ppm or 100,000 ppm increased the efficacy of the anti-rPD-Ll mAb as demonstrated by the improved tumor inhibition (FIG. 8B).

[0315] EXAMPLE 6

[0316] TREATING HUMANS WITH LOW VOLUMETRIC FLOW RATE OF UNO

[0317] The treatment of humans having relap sed / refractory tumors or prolonged stable disease (> 12 weeks) after previous treatment with single agent PD-1 inhibitor using a combination of (i) UNO at a dose of 25,000 ppm ± 10% or 50,000 ppm ± 10% or 100,000 ppm ± 10% administered intratumorally for a duration of 8 minutes and a volumetric flow rate between .2 mLPM and 2 mLPM and (ii) a PD-1 inhibitor, e.g., pembrolizumab, nivolumab, or cemiplimab, at a therapeutically effective dose will be studied. UNO will be administered as a single dose 48-96 hours prior to the PD-1 inhibitor, which will subsequently be administered every 14 or 21 days (depending on the PD-1 inhibitor).

[0318] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a tumor in a subject in need thereof, the method comprising: locally administering to the tumor a therapeutically effective amount of ultra-high concentration gaseous nitric oxide (UNO), wherein the UNO is administered at a volumetric flow rate of less than 10 mL / min (mLPM).

2. The method of claim 1, wherein the UNO is administered at a volumetric flow rate between .01 mLPM and 20 mLPM.

3. The method of claims 1 or 2, wherein the UNO is administered for a time period of about 30 seconds to about 30 minutes.

4. The method of any one of claims 1-3, wherein the UNO is administered at a dose between 1,000 ppm and 1,000,000 ppm.

5. The method of any one of claims 1-4, wherein locally administering the UNO is by intra-tumoral injection.

6. The method of any one of claims 1-5, further comprising: monitoring a pressure parameter associated with the tumor; and controlling the volumetric flow rate of the locally administered UNO such that the monitored pressure parameter remains below a predetermined value.

7. The method of claim 6, wherein the pressure parameter is the intra-tumoral pressure of the tumor.

8. The method of any one of claims 6-7, wherein the predetermined value is an absolute pressure between 1 atmosphere (ATM) and 2 ATM.

9. The method of any one of claim 6-8, further comprising: upon determining that the monitored pressure parameter has reached the predetermined value, ceasing or slowing the volumetric flow rate of the UNO.

10. The method of any one of claims 1-9, wherein the volumetric flow rate is controlled until (i) a total UNO administration time has elapsed, (ii) a total operation time has elapsed, or (iii) a total volume of UNO has been administered.

11. The method of claim 10, wherein UNO is administered either continuously or intermittently during the total operation time.

12. The method of any one of claims 1-11, wherein UNO can be administered in combination with one or more checkpoint inhibitors.

13. The method of claim 12, wherein the one or more checkpoint inhibitors are selected from the group consisting of an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, and an anti-LAG-3 antibody.

14. The method of claims 12 or 13, wherein the tumor is refractory to treatment with the checkpoint inhibitor.

15. The method of claims 12 or 13, wherein the tumor relapsed after previous treatment with the checkpoint inhibitor.

16. The method of claims 12 or 13, the subject achieved a prolonged stable disease state after previous treatment with the checkpoint inhibitor.

17. The method of any one of claims 12-16, wherein the combination of UNO and the one or more checkpoint inhibitors results in a reduction of at least one of tumor size and volume.

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