Cancer treatment using 5,6-dihydro-4h-benzo[de] quinoline-camptothecin

Abstract

The anti-cancer use of a camptothecin derivative which demonstrates a surprising resistance to the degrading effects of Human Serum Albumin ("HSA") in vivo is disclosed. Since HSA represents about half of serum protein in human blood, previous camptothecins generally worked poorly or not at all in vivo due to inactivation by HSA among other factors. Since NSS-01 is similar in mechanism of action to other camptothecins, and as demonstrated herein has both: (i) greater anti-tumor activity against various human cancers and (ii) less toxicity at therapeutic dosing - than topotecan and irinotecan; and without wishing to be bound by theory, NSS-01 demonstrated desirable antitumor activity against adenocarcinomas, it therefore should demonstrate desirable antitumor activity against other cancers that are susceptible to Topoisomerase I inhibitors.

Description

CANCER TREATMENT USING 5,6-DIHYDRO-4H-BENZO[DE] QUINOUNE- CAMPTOTHECIN[00011 This application is an International Application which claims priority from U.S. provisional patent application no.63 / 683,843, filed on August 16, 2024, and U S. provisionar patent application no. 63 / 785,751, filed on April s, 2025, the entire contents of each which are incorporated herein by reference.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known tothose skilled therein as of the date of the invention described and daimed herein.COPYRIGHT NOTICE

[0003] A portion of tire disclosure of this patent contains material that is subject to copyright protection. The copyright owner has no objection to the reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever.HELD OF THE INVENTION

[0004] The present invention relates to a method of beating cancer with a camptothecin derivative, 5,6-dihydro-4H-benzo[de] quinoline-camptothecin ("NSS-01”), that is active against cancer in vitro and in vivo in the presence of Human Serum Albumen. lt also relates to a novel method(s) of making and formulating NSS-01.BACKGROUND OF THE INVENTION

[0005] Camptothecin (or "CPT”) is a naturally occurring alkaloid that was first isolated in 1966 from the bark and stem of the Camptotheca acuminata, a tree native to China. The compound was found to have potent anti-tumor activity in animal models and was subsequently investigated as a potential cancer treatment

[0006] The initial research on camptothecin was hampered by its low solubility and stability, as well as its toxicity. In the 1970s, researchers synthesized a number of derivatives of camptothecin in an attempt to improve its properties. One of these derivatives, topotecan (sold under the brand name ‘Hycamtin', among others), was approved by the FDA in 1996 for the treatment of ovarian cancer as wen as small cell lung cancer. Another CPT derivative, irinotecan hydrochloride (or “irinotecan”), also known as “Gamptothecin-11” or “CPT-11", was approved by the FDA in 1996 for the treatment of colorectal cancer. Irinotecan (sold under the trade name “Camptosar”), Is also used (often adjunctivety with other therapeutic agents) to treat other solid tumor types of cancer induding lung, pancreatic, ovarian, andoervkal cancer. Irinotecan is a prodrug for its active metabolite molecule, ‘SN-38'.

[0007] In addition to their anti-tumor activity, camptothecins have also been found to have antiviral and anti-inflammatory effects.

[0008] Although camptothecin and its derivatives, both individually and combined with other therapeutic agents, have shown demonstratable anti-tumor activity, they also typically have several limitations and potential problems.

[0009] First, CPT and its anti-cancer derivatives (including topotecan and irinotecan / SN- 38) are hampered to varying degrees in the presence of Human Serum Albumin ("HSA"). CPT, espedalty in its carboxylate form, has an affinity for, and is hampered by, HSA. Because of that, the equilibrium between the lactone ring and the carboxylate form is driven toward the carboxylate form. The carboxylate form of CPT is an inactive form against cancer cells as it does not interact with Topoisomerase I. Irinotecan also binds to HSA, altering its bioavaitebHity and thus decreasing its antitumor effect This HSA binding necessitates the need for increased amounts of irinotecan / SN-38 to be used in treatment, with a concomitant increase in toxicity (See

[0012] below). Topdecan was synthesized to reduce th'® HSA induced limitation, however topotecan has markedly less anti-cancer effect than irinotecan / SN-38.

[0010] Another of the main issues with camptothecins is their low solubility in water, which makes them difficult to formulate into a drug that can be easily administered. While topotecan is synthesized to improve its water solubility, in so doing its structuretrades some improvement in water solubility for a decrease in the overall anti-cancer activity.

[0011] Camptothecin and its derivatives (e.g., topotecan and irinotecan / SN-38) are also unstable and rapidly degrade in the presence of light, heat, and acidle conditions, which limite their shelf life and makes them difficult to store and transport and also limits available dosing routes.

[0012] Another challenge with camptothecins commonly included in chemotherapy protocols (e.g., topotecan and irinotecan / SN-38) is their toxicity. These compounds can cause serious side effects, induding myelosuppression (a decrease in the production of blood cells), gastrointestinal toxidty, and severe diarrhea. The toxicity of camptothecin and its derivatives has limited their effectiveness as cancer treatments.SUMMARY OF THE INVENTION

[0013] The present invention relates to a method of treating a cancer patient with a camptothecin analogue composition comprising 5,6-dihydro-4H-benzo[de] quinoline- camptothecin ("NSS-01 ") which retains its superior anti-cancer activity despite HSA presence in vitro and in vivo, as it is resistant to suppression, deactivation, and / or inactivation by HSA in vivo, while also being surprisingly less toxic to the redpientthan certain current "standard of care” camptothecins (induding topotecan and irinotecan). "Standard of care" can refer to treatment that is accepted by medical experts as a proper treatment for a certain type of disease and that is widely used by health care professionals, and It may also be considered a "best practice", "standard medical care", or a “standard therapy".

[0014] Accordingly, in one embodiment, there is a method of treating a patient with cancer comprising administering to the patient a therapeutically effective amount of a composition containing 5,6-dihydro-4H-benzo[de] quinoline-camptothecin (NSS-01) or a pharmaceutically acceptable salt thereof which inhibits Topoisomerase I and is resistant to suppression, deactivation, and / or inactivation by human serum albumin (HSA) in vivo.

[0015] Other embodiments comprise methods or protocols for synthesis of 5,6-dihydro- 4H-benzo[de] quinoline-camptothecin (NSS-01). For example, the method comprises (a) employing a Friedlander's condensation wherein synthone 1 ("S1”): ((S)-4-Ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano[3,4-fJindolizine- 3,6,10(4H)-trione); and synthone 2 ("S2"): ((8 - amino - 5 - hydroxy - 1 ,2,3,4 - tetrohydronaphtalene -1 - one)) are dissolved in an organic solvent in the presence of a levi’s catalyst and heated to between about 60° to about 180° C for a period of about 10 minutes to about 24 hours, or (b) utilizing the same reaction mixture subjected to microwave energy (100W to 2200W) for 0.1 minutes to 60 minutes; which product is then purified by one of three methods. For example, the purification methods can comprise (i) washing the crude powderwifo a series of solvents, or by (ii) preparative HPLC on C18 column, with a gradient of 40-100% MeOH or 10-30% ACN as the mobile phase to elute the product, Which was then evaporated to near dryness under a vacuum and then lyophilized to a dry powder, or by (iii) the addition of cold water, precipitated for one minute to 12 hours at temperatures from 1° C to 100°C, then pelletized by centrifugation, and then lyophilized.

[0016] In yet another embodiment, a formulation for treatment of cancer wherein the active ingredient is 5, 6-dihydro-4H-benzo[de] quinoline-camptothecin (NSS- 01),

[0017] In one embodiment, the 5,6-dihydro-4H-benzo[de] quinoline-camptothecin (NSS- 01) prevents or reduces cancer cefi proliferation and / or increases cancer cell death in the presence of HSA in vivo.

[0018] In another embodiment, the cancer is a solid tumor or a liquid cancer. For example, the solid tumor can be located in or near one or more of the bladder, breast, colon, ovaries, pancreas, or brain.

[0019] In another embodiment, aspects of the invention are drawn toward a method of inhibiting or preventing the growth of one or more tumors in a subject, the method comprising administering to the subject a composition comprising 5,6-dihydro-4H- benzofde] quinoline-camptothecin (NSS-01) or a pharmaceutically acceptable salt thereof.

[0020] In another embodiment, the cancer is an adenocarcinoma or a sarcoma.

[0021] in another embodiment, the cancer has metastasized.

[0022] in another embodiment, the composition further comprises a pharmaceutically acceptable carrier, exdpient, or diluent

[6023] In another embodiment, the composition is administered to the patient by infusion, oral or injection.

[0024] In another embodiment, the composition is administered locally, regionally, or systemically.

[0025] In another embodiment, the composition comprises the S isomer of 5,6-dihydro- 4H-benzo[de] quinoline-camptothecin, the R isomer of 5,6-dihydro-4H-benzo[de] quinoline-camptothecin, or both.

[0026] in other embodiments, the subject has been, is presently, or will be administered one or more additional anti-cancer therapies. For example, the one or more additional anti-cancer therapy comprises surgery, chemotherapy, immunotherapy, radiotherapy, or any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1a and FIG. 1b represent tile structure of NSS-01 including both R (FIG. 1a) and S (FIG. 1b) stereoisomers.

[0028] FIG. 2 represents the structure of NSS-01 not accounting for stereoisomers.

[0029] FIG. 3 shows therapy of LS174T human colon cancer tumors in NOD SCID mice with and without HSA influence.

[0030] FIG. 4 shows thenrapy of LS174T human colon cancer tumors in NOD SCID mice with and without HSA influence (normalized).

[0031] FIG. 5 shows antitumor effect of NSS-01 on LS174T human colon cancer xenografts in nude mice in the presence of HSA (tumor volume).

[0032] FIG. 6 shows antitumor effect of NSS-01 on LS174T human colon cancer xenografts in nude mice in the presence of HSA (tumor mass).

[6033] FIG. 7 Shows antitumor effect of TOPO on LS174T human colon cancer xenografts in nude mice in the presence of HSA (tumor volume).

[6634] FIG. 8 shows antitumor effect of TOPO on LS174T human colon cancer xenografts in nude mice in the presence of HSA (tumor mass).

[0035] FIG. 9 shows antitumor effect of IRINO on LS174T human colon cancer xenografts in nude mice in the presence of HSA (tumor volume).

[0036] FIG. 10 shows antitumor effect of IRINO on LS174T human colon cancer xenografts in nude mice in the presence of HSA (tumor mass).

[0037] FIG. 11 shows antitumor effect of NSS-01 on LS174T human coion cancer xenografts in HSA+, hFcRn+, Rag- / - mice (tumor volume).

[0038] FIG. 12 shows antitumor effect of NSS-01 on LS174T human colon cancer xenografts in HSA+, hFcRn+, Rag - / - mice (image of femora).

[0030] FIG. 13 shows therapy on LS174T human colon tumors in HSA+ Rag - / - mice (tumor volumes).

[0040] FIG. 14 shows general toxicity of therapy on LS174T human colon cancer tumors in HSA+ Rag - / - mice (body weight).

[0041] FIG. 15 shows images of therapy on LS174T human colon cancer tumors in HSA+ Rag - / - mice.

[0042] FIG. 16 shows antitumor effect of NSS-01 and SN-38 therapy on LS174T human colon cancer xenografts in HSA+, hFcRn+, Rag- / - older mice (sdp).

[0043] FIG. 17 shows general toxicity of NSS-01 and SN-38 therapy on LS174T human colon cancer xenografts in HSA+, hFcRn+, Rag- / - older mice (sdp).

[0044] FIG. 18 shows antitumor effect of NSS-01 and SN-38 therapy on LS174T human colon cancer xenografts in HSA+, hFcRn+, Rag- / - older mice (sdp).

[0045] FIG. 19 shows images of antitumor effect of NSS-01 and SN-38 therapy on LS174T human colon cancer xenografts in HSA +, hFcRn+, Rag - / - older mice.

[0046] FIG. 20 shows antitumor effect of NSS-01 and SN-38 therapy on LS174T human colon cancer xenografts in HSA+, hFcRn+, Rag- / - older mice (oral gavage).

[0047] FIG. 21 shows general toxicity of NSS-01 and SN-38 therapy on LS174T human colon cancer xenografts in HSA+, hFcRn+, Rag- / - older mice (oral gavage).

[0048] FIG. 22 shows images of control group (left) and NSS-01 Therapy 2 group (right) for Therapy 2 (day zero).

[0040] FIG. 23 shows growth of CF-Pac1-Luc (human pancreatic cancer) tumors in nude mice in the presence of HSA (Therapy 1 non-treated / control group).

[0050] FIG. 24 shows growth of CF-Pac14.uc (human pancreatic cancer) tumors in nude mice in the presence of HSA (Therapy 2 non-treated / control group).

[0051] FIG. 25 shows images of excised tumors of treated (NSS-01 ) and non-treated (control) group mice (Therapy 1 and 2 experiments).

[0052] FIG. 26 shows a NSS-01 Therapy 1 treated group mouse on day 28, showing no progression, no metastases, reduced disease bulking.

[0053] FIG.27 shows images of a rion-treated (control) Therapy 1 group mouse on day 28 (showing metastases, progression and increased disease bullring).

[0054] FIG.28 shows the effect of NSS-01 on CF-Pac1-Luc tumors in nude mice in the presence of HSA (Therapy 1 treated group).

[0055] FIG.29 shows the effect of NSS-01 Therapy 1 on CF-Pac1-Luc tumors in nude mice in the presence of HSA (average tumor volume).

[0056] FIG. 30 shows the effect of NSS-01 on CF-Pac1-Luc tumors in nude mice in the presence of HSA (Therapy 2 treated group).

[0057] FIG. 31 shows the effect of NSS-01 Therapy 2 oh CF-Pac1-Luc tumors in nude mice in the presence of HSA (average tumor volume).

[0058] FIG. 32 shows images of a Therapy 2 control group mouse on day 21.

[0059] FIG 33 shows images of an NSS-01 Therapy 2 treated group mouse on day 21.

[0060] FIG. 34 shows a summary of NSS-01 therapy (1 and 2) on CF-Pac1-Luc tumors in nude mice in the presence of HSA (average final tumor mass - treated and non- treated / control groups).

[0061] FIG. 35 shows a summary of NSS-01 Therapy (1 and 2) on CF-Pac1-Luc tumors in nude mice in the presence of HSA (average final tumor volume- treated and non- treated / control groups).

[0062] FIG. 36 shows general toxicity of NSS-01 on CF-Pac1-Luc bearing nude mice in the presence of HSA (Therapy 1 non-treated / control group).

[0063] FIG. 37 shows general toxicity of NSS-01 on CF-Pac1-Luc bearing nude mice in the presence of HSA (body weight). (Therapy 1 treated group).

[0064] FIG. 38 shows general toxicity of NSS-01 on CF-Pac1-Luc bearing nude mice in the presence of HSA (body mass); (Therapy 1 versus Control Groups).

[0065] FIG. 39 shows general toxicity of NSS-01 on CF-Pac1-Luc bearing nude mice in the presence of HSA (Therapy 2 non-treated / control group).

[0066] FIG. 40 shows general toxicity of NSS-01 on CF-Pac1-Luc bearing nude mice in the presence of HSA (body weight). (Therapy 2 treated).

[0067] FIG. 41 shows general toxicity of NSS-01 of CF-Pac1-Luc bearing nude mice in presence of HSA (body mass %) (Therapy 2 versus Control Groups).

[0068] FIG.42 shows NSS-01 Therapy (1 and 2) on CF-Pac1-Luc tumors in nude mice in the presence of HSA (correlation of final tumor volume and mass - treated and non- treated / control).

[0069] FIG. 43 provides a summary data table of the concentrations of NSS-01 and drag comparators required to reduce by 50% the biological activity (proliferation) of various cancer cell lines (EJ-1 human bladder cancer, MCF-7 human breast cancer, and A2780 human ovarian cancer), with and without (or in the presence or absence of) HSA, as reflected in mean IC50 data.

[0070] FIG. 44 shows the concentration of NSS-01 and drag comparators required to reduce, by 50%, the biological activity (proliferation) of EJ-1 human bladder cancer cell line without (in the absence of) HSA.

[0071] FIG.45 shows the concentration of NSS-01 and drug comparators required to reduce, by 50%, the biological activity (proliferation) of EJ-1 human bladder cancer cell line with (in the presence of) HSA.

[0072] FIG. 46 shows the concentration of NSS-01 and drug comparators required to reduce, by 50%, the biological activity (proliferation) of MCF-7 human breast cancer cell line without (in the absence of) HSA.

[0073] FIG. 47 shows the concentration of NSS-01 and drag comparators required to reduce, by 50%, the biological activity (proliferation) of MCF-7 human breast cancer cell line with (in the presence of) HSA.

[0074] FIG. 48 shows the concentration e of NSS-01 and drag comparators required to reduce, by 50%, the biological activity (proliferation) of A2780 human ovarian cancer cell line without (in the absence of) HSA by 50%.

[0075] FIG. 49 shows the concentration of NSS-01 and drug comparators required to reduce, by 50%, the biological activity (proliferation) on A2780 human ovarian cancer cell line with (in the presence of) HSA.DETAILED DESCRIPTION OF THE INVENTION

[0076] Camptothecin derivatives are widely recognized for their potent anticancer properties, primarily through the inhibition of topoisomerase I. However, their clinical efficacy is frequently limited due to instability and reduced potency in physiological environments, particularly in the presence of serum proteins like HSA. When evaluating the impact of HSA on chemotherapy performance, in vivo studies can provide dynamic systems that account for dynamic protein binding, FcRn-mediated recycling, drug distribution, and clearance-factors that critically influence the real-world efficacy of albumin-binding Chemotherapeutics. Therefore, our research included in vivo models in the presence of HSA. Described herein provides a camptothecin derivative which demonstrates a surprising resistance to the degrading effects of Human Serum Albumin ("HSA") / n vivo.[0077} While this invention is susceptible to embodiment in many different forms, there is shown in the drawings, and will herein be described in detail, specific embodiments with the understanding that the present disclosure of such embodiments is to be considered as an example of the principles and not intended to limit the invention to the Specific embodiments shown and descrfoed. In toe description below, reference numerals are used to describe the same, similar, or corresponding parts in the several views of the drawings. This detailed description defines the meaning of the terms used herein and specifically describes embodiments in order for those skilled in the art to practice the invention. The terms "about" and "essentially" mean ±10 percent.

[0078] The terms "a" or "an", as used herein, are defined as one or as more than one. The term "plurality", as used herein, is defined as two or as more than two. The term "another, as used herein, is defined as at least a second or more. The terms "including" and / or "having", as used herein, are defined as comprising (i.e., open language). The term "coupled", as used herein, is defined as connected, although not necessarily directly, and not necessarily mechanically.

[0679] The term "comprising" is not intended to limit inventions to claiming only the present invention with such comprising language. Any invention using the term comprising could be separated into one or more claims using "consisting" or "consisting of" claim language and is so intended.

[0080] Reference throughout this document to "one embodiment", "certain embodiments", "an embodiment", or similar terms means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment Furthermore, the features, structures, or characteristics may be combined in any suitable manner in one or more embodiments without limitation.

[0081] The term "or", as used herein, is to be interpreted as an inclusive, or meaning any of, or any combination hereof. Therefore, "A, B, or C" means any of the following: "A; B; C; Aand B; A and C; B and C; or A, B, and C". An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in some way inherently mutually exclusive.

[0082] The drawings featured in the figures are for the purpose of illustrating certain convenient embodiments of the present invention and are not to be considered as limitation thereto. The term "means" preceding a present participle of an operation indicates a desired function for which there is one or more embodiments, ( / .e., one or more methods, devices, or apparatuses for achieving the desired function) and that one skilled in the art could select from these or their equivalent in view of the disclosure herein, and use of the term “means’ is not intended to be limiting.

[0083] As used herein, the term "cancer tumor or cancer" can refer to the presence of ceils with typical oncogenic cell characteristics {e.g., uncontrolled proliferation, loss of specialized function, immobility, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rates, and certain characteristic morphologies and cell markers). In some cases, the cancer cells will be in the form of a tumon such cells may be present locally within the animal or circulate in the bloodstream as independent cells, such as leukemia cells.

[0084] As used herein, "tumor" can refer to all neoplastic cell growth and proliferation, whether malignant or benign, as well as all pre-cancerpus and cancerous cells and tissues. As used herein, the tumor may be a "primary tumor” (e.g., the tumor begins in the tissue where it is detected) or the tumor may be a "secondary tumor" (e.g., the tumor begins in one tissue and fiien spreads to the other parts of the body).As used herein, a "solid tumor" can refer to an abnormal tissue mass that generally does not contain cysts or fluid areas. By way of example, solid tumors can be in the brain, colon, breast, prostate, liver, kidney, lung, esophagus, head and neck, ovary, cervix, stomach, colon, rectum, bladder, uterus, testis, and pancreas. In some embodiments, the solid tumor regresses or its growth slows or stagnates (arrests) after treatment of the solid tumor with the methods disclosed herein. In other embodiments, the solid tumor is malignant In some embodiments, the cancer comprises stage 0 cancer, in some embodiments, the cancer comprises a Stage I cancer. In some embodiments, the cancer comprises a- Stage II cancer. In some embodiments, the cancer comprises a Stage III cancer. In some embodiments, the cancer comprises Stage IV cancer, in some embodiments, the cancer is refractory and / or metastatic, For example, cancer may be refractory to monotherapy with radiotherapy, chemotherapy, or immunotherapy, yet the cancer may be responsive to (kitted by) administration of a topoisomerase 1 inhibitor. As used herein, the term "liquid cancer" can referto a cancer in which the abnormal and proliferating cells are located in a liquid body fluid, for example, in the blood fl.e., leukemia) or in the lymph. For example, a liquid cancer may be selected from the group consisting of lymphoma, leukemia, and hematopoietic cancer, all such cancers susceptible to a topoisomerase I interaction and stabilization of DNA.

[0085] In embodiments, the cancer can be (but is not limited to) a newly diagnosed, relapsed and / or refractory cancer selected from the group consisting of: nasopharyngeal carcinoma (celnospherygeal cancer), synovial carcinoma, hepatocellular carcinoma, renal carcinoma, connective tissue cancer, melanoma, colon cancer, colorectal cancer, rectal cancer, brain cancer, laryngeal cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T- cell leukemia / lymphoma, neuroma, von HippeHJndau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondrosoma, chondrosarcoma, Ewing's sarcoma, primary site-unknown cancer, carcinoid cancer, gastrointestinal carcinoid cancer, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer,eye cancer, neck cancer, kidney cancer, Wilms 'tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin’s lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagon tumor, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus gland cancer, thyroid cancer, trophoblastic ceil cancer, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fangoides, pancreatic islet tumors, carcinoid syndrome, Somatostatin tumors, gingival cancer, heart cancer, lip cancer, meningeal cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

[0086] As used herein, the term "treating", or the like, can refer to alleviating or ameliorating (or attempting to alleviate or ameliorate) a disease or condition and / or symptoms associated with a mammal, such as a human, having cancer. It is to be understood that, although not excluded, treating a disease or condition does not require that the disorder, condition, or symptoms associated therewith be completely cured.

[0087] Any of the therapeutic applications described herein can be applied to any subject to need of such therapy. As used herein, the terms "subject” or "patient" can refer to a mammal, such as a rodent, a feline, a canine, and a primate. In an embodiment, the subject according to the invention is a human. In some embodiments, the patient is an adult In some embodiments, the subject is more than 10 years old. In some embodiments, the subject is more than 20 years old. In some embodiments, the subject is mere than 35 years old. In some embodiments, the subject is more than 50 years old. In some embodiments, the subject is more than 65 years old. In some embodiments, the patient Is elderly. In some embodiments, the therapeutic applications described hereto can be applied to a veterinary setting. For example, the subject may be a cat or a dog.

[0088] As used hereto, the term "administering” can refer to the actual physical introduction of the composition of the invention into or onto (as appropriate) a cancer cell or cancerous tumor in a patient. Any and all methods of introducing the compositioninto the host or cell are contemplated according to the invention; the method is not dependent on any particular means of introduction and is not to be so construed. Means of introduction are well-known to those skilled in the art and are also exemplified herein. In one embodiment, administration is by injection in the in the forms of solution, suspension nanocrystals, by infusion, etc. and the places of interjection are intravenous, subcutaneous, and intraperitoneal. In other embodiments, oral administration is contemplated.

[0089] The compounds of the present invention may crystallize in more than one form, a characteristic known as "polymorphism", and such polymorphic forms ("polymorphs") are within the scope of the present invention. Polymorphism generally can occur as a response to changes in temperature, pressure, or both. Polymorphism can also result from variations in the crystallization process. Polymorphs can be distinguished by various physical characteristics known in the art such as x-ray diffraction patterns, solubility, and melting point.

[0098] The composition described herein contains a chiral center or may otherwise be capable of existing as multiple stereoisomers. NSS-01 exists or can exist as an R and as an S stereoisomer. Both are active forms, with the S isomer being the most active. The methods disclosed herein of synthesis (construction) will, on demand, produce the more active S form in excess and with a form purity greater or equal to 90%.Additionally, the R form can be produced in excess, at will, as well as any desired ratio of S:R, and with the same goodness of purity. The scope of the present invention includes mixtures of stereoisomers as well as purified enantiomers or enantiomerically / diastereomerically enriched mixtures. Also included within the scope of the invention are the individual isomers of the compounds of the present invention, as well as any wholly or partially equilibrated mixtures thereof. The present invention also includes toe individual isomers of the compound(s) represented by the formulas in Fig. 1a and 1b (the R arid S stereoisomers / enantiomers) as mixtures with / of isomers thereof, in which one or more chiral centers are inverted. The present invention also includes chemical modifications of the compound by means known in the art which do not change or modify its activity or pharmacological properties to a significant extent

[0091] The composition herein indudes the salts of the present composition arid includes the pharmaceutically acceptable salts. Sats encompassed within the term "pharmaceutically acceptable salts" can refer to non-toxlc sate of the compounds of this invention. Sate of the compounds of the present invention may include acid addition salts. Representative sate indude acetate, benzenesulfonate, benzoate, bicarbonate, bisulfete, bitartrate, borate, calcium edetete, camsyiate, carbonate, davulanate, titrate, dihydrochloride, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfete, monopotassium maleate, mucate, napsylate, nitrate, N-methylglucamirie, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate / diphosphate, polygalacturonate, potassium, salicylate, sodium, stearate, subacetete, succinate, sulfate, tannate, tartrate, teodate, tosylate, thethiodide, thmethylammonium, and valerate salts. Other salts, which are not pharmaceutically acceptable, may be useful in the preparation of compounds of this invention and these should be considered to form a further aspect of the invention.

[0092] The formulations of the invention indude those suitable for oral, redal, buccal (e.g., suMingual), vaginal, infusion, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated.

[0093] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, Salves, gels, dr creams as generally known in the art.

[0094] Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tables, each containing a predeterminedamount of the extract; as a powder or granules; as a solution or a suspension in ah aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association of the isolated extract and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the formulations of the invention are prepared by uniformly and intimately admixing the isolated extract with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a powder or granules containing the isolated extract, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and / or surface active / dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.

[0095] Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the isolated extract in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin, or Sucrose and acacia.

[0096] Formulations of the present invention that are suitable for parenteral or infosion administration comprise sterile aqueous and non-aqueous injection solutions of the isolated extract, which preparations are preferably isotonic with the blood of the intended recipient These preparations may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents. The formulations may be presented in unifldose dr multidose containers, for example sealed ampoules and vtels, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the stenle liquid carrier, for example, saline or water-for-injection immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the Idnd previously described.

[0097] Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by mixing the isolated extract with one or more conventional solid carriers, for example, cocoa butter, arid then shaping the resulting mixture.

[0098] Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Cartiers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

[0099] Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of toe recipient for a prolonged period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis.

[0100] It is understood that the teaching provided herein is for examples only, and that a metronomic dosing regimen can be routinely designed in accordance with the teachings provided herein and based upon toe individual standard maximum tolerable dose ("MTD") schedule, and that the metronomic dosing regimen used in these experiments merely serves as one example of possible changes in dosing interval and duration which are made to a standard MTD schedule to arrive at an optimal metronomic dosing regimen.

[0101] Embodiments described herein may be used alone as a treatment of a proliferative disease (e.g., cancer), or carried out in a combination therapy context, such as the combination therapies described herein. For example, NSS-01 may be administered in a combination therapy with at least one additional anti-cancer therapy, such as an anti-cancer immunotherapy (e.g., checkpoint blockade targets) or an inhibitor. By "combination or in conjunction with" it is meant that the administration of the present invention is conducted either at the same time as the standard MTD regimen of established therapies, or between courses of induction therapy to sustain the benefit accrued to the individual by the induction therapy, the intent of which is to continue to inhibit tumor growth white not unduly compromising the individual's hearth or toe individual's ability to withstand the next course of induction therapy.

[0102] The topoisomerase 1 inhibitor (e.g., NSS-01) as described herein can be administered to the subject in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier.

[0103] "Pharmaceutically" or "pharmaceutically acceptable" can refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient can refer to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary Of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone of in combination with another active principle, can be administered in a unit administration form, or as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the tike or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptablesalts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nudeic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts indude the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the Wee carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifongal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride, Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered dentation. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectabte soutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially Suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 mL of isotonic NaCI solution and either added to 1000 mL of hypodermodysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject

[0104] Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as prolamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-pdyoxypropylene- block polymers, polyethylene glycol and wool fat

[0105] Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent forexample as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and Its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0106] The compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents indude, e.g., lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

[0107] The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0108] NSS-01 as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

[0109] A method for controlling the duration of action comprises incorporating the active compound into particles of a polymeric substance such as a polyester, peptide, hydrogel, polylactide / glycolide copolymer, or ethylenevinylacetate copolymers. Alternatively, an active compound can be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin- microcapsules or pdy(methylmethacrylate) microcapsules, respectively, or in a colloid drug delivery system. Colloidal dispersion systems Include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0110] in some embodiments, sterile injectable solutions can be prepared by incorporating the compound in the required solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useM preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0111] As used herein, the term "human serum albumin" (or “HSA") can refer to the serum albumin constitutively found in human blood. It isrthe most abundant protein in human blood plasma; it constitutes about half of serum protein. It Is produced in the liver. It is soluble in water, and it is monomeric and dimeric.HSA transports hormones, fatty adds, and other compounds, buffers pH, and maintains oncotic pressure, among other functions. It is also known to degrade or inhibit camptothecins (e.g., topotecan and irinotecan / SN-38) and is the reason previous carhptothecins often worked quite well kflling certain cancer cells in xritro (in the absence of HSA) but not as well in vivo (in the presence of HSA).

[0112] HSA is synthesized in the liver as preproalbumin, which has an N-terminal peptide that is removed before the nascent protein is released from the roughendoplasmic reticulum. The product, proalbumin, is in turn cleaved in the Golgi apparatus to produce the secreted albumin.

[0113] As used herein, the term “5,6-dihydro-4H-benzo[de] quinoline- camptothecin” ("NSS-01") can refer to the R and / or S structure shown in FIG. 1a and FIG. 1b, respectively, and / or the racemic structure shown in FIG. 2. It has been discovered that, surprisingly, this camptothecin (NSS-01) is resistant to degradation by HSA in vivo and thereby preserves its bioactivity and / or bioavailability. Furthermore, NSS-01 inhibits nuclear enzyme DNA topoisomerase I ("TOP1") by the formation of a covalent bond with an active site of TOP1 during TOP1's interaction, with DNA. This interaction with the cellular DNA permanently stops further reaction and thus causes the cell to die due to DNA damage. It was also observed that markedly less treatment related toxicity indicators occurred in NSS-01 study groups of mice versus all other standard of care camptothecin study groups tested.

[0114] As used herein, the term ‘classical method” (or "Classic synthesis") can refer to a synthesis employing a Friedlander’s condensation of synthone 1 ("S1"): ((S)- 4-Ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine- 3,6,10(4H)-trione); and synthone 2 ("S2"): (8 - amino -5- hydroxy- 1,2, 3,4 -tetrohydronaphtalene -1 -one) which are dissolved in a suitable organic solvent (e.g., DMSO) in the presence of a levi’s catalyst (e.g., toluensulfonic), heated to about 60°-180° C for a period of about 10 minutes to about 24 hours. In such installment, the compounds S1 and S2 are mixed in a ratio of 1 :1 (mol:mol) or 6:4 (mass:mass) into an organic solvent concentration of 10mg / ml DMSO with 40mg / ml p-toluensulfonic acid ("pTSA") added.

[0115] As used herein, the term "microwave assisted method" or the"Microwave synthesis", the sane reaction mixture of S1 and S2 was subjected to microwave energy (100 W to 2200 W) for 0.1 minutes to 60 minutes. The reaction was stopped and the crude product NSS-01 was separated by the addition of cold water arid predpitated for one minute to 12 hours at temperatures from 1° C to 100° C. The mixture was then centrifaged, then pelletized, and then lyophilized to yield NSS-01.

[0116] As used herein, the term “topoisomerase I" can refer to topoisomerase 1 ("TOP1") a highly conserved enzyme that can be found in both prokaryotes and eukaryotes. In the mammalian system, TOP1 is an essential enzyme for normaldevelopment A major function of T0P1 is to relax supercoiled DNA and alleviate the DNA helical constraints. In cancer cells it is a necessary enzyme to support cancer cell growth, or its inhibition of the growth. Its conversion to a cancer cell poison provides the general mechanism of the camptothecin of the present invention in addition to the surprising benefit of NSS-01 activity being resistant to HSA hindering or hampering.

[0117] As used herein, the term “resistance" can refer to development of the ability to withstand the previously destructive effect on a drug by a molecule, physical condition or tumor cells.

[0118] Aspects of the invention are drawn towards compositions and methods for treating cancer. As used herein, the term “tumor" or "cancer" has its general meaning in the art and can refer to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of toe body. The term "cancer" can farther encompass both primary arid metastatic cancers.

[0119] As used herein, toe term "aggressiveness of a cancer" can refer to the capacity of a cancer to lead to the formation of metastasis by improving cancer cell dissemination, cancer cell migration and invasion abilities, modifying their adhesive capacities and favoring pre-metastatic and metastatic niche formation. Metastasis can referto toe growth of cancer cells in a secondary site / organ following cancer cell dissemination from a primary site / organ. In vitro invasion and migration abilities can be monitored by visualization methods. The skilled person will know how to detect metastasis in vivo.

[0120] A cancer can be characterized by the presence of cells with typical oncogenic cell characteristics (e.g., uncontrolled proliferation, loss of specialized function, immobility, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rates, and certain characteristic morphologies and cell markers), in some cases, the cancer cells win be in the form of a tumor; such cells may be present locally within the animal or circulate in toe bloodstream as independent cells, such as leukemia cells.

[0121] In embodiments, the cancer may be metastatic cancer. The cancer cells and or tumors toat are treated may or may not be resistant to conventional cancer therapy, i.e. toe cells in a tumor may exhibit either primary or acquired resistance toconventional cancer therapy and yet they are responsive to (killed by) administration of a topoisomerase 1 inhibitor.

[0122] In another embodiment, the subject has, or is at risk of developing, colon cancer. As used herein, terms like “colon cancer” and “colorectal cancer” can refer to malignant growth arising in the colon, a segment of toe large intestine. Colon cancer most commonly occurs in older adults but can develop at any age. The disease often originates from small clusters of cells, known as polyps, that form on the inner lining of the colon. While most polyps are benign, certain types can undergo malignant transformation over time, resulting in colon cancer. Certain colorectal cancers, particularly those characterized by high proliferative activity or specific genetic alterations, may be Susceptible to chemotherapeutic agents that target DNA topoisomerase I, such as camptotoecin derivatives, which interfere with DNA replication and transcription in rapidly dividing tumor cells.

[0123] In another embodiment, the subject has, or is at risk of developing, breast cancer. As used herein, the term “breast cancer" can refer to a malignant growto originating in the breast tissue, most often in the ducts (breast adenocarcinoma) or lobules (lobular carcinoma) that produce and transport milk. Although breast cancer can occur in both men and women, it Is far more common in women. The disease may begin as abnormal cells that proliferate uncontrollably, forming a lump or mass. While some breast abnormalities are benign, certain lesions can progress to invasive cancer over time if left untreated. Specific subtypes of breast cancer, including those with high cell turnover rates or particular molecular markers, may exhibit susceptibility to DNA topoisomerase l-targeting dtemotherapeutics, such as camptotoecin derivatives, which disrupt DNA replication and tiansaription, leading to tumor cell death.

[0124] In another embodiment, toe subject has, or is at risk of developing, bladder cancer. As used herein, the term “bladder cancer" can refer to a malignant growth arising in the tissues of the bladder, toe organ responsible for storing urine. The most common type, urothelial carcinoma (also known as transitional cell carcinoma), originates in toe cells lining the inner surface of the bladder. Bladder cancer can occur at any age but is more frequently diagnosed in older adults. The disease may begin as abnormal cells in the bladder lining that multiply uncontrollably, forming tumors. Whilesome bladder growths are noninvasive, certain forms can progress and Invade deeper bladder tissues or metastasize to other organs if left untreated. Certain bladder cancers, particularly those with high proliferative activity or specific molecular profiles, may be susceptible to chemotherapeutic agents that target DNA topoisomerase I, such as camptothecin derivatives, which exert cytotoxic effects by interfering with DNA replication and transcription in rapidly dividing tumor ceils.

[0125] In another embodiment, the subject has, or is at risk of developing, ovarian cancer. As used herein, the term “ovarian cancer" can refer to a malignant growth arising in the tissues of the ovary. The most common type, epithelial ovarian adenocarcinoma, originates from the surface epithelium of the ovary and includes histologic subtypes such as serous, endometrioid, mucinous, and dear cell carcinoma. Ovarian cancer can occur at any age but is most frequently diagnosed in postmenopausal women. The disease may begin as abnormal epithelial cells that proliferate uncontrollably, forming tumors capable of local invasion within the pelvis and metastasis to distent sites via peritoneal dissemination or hematogenous spread. Certain ovarian cancers, particularly those with high proliferative activity or defined molecular alterations, may be susceptible to chemotherapeutic agents that target DNA topoisomerase I, such as camptothecin derivatives, which disnipt DNA replication and transcription in rapidly dividing tumor cells, resulting in cell cycle arrest and apoptosis.

[0126] In another embodiment, tile subject has, or is at risk of developing, brain cancer. As used herein, the term “brain cancer" can refer to a malignant growth arising in the brain parenchyma, meninges, or assodated central nervous system structures. Brain cancers encompass a range of histologic types, inducting primdry tumors such as glioblastoma, astrocytoma, oligodendroglioma, and ependymoma, as well as metastatic brain tumors originating from primary cancers elsewhere in the body, such as adenocarcinomas of the lung, breast, or coion . Brain cancers may occur at any age, with certain subtypes more prevalent in children and others in adults. The disease may begin as abnormal glial or epithelial cells that proliferate uncontrollably, forming tumors capable of infiltrating surrounding neural tissue and disrupting neurological function. Certain brain cancers, particularly those characterized by high mitotic activity dr specific molecular signatures, may be susceptible to chemotherapeutic agents that target DNAtopoisomerase I, such as camptothecin derivatives, which interfere with DNA replication and transcription in rapidly dividing tumor cells, leading to cell cycle arrest and apoptosis.

[0127] Many different types of brain tumors exist. Types of brain tumors include, but are not limited to, gliomas and related brain tumors (e.g., astrocytoma, glioblastoma, oligodendroglioma, arid ependymoma), choroid plexus tumors, embryonal tumors (e.g., medulloblastoma), germ cell tumors, pineal tumors (e.g., pineoblastoma), meningiomas, nerve tumors (e.g., schwannoma), pituitary tumors, among others.

[0128] In another embodiment, the subject has, or is at risk of developing, pancreatic cancer. As used herein, the term “pancreatic cancer" can refer te a malignant growth arising in the tissues of the pancreas, most commonly in the exocrine component as pancreatic adenocarcinoma originating from the ductal epithelium.Pancreatic cancer can occur at any age but is more frequentiy diagnosed in older adults and is often associated with a poor prognosis due to late-stage detection. The disease may begin as abnormal epithelial cells within the pancreatic ducts that proliferate uncontrollably, forming tumors capable of local invasion and distant metastasis. While less common, tumors may also arise from the endocrine pancreas, such as pancreatic neuroendocrine tumors (PanNETs). Certain pancreatic cancers, particularly those with high proliferative activity or specific molecular features, may be susceptible to chemotherapeutic agents that target DNA topoisomerase I, such as camptothecin derivatives, which interfere with DNA replication and transcription in rapidly dividing tumor cells, leading to cell cycle arrest and apoptosis.

[0129] intercellular communication within pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment dramatical^ contributes to metastatic processes. As used herein, the term “stroma" can refer to the intra-tumoral microenvironment Tumors are composed of neoplastic cells and non-neoplastic cells in various ratios depending on tumors type and grade. The total amount of non-neoplastic cells composed the stroma, which are mainly cancer-assodated fibroblasts (CAFs) and immune cells.

[0130] As used herein, the terms “cancer-assodated fibroblast" (CAF) or “tumor-associated fibroblast” or ‘carcinogenic- associated fibroblast” or “activated fibroblast" can refer to a cell type within the tumor microenvironment that promotestumorigenic features by initiating the remodeling of the extracellular matrix or by secreting cytokines. CAFs are a complex and abundant cell type within the tumor microenvironment; the number cannot decrease, as they are unable to undergo apoptosis. CAFs have been found to be abundant in a tumor stroma. Myofibroblasts and fibroblasts make up CAFs. The functions of these CAFs have been known to stimulate angiogenesis, supporting the formation of tumors and thus proliferation of cancer cells and metastasis. CAFs are derived from either normal fibroblasts, pericytes, smooth muscle cells, fibrocytesor mesenchymal stem cells. CAFs then go on to support tumor growth by secreting growth factors such as Vascular Endothelial Growth Factor (VEGF), Platelet Derived Growth Factor (PDGF) and Fibroblast Growth Factor (FGF) and other chemokines to stimulate angiogenesis and thus the growth of a tumor.

[0131] The terms "treating" or “treatment" can also refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0132] The term "therapeutic regimen" can refer to the pattern of treatment of an illness, e g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" can refer to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (to part or in whole) a "loading regimen", which may include administering a greater dose of the drag than a physician would employ during a maintenance regimen, administering a drag more frequently than a physician would administer the drag during a maintenance regimen, orboth. The phrase "maintenance regimen" or "maintenance period" can refer to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for tong periods of time (months or years). A maintenance regimen may employ continuous therapy (e g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria (e.g., pain, disease manifestation, etc.);

[0133] Embodiments as described herein can be administered to a subject in a therapeutically effective amount A "therapeutically effective amount" can refer to an amount of active agent which is necessary to impart therapeutic benefit to a subject For example, a "therapeutically effective amount" can refer to an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. Referring to the examples herein, therapeutically effective amounts of NSS-01 are shown to achieve an objective of reducing or downregulating, or eliminating, tumor growth. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment The specific therapeutically effective dose level for any particular subject will depend upon a variety of fedora including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and did of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors wen known in the medical arts. For example, it is wen within the skill of the art to start doses of the compound at levels tower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied oyer a wide range from 0.01 to 1,000 mg per adult per day. Typically, compositions contain about 0.01 , 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 or 500 mg of the active ingredient for the symptomatic adjustment of thedosage to the subject to be treated. An effective amount of the drug can be supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.01 mg / kg to 10 mg / kg of body weight per dosing day.

[0134] A therapeutically effective dose can depend upon a number of factors known to those of ordinary skill in the art. The dose(s) can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, farther depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires, these amounts can be readily determined by the skilled artisan .

[0135] In embodiments, the dose can bee human equivalent dose. The term "human equivalent dose" can refer to a dose of a composition to be administered to a human that is calculated from a specific dose used in an animal study.

[0136] In some embodiments, the therapeutically effective amount of a compound of the invention administered to a subject is at toast about 0.0001 mg / kg body weight, 0.0005 mg / kg body weight, 0.001 mg / kg body weight, 0.005 mg / kg body weight, 0.01 mg / kg body weight, 0.05 mg / kg body weight, 0.1 mg / kg body weight, at least about 0.25 mg / kg body weight, at toast about 0.5 mg / kg body weight, at least about 0.75 mg / kg body weight, at feast about 1 mg / kg body weight, at least about 2 mg / kg body weight, at leastabout 3 mg / kg body weight, at least about 4 mg / kg body weight, at least about 5 mg / kg body weight, at least about 6 mg / kg body weight, at least about 7 mg / kg body weight, at least about 8 mg / kg body weight, at least about 9 mg / kg body weight, at least about 10 mg / kg body weight, at least about 15 mg / kg body weight, at least about 20 mg / kg body weight, at least about 25 mg / kg body weight, at toast about 30 mg / kg body weight, at least about 40 mg / kg body weight, at least about 50 mg / kg body weight, at least about 75 mg / kg body weight, at least about 100 mg / kg body weight, at least about 200 mg / kg body weight, at least about 250 mg / kg body weight, at least about 300 mg / kg body weight, at least about 350 mg / kg body weight, at least about 400 mg / kg body weight, at least about 450 mg / kg body weight, at least about 500 mg / kg body weight, at feast about 550 mg / kg body weight, at least about 600 mg / kg body weight, at least about 650 mg / kg body weight, at least about 700 mg / kg bodyweight, at least about 750 mg / kg body weight, at least about 800 mg / kg bodyweight, at least about 900 mg / kg body weight, oratleast about 1000 mg / kg body weight

[0137] In some embodiments, the therapeutically effective amount of a compound of the invention (e.g., the NSS-01 / or additional therapeutic agent) administered to a subject is from 0.0001 mg / kg body weight to 0.0005 mg / kg body weight, from 0.0005 mg / kg body weight to 0.001 mg / kg body weight from 0.001 mg / kg body weight to 0.005 mg / kg body weight from 0.01 mg / kg body weight to 0.05 mg / kg body weight, from 0.05 mg / kg body weight to 0.1 mg / kg body weight, from 0.1 mg / kg body weight to 0.5 mg / kg body weight from 0.5 mg / kg body weight to 1.0 mg / kg body weight, from 1.0 mg / kg body weight to 2.0 mg / kg body weight, from 2.0 mg / kg body weight to 3.0 mg / kg body weight from 3.0 mg / kg body weight to 4.0 mg / kg body weight, from 4.0 mg / kg body weight to 5.0 mg / kg body weight, from 5.0 mg / kg body weight to 7.5 mg / kg body weight from 7.5 mg / kg body weight to 10 mg / kg body weight, from 10 mg / kg body weight to 25 mg / kg body weight from 25 mg / kg body weight to 50 mg / kg body weight, from 50 mg / kg body weight to 100 mg / kg body weight, from 100 mg / kg body weight to 250 mg / kg body weight from 250 mg / kg body weight to 500 mg / kg body weight, or from 500 mg / kg body weight to 1000 mg / kg body weight

[0138] In some embodiments, the NSS-01 compound is administered to a subject in a low dose (e.g., a sub-perceptive dose, e.g., such that the subjects behavior is not altered). For example, a sub-perceptive dose can be less than about 100 μg / kg, less than about 75 μg / kg, less than about 50 μg / kg, less than about 25 μg / kg, less than about 10 μg / kg, less than about 7.5 μg / kg, less than about 5.0 μg / kg, less than about 2.0 μg / kg, less than about 1.5 μg / kg, less than about 1.0 μg / kg, less than about 0.5 μg / kg, less than about 0.1 μg / kg, or lower.

[0139] In another embodiment, intermittent dosing is as effective as regular dosing. In further or alternative embodiments, the NSS-01 compound is administered only when the patient exhibits a particular symptom, e.g., the onset of pain, the onset of a fever, the onset of an inflammation, or the onset of a skin disorder, if two or more compounds are administered, doing schedules of each compound can depend on the other or can be independent of the other.

[0140] In another embodiment, the administration of the NSS-01 compound can be administered chronically, that is, for an extended period of time, including throughout the duration of the subject's life in order to ameliorate or otherwise control or limit the symptoms of the subject's disorder.

[0141] In another embodiment, the administration of the NSS-01 compound can be given continuously; alternatively, the dose of drug being administered can be temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday can vary between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday may be from 1%-100%, including, by way of example only, 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0142] Embodiments as described herein can be administered to a subject as a prodrug. A “prodrug" can refer to a medication or compound that, after administration, is metabolized into a pharmaceutically active drug. Inactive prodrugs are pharmacologically inactive medications or compounds that are metabolized into an active form within the body.

[0143] The compound of FIG. 1a, FIG. 1 b, and / or FIG.2 can be administered to the subject one time (e.g., as a single injection or deposition), Alternatively, administration can be once or twice daily to a subject in need thereof for a period of from about 2 to about 28 days, or from about 7 to about 10 days, or from about 7 to about 15 days. It can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 times per year, or a combination thereof. The dosage can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health and weight of the recipient nature and extent of symptoms; kind of concurrent treatment frequency of treatment and the effect desired; and rate of excretion.

[0144] In some embodiments, the NSS-01 compound is administered to the subject on a regular basis, e.g., three times a day, two times a day, once a day, every other day or every 3 days. In other embodiments, the NSS-01 compound is administered to the subject on an intermittent basis, e.g., twice a day followed by once a day followed by three times a day; or the first two days of every week; or the first, second and third day.

[0145] In some variations, the NSS-01 is administered at least about any of 1x,2x, 3x, 4x, 5x, 6x, 7x (i.e. , daily) a week. In some variations, the intervals between each administration are less than about any of 28 days, 21 days, 14 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, and 1 day. In some variations, the NSS-01 is administered over a period of at least about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 and 36 months.

[0146] the invention also provides for metronomic dosing regiments). There is provided a method of administering te a subject a composition comprising a dose of NSS-01 based on a metronomic dosing regimen. The methods are applicable to methods of treatment thatcan provide clinical benefit, defined broadly as any of the following: inhibiting an increase in tumor volume, stowing or inhibiting worsening or progression of cancer cell proliferation, inducing tumor regression, reducing primary tumor size, reducing occurrence or size of metastasis, reducing or stopping tumor growth, inhibiting tumor cell division, killing a tumor cell, inducing apoptosis in a tumor cell, reducing or eliminating tumor recurrence.

[0147] "Metronomic dosing regimen" can refer to frequent administration ofNSS-01 and or a compound containing NSS-01 without prolonged breaks (or drug holidays) at a dose below the established maximum tolerated dose (MTD) via a traditional schedule with breaks (hereinafter also referred to as a "standard MTD schedule" or a "standard MTD regimen"). In metronomic dosing, the same# lower, or higher cumulative dose over a certain time period as would be administered via a standard MTD schedule may ultimately be administered. In some Cases, this is achieved by extending the time frame and / or frequency during which the dosing regimen is conducted while decreasing the amount administered at each dose. Generally, the NSS-01 and or a compound containing NSS-01 administered via themetronomic dosing regimen of the present invention is better tolerated by the individual. Metronomic dosing can also be referred to as maintenance dosing or chronic dosing.

[0148] Referring to the Examples herein, such as Example 8, a metronomic dose of NSS-01 of 2.5 mg / kg given every 12 hours was administered for a period of 48 hours.

[0149] Referring to the Examples herein, Such as Example 9, a metronomic dose of NSS-01 Of 5 mg / kg 2x / week was administered for a period of 3-4 weeks.

[0150] The sldlled artisan will recognize that the metronomic dosing regimen can depend upon a number effectors known to those of ordinary skill in the art. The metronomic dosing regimen can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon tire route by which the composition is to be administered, if applicable, and the effect which the practitioner desires. These amounts can be readily determined by the drilled artisan.

[0151] In embodiments, the metronomic dosing of the NSS-01 at each administration is about 0.25% to about 50% of its maximum tolerated dose following a traditional dosing regimen. In embodiments, the metronomic dose of the NSS-01 at each administration is about 0.25% to about 35% of its maximum tolerated dose following a traditional dosage regimen. In embodiments, the metronomic dose of the NSS-01 at each administration is about 0.25% to about 25% of its maximum tolerated dose following a traditional dosage regimen. In embodiments, the metronomic dosing of the NSS-01 Is between about 0.25% to about 25% of the corresponding MTD value, including for example any of about 0.25% to about 20%, about 0.25% to about 15%, about 0.25% to about 10%, of the corresponding MTD value. In some variations, the dosing of the NSS-01 per administration is less than about any of 1%, 2%, 3&, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 18%, 20%, 22%, 24%, 25%, 27%, 30%, 32%, 35%, 40%, 45%, or 50% of the MTD for the same NSS-01 in the same formulation following a given traditional dosing schedule. Traditional dosing schedule can refer to the dosing schedule that is generally established in a clinical setting.

[0152] In embodiments, the metronomic dosing of the NSS-01 comprises administration over a period of at least one month. In embodiments, the interval between each administration is less than about a week, such as less than about any of 6, 5, 4, 3, 2, or 1 day. For example, the interval between each administration may be nomore than about 1 day, no more than about 2 days, no more than about 3 days, no more than about 4 days, nd more than about 5 days, no more than about 6 days, or no more than about 7 days. In embodiments, the interval between each administration is more than about a week, such as more than about 7 days, about 14 days, about 21 days, or about 28 days.

[0153] lh some variations, the interval between each administration is constantFor example, the administration can be carried out daily, every two days, every three days, every four days, every five days, or weekly. In some variations, the administration can be carried otittwice daily, three times daily, or more frequently.

[0154] the metronomic doting regimens described herein can be extended over an extended period of time, such as from about a month, up to about three years, or longer than 3 years. For example, the doting regimen can be extended over a period of any of about 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 18, 24, 30, and 36 months. Generally, there are no breaks in the doting schedule.

[0155] The cumulative dose of the NSS-01 administered by the metronomic regimen may be higher than that administered according to a standard MTD dosing schedule over the same time period. In some variations, the cumulative dose of the NSS-01 administered by the metronomic regimen equals to or is lower than that of the NSS-01 administered according to a standard MTD doting schedule over the same time period.

[0156] NSS-01 would act to directiy block the translation of the targeted mRNA by binding thereto and thus preventing protein translation dr increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art. Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, thevector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and indude, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known in the art

[0157] As used herein, the term “simultaneous use" can refer to the use of a topoisomerase 1 inhibitor and at least one anti-cancer agent occurring at the same time.

[0158] As used herein, the term “separate use" can refer to the use of a topoisomerase! inhibitor ligand and at least one anti-cancer agent not occurring at the same time.

[0159] As used herein, the term “sequential use" can refer to the use of a topoisomerase 1 inhibitor and at least one anti-cancer agent occurring by following an order.

[0150] As used herein, the terms “combined treatment", “combined therapy" or “therapy combination" can refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.

[0161] As used herein, the term “administration simultaneously" can refer to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term "administration separately" can refer to an administration of 2 active ingredients at the same time or at substantially the same tone by different routes. The term “administration sequentially" can refer to an administration of 2 active ingredients at different times, the administration route being identical or different

[0162] In some embodiment, the dassical treatment is selected from the group consisting of radiotherapy, chemotherapy or immunotherapy.

[0163] In some embodiments, the classical treatment consists of chemotherapy.As used herein, the term “chemotherapy" can refer to use of chemotherapeutic agents to treat a" subject As used herein, the term "chemotherapeutic agent" can refer to chemical compounds that are effective in inhibiting tumor growth. Bcamples of chemotherapeutic agents indude alkylating agents such as thiotepa and cydosphosphamide; alkyl sulfonates such as busulfen, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines induding altretamine, triethylenemelamine, trietylenephosphoramide, triethytenethiophosphaoramide and trimethytotomelamine; acetogenins (espedally bullatacin and bullatacinone); a camptothecin (induding the synthetic analogue topotecan); bryostetin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dotestatin; duocarmycin (induding the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofbsfamide, uradl mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Angew Chem Int. Ed. Engl. 33: 183-186 (1994); dynemicin, induding dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), adacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diaro-5-oxo-L-norleucine, doxorubicin (induding morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxontoicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peptomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tuberddin, ubenimex, zinostatin,zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as deriopterin, methotrexate, pferopterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as andtabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enodtabine, floxuridlne, 5-FU ; androgens such as calusterone, dromostanolone propionate, epitidstanol, mepitiostane, testoiactone; anti- adrenals such as aminoglutethimide, mitotahe, trilostane; folic acid replenisher such as frollnic acid ; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabudl; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etogludd; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mltoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2'. 2"-trichlorotriethylarnine; trichothecenes (espedally T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cydophosphamide; thiotepa; taxoids, e.g. paditaxei (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); the taxane drug family; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfemide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capedtab'me; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens iriduding for example tamoxifen, raloxifene, aromatase inhibiting 4(5)- imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0164] In some embodiments, the classical treatment consists of adminfetering to the subject an immunotherapeutic agent The term "Immunotherapeutic agent" as used herein, can refer to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and / or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and / or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the ait as immunologic therapy, biological therapy biological response modifier therapy and biotherapy. Examples of common immunotherapeutic agents known in the art include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants. Alternatively, the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (e.g., T cells, NK, cells, dendritic cells, B cells).

[0165] Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally sb that the human body becomes more effective in fighting the growth and / or spread of cancer cells, or they can be specific, l.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents.

[0166] Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system. Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines). Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents. Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines. Non-specific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants.

[0167] A number of cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins and colony-stimulating factors.

[0168] Interferons (IFNs) indude the common types of IFNs, IFN-alpha (IFN-a),IFN-beta (IFN-P) and IFN-gamma (IFN-y). IFNs can act diredly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behavior and / or increasing their production of antigens thus making the cancer cells easier for the immune system to recognize and destroy. IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and / or stimulating natural Idler (NK) cells, t ceils and macrophages. Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation).

[0169] Interleukins indude IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins indude Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL-12; Wyeth Pharmaceuticals). Zymogenetics, Inc. (Seattle, Wash.) is currently testing a recombinant form of IL-21 , which Is also contemplated for use in the combinations of the present invention.

[0170] Colony-stimulating factors (CSFs) include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM- CSF or sargramostim)and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy. Accordingly, treatment with CSFs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemotherapeutic agents to be used. Various-recombinant colony stimulating factors are available commercially, for example, Neupogen® (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Beriex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), Amesp (erytropoietin).

[0171] In addition to having specific or non-specific targets, immunotherapeutic agents can be active, i.e. stimulate the body's own immune response, or they can bepassive, i.e. comprise immune system components that were generated external to the body.

[0172] Passive specific immunotherapy typically involves the use of one or more monoclonal antibodies that are specific for a particular antigen found on the surface of a cancer cell or that are specific for a particular cell growth factor. Monoclonal antibodies may be used in the treatment of cancer in a number of ways, for example, to enhance a subject's immune response to a specific type of cancer, to interfere with the growth of cancer cells by targeting specific cell growth factors, such as those involved in angiogenesis, or by enhancing the delivery of other anticancer agents to cancer cels when linked or conjugated to agents such as chemotherapeutic agents, radioactive particles or toxins.

[0173] In some embodiments, the immunotherapeutic agent is an indirect immune checkpoint inhibitor and can trigger ICD or immune checkpoint death.

[0174] The term "Immune checkpoint inhibitor" can refer to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins. The term "immune checkpoint protein" has its general meaning in the art and can refer to a molecule ttiat is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman el al. 2011. Nature 480:480- 489). Examples of stimulatory checkpoint include CD27, CD28, CD40, CD122, CD137, 0X40, G1TR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The Adenosine A2A receptor (A2AR) is regarded aS an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also called CD276, was originally understood to be a co-stimulatory molecule but is now regarded as co-inhibitory. B7-H4, also called VTCN1 , isexpressed by tumor cells and tumor- associated macrophages and plays a role in tumor escape / B and T LymphocyteAttenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. Surface expression of BTLA is gradually dpwnregufated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumorspecific human CD8+ T cells express high levels of BTLA. CTLA-4, Cytotoxic T- Lymphocyte- Associated protein 4 and also called CD 152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDO. also known as Indoleamine 2,3- dioxygenase, is a tryptophan catabolic enzyme and a key immunoregulatory molecule that suppresses T cell responses by depleting tryptophan and generating immunosuppressive metabolites, such as kynurenines, which contribute to immune tolerance and the maintenance of regulatory T cell function. Another important molecule is TDO, tryptophan 2,3-dfoxygenase. IDO is known to suppress T arid NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. KIR, Killer-cell Immunoglobulin-like Receptor, is a receptor for MHO Class I molecules on Natural Killer cells. LAGS, Lymphocyte Activation Gene-3, works to suppress an immune response by action to tregs as well as direct effects on CD8+ T cells. PD-1, Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Go 's melanoma drug Keytruda, which gained FDA approval in September 2014. An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment. TIM-3, short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines. TIM-3 acts as a negative regulator of Thl / Tcl function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, Short for V- domain Ig suppressor of T cell activation, is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, NSS-01 inhibiting a checkpoint protein on the immune system may enhance the antitumor T-cell response through cGAS-STING, PD_L1 , DAMPS, and ICB.

[0175] In some embodiments, an immune checkpoint inhibitor can refer to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In some embodiments, the immune checkpointinhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands.

[0176] In a particular embodiment, the immune checkpoint inhibitor is an antibody. Typically, antibodies are directed against but not limited to A2AR, B7-H3, B7- H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 VISTA, DDR, P53, NF-kB, PJ3K / AKT, JNK pr AMPK.

[0177] In a particular embodiment, small organic molecules interfere with transduction pathways. For example, they can interfere wife molecules, receptors or enzymes involved in topoisomerase 1 pathway.

[0178] In a particular embodiment, the topoisomerase 1 inhibitor is a non- traditional RNA silencer and / or a oligonucleotide, through conjugation.

[0179] In a particular embodiment, the topoisomerase 1 inhibitor to an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capadty to recognize virtually any class of target molecules with high affinity and specificity.

[0180] tn a particular embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, W02008156712, W02011110621, WO2014055648 and WO2014194302. Examples of anti-PD-1 antibodies which are commercialized: Nivdumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK).

[0181] In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody such as described ih WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897. Examples of anti-PD-L1 antibodies which are on clinical trial: Atezdizumab (MPDL3280A, Genentech / Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS).

[0182] in another embodiment, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.

[0183] In the context of the invention, the immune checkpoint inhibitor inhibits topoisomerase 1 or its ligand modulating immune checkpoint pathways. Studies indicate that CPT-induced DNA damage can trigger immunogenic cell death (ICD), leading to increased tumor antigen presentation and stimulation of the immune system. Additionally, CPT may influence the tumor microenvironment by reducing immunosuppressive cells, such as regulatory T ceils (Tregs) and myeloid-derived suppressor cells (MDSCs), thereby enhancing anti-tumor immunity. Some studies also suggest that CPT-derived chemotherapy may downregulate PD-L1 expression in tumor cells, potentially increasing their susceptibility to immune checkpoint blockade. However, CPT itself does not directly block immune checkpoints like ICIs but may be used synergistically with them to enhance therapeutic outcomes. Thus, while camptothecin does not function as a classical immune checkpoint inhibitor, its immune- modulatory effects may complement Checkpoint blockade therapies in cancer treatment

[0184] In another embodiment, the immune checkpoint inhibitor is a small organic molecule. The term "small organic molecule" as used herein, can refer to a molecule of a size comparable to those organic molecutes generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size upto about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0185] Others classical treatment used as anti-cancer agents may be tor example cytarabine, anthracydines, fludarabine, capedtabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfemide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomydri, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L- asparaginase, doxorubicin, epimbicm, 5 -fluorouradl, taxanes such as docetaxel and paditaxel, leucovorin, tevamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BOND, nitrosoureas such as carmustme and tomustine, vinca alkaloids such as vinblastine, Vincristine and vinorelbine, imatimb mesylate, hexamethyhnetemine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins,protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. As example, a p38MAPK inhibitor may be SB203580, SB202190, SB202474.

[0186] In some embodiments, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, texanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.

[0187] Additional anti-cancer agents may be selected from, but are not limited to, cytokines, chemokines, growth fectors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bispecific or multi-specific antibodies, monobodies, polybodies. Other additional anticancer agent may be selected from, but are not limited to, growth or hematopoietic fectors such as erythropoietin and thrombopoietln, and growth factor mimetics thereof. For example, the additional anti-cancer agent can be Bevacizumab, a monoclonal antibody that binds to VEGF protein and prevents new blood vessels from farming.

[0188] In the present methods for treating cancer the further therapeutic active agent can be an antiemetic agent Suitable antiemetic agents include, but are not limited to, metoclopramide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acetylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, budizine, debopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, medizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine and trcplsetron. In a preferred embodiment the antiemetic agent is granisefron or ondansetron.

[0189] In another embodiment the further therapeutic active agent ca n be a hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating fectors indude, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.

[0190] In still another embodiment, the other therapeutic active agent can be an opioid or nonopioid analgesic agent. Suitable opioid analgesic agents indude, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, bpermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cydazocine, methadone, isomethadone and propoxyphene. Suitable non-opldd analgesic agents indude, but are not limited to, aspirin, celecoxib, rofecoxib, didofinac, diflusinal, etodolac, fenoprofen, flurbiprofen; ibuprofen, ketoprofen, indomethacin, ketorolac, medofenamate, mefenamic acid, nabumetone, naproxen, piroxicam and suKndac.

[0191] In yet another embodiment, the further therapeutic active agent can be an anxiolytic agent Suitable anxiolytic agents indude, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.(00192]DETAILED DESCRIPTION OF THE FIGURES

[0193] Referring to FIG. 1 A and FIG. 1B, the topoisomerase I inhibitor can be 5,6-dihydro-4H-benzo[de] quinoline-camptothecin ("NSS-01"). As used herein, the term “5,6-dihydro-4H-benzo[de] quinoline-camptothecin" can refer to the R and / or S stmcture shown in Fig 1a and 1b, respectively, or to the racemic structure according to FIG. 2. It has been discovered that, surprisingly, this camptothecin (NSS-01) is resistant to degradation by HSA in vivo and thereby preserves its bioactivity and / or bioavailability. Furthermore, NSS-01 inhibits nuclear enzyme DNA topoisomerase I (“TOP1") by the formation of a covalent bond with an active site of TOP1 during TOPfs interaction with DNA This interaction with the cellular DNA permanently stops further reaction and thus causes the cell to die due to DNA damage. It was observed that NSS-01 has superior anti-tumor properties, to comparators, and it was also observed that markedly less treatment related toxicity indicators occurred in NSS-01 study groups of mice versus all other standard of care camptothecin study groups tested.

[0194] FIG. 2 represents the chemical structure of 5,6-dihydro-4H-benzo[de] quinoline-camptothecin.

[0195] FIG. 3 presents data on the effects of NSS-01 versus SN-38 and a nontreated (control) group on LS174T human colon cancer tumors in NOD SCID mice with and without HSA influence.

[0196] FIG. 4 presents data on the effects of various therapies, including SN-38 and NSS-01, on LS174T human colon cancer tumors in NOD SCID mice With and Without HSA influence.

[0197] FIG. 5 presents data on the antitumor effects of NSS-01 on LS174T human colon cancer xenografts in nude mice, in the presence of HSA, comparing various administration routes and their impact on tumor volume over time versus a control (non-treated) group. Tumor mass was measured over time.

[0198] FIG. 6 presents data on the antitumor effects of NSS-01 on LS174T human colon cancer xenografts in nude mice, in the presence of HSA, comparing different administration routes and their impact on tumor mass versus a control (nontreated) group. Tumor volume was measured over time.

[0199] FIG. 7 presents data on the antitumor effects of TOPO on LS174T human colon cancer xenografts in nude mice, in the presence of HSA, comparing various administration routes and their impact on tumor volume over time, versus a control (non-treated) group, (tumor volume)

[0200] FIG. 8 presents data on the antitumor effects of TOPO on LS174T human colon cancer xenografts in nude mice, in the presence of HSA comparing different administration routes and their impact on tumor mass, versus a control (non-treated) group, (tumor mass)

[0201] FIG. 9 presents data on the antitumor effects of IRINO on LS174T human colon cancer xenografts in nude mice, in the presence of HSA, comparing tumor volumes across different treatment methods over 14 days, versus a control (nontreated) group, (tumor volume)

[0202] FIG. 10 presents data on the antitumor effects of IRINO on LS174T human colon cancer xenografts in nude mice, in the presence of HSA, comparingdifferent administration routes and their impact on tumor mass, versus a control (nontreated) group, (tumor mass)

[0203] FIG. 11 presents data on the antitumor effects of NSS-01 on LS174T human colon cancer xenografts in a specified mouse model containing HSA, detailing tumor volume changes over 14 days, versus a control (non-treated) group.

[0204] FIG. 12 presents images of the tumors at day 14, related to FIG. 11.

[0205] FIG. 13 presente data on the effects of NSS-01 and comparator cancer therapies on tumor volumes in LS174T human colon cancer tumors in HSA+ Rag V- mice over a 14-day period, versus a control (non-treated) group.

[0206] FIG. 14 presents data on the general toxicity of NSS-01 and comparator cancer therapies on LS174T human colon cancer tumors in HSA + Rag - / - mice, measured by body weight over a period of 14 days and versus a control (non-treated) group.

[0207] FIG. 15 presents images of tumors under various therapies at day 14, related to FIG. 14.

[0208] FIG. 16 presents data on the antitumor effects of NSS-01 and SN-38 therapy on LS174T human colon cancer xenografts in a specified mouse model containing HSA, detailing tumor volume changes over 14 days, versus a control (nontreated) group.

[0209] FIG. 17 presents data on the general toxicity of NSS-01 and SN-38 therapy in LS174T human colon cancer xenografts using HSA+, hFcRn+, Rag - / -older mice, focusing on body weight changes overtime related to FIG. 16.

[0210] FIG. 18 presents data on the anti-tumor effects of NSS-01 and SN-38 therapies on LS 174T human colon cancer xenografts in HSA+, hFcRn+, Rag - / - older mice, versus a control (non-treated) group.

[0211] FIG. 19 presents images of tumors under NSS-01 and SN-38 therapies at day 14, versus a control (non-treated) group, related to FIG. 18.

[0212] FIG. 20 presents data on the antitumor effect of NSS-01 and SN-38 therapies on LS174T human colon cancer xenografts using HSA+, hFcRn+, Rag- / - older mice, detailing tumor volume changes over 14 days, versus a control (non-treated) group.

[0213] FIG. 21 presents data on the general toxicity of NSS-01 and SN-38 therapies in LS174T human colon cancer xenografts using HSA+, hFcRn+, Rag- / - older mice, detailing body weight changes over time, versus a control (non-treated) group.

[0214] FIG. 22 presents images of control group mice and therapy 2 group mice on therapy day 0, related to Experiment 9.

[0215] FIG. 23 presents data on the growth of CF-Pac1-Luc (human pancreatic cancer) tumors in nude mice, in the presence of HSA, detailing tumor volume changes over 28 days in a control (non-treated) group for Therapy 1.

[0210] FIG. 24 presents data on the growth of CF-Pac1-Luc (pancreatic) tumors in nude mice, in the presence of HSA, including tumor volume and metastasis volume measurements over time in a control (non-treated) group for Therapy 2.

[0217] FIG. 25 presents images of excised tumors of treated and non-treated (control) group mice in Therapy 1 and Therapy 2 experiments.

[0218] FIG. 26 presents images of an NSS-01 Therapy 1 group treated mouse on day 28.

[0219] FIG. 27 presents images of a non-treated / control group mouse in Therapy1 group on day 28.

[0220] FIG. 28 presents experimental results ofthe effects of NSS-01 Therapy 1(5 mg / kg s.c. twice weekly) on CF-Pac1-Luc tumor growth in nude mice, in the presence of HSA, detailing tumor volumes over a period of 28 days.

[0221] FIG. 29 presents the experimental results of the effect of NSS-01 Therapy1 (5mg / kg s.c. twice weekly for 4 weeks) on GF-Pacl -Luc tumors in nude mice, in the presence of HSA, detailing average tumor volume changes over 28 days, versus non- treated / control group.

[0222] FIG. 30 presents the experimental results of the effect of NSS-01 Therapy2 (5 mg / kg s c. twice weekly for 3 weeks) on CF-Pac1-Luc tumors in nude mice, in the presence of HSA, including tumor volume measurements over 21 days.

[0223] FIG. 31 presents the experimental results of the effect of NSS-01 Therapy2 on CF-Pac1-Luc tumor growth in nude mice, in the presence of HSA, comparing average tumor volumes between treated and control (non-treated) groups over 21 days.

[0224] FIG. 32 presents images of a Therapy 2 control group mouse on day 21

[0225] FIG. 33 presents images of a Therapy 2 group treated mouse on day 21.

[0226] FIG. 34 presents summary data on average final tumor mass (weight) after NSS-01 Therapy 1 or Therapy 2 on CF-Pac1-Luc (human pancreatic cancer) tumors in nude mice in the presence of HSA, versus control (non-treated) groups.

[0227] FIG. 35 presents summary data on average final tumor volume after NSS-01 therapy 1 or Therapy 2 on CF-Pac1-Luc (human pancreatic cancer) tumors in nude mice in the presence of HSA, versus control (non-treated) groups.

[0228] FIG. 36 presente data on the general toxicity in non-treated / control group of CF-Pac1-Luc (human pancreatic cancer) bearing nude mice in the presence of HSA, including body weight measurements over time (28 days) (Therapy 1 Control Group).

[0229] FIG. 37 presents data on the general toxicity of NSS-01 Therapy 1 in CF- Pac1-Luc bearing nude mice, in the presence of HSA, detailing body weight changes overtime in response to treatment (5mg / kg twice weeldy for 4 weeks).

[0230] FIG. 38 presents data on the general toxictty of NSS-01 Therapy 1 in CF- Pad-Luc bearing nude mice, in the presence of HSA, comparing average body mass changes over time (28 days) between the Therapy 1 treatment and control (non-treated) groups.

[0231] FIG. 39 presents data bn the general toxicity in non-treated / control group of CF-Pac1-Luc (human pancreatic cancer) bearing nude mice in the presence of HSA, including body weight measurements overtime (21 days) (Therapy 2 control group).

[0232] RG. 40 presents data on the general toxicity of NSS-01 Therapy 2 in CF-Paci -Luc bearing nude mice, in the presence of HSA, detailing body weight changes over time in response to treatment (5mg / kg twice weeldy for 3 weeks).

[0233] FIG.41 presents data on the general toxicity of NSS-01 Therapy 2 in CF-Pac1-Luc bearing nudemice, in the presence of HSA, comparing body weight changes oyer time (21 days) between toe therapy and control (non-treated) groups.

[0234] FIG. 42 presents data highlighting the correlation between tumor volume and mass (weight) for treated and non-treated / control groupsfor Therapy 1 and Therapy 2.

[0235] FIG. 43 provides a summary data table of the concentrations of NSS-01 and drag comparators required to reduce by 50% the biological activity (proliferation) of various cancer cell tines (EJ-1 human bladder cancer, MCF-7 human breast cancer, and A2780 human ovarian cancer), with and without (or in the presence or absence of) HSA, as reflected in mean IC50 data.

[0236] FIG.44 Shows the concentration of NSS-01 and drug comparators required to reduce, by 50%, the biological activity (proliferation) of EJ-1 human bladder cancer cell line without (in toe absence of) HSA.

[0237] FIG. 45 shows the concentration of NSS-01 and drag comparators required to reduce, by 50%, the biological activity (proliferation) of EJ-1 human bladder cancer cell line with (in the presence of) HSA.

[0238] FIG.46 showsthe concentration of NSS-01 and drag comparators required to reduce, by 50%, toe biological activity (proliferation) of MCF-7 human breast cancer cell line without (in toe absence of) HSA.

[0239] FIG.47 shows toe concentration of NSS411 and drag comparators required to reduce, by 50%, toe biological activity (proliferation) of MCF-7 human breast cancer cell line with (in the presence of) HSA.

[0240] FIG. 48 shows the concentration e of NSS-01 and drag comparators required to reduce, by 50%, toe biological activity (proliferation) of A2780 human ovarian cancer cell line without (in the absence of) HSA by 50%.

[0241] FIG. 49 shows the concentration of NSS-01 and drug comparators required to reduce, by 50%, toe biological activity (proliferation) on A2780 human ovarian cancer cell line with (in the presence of) HSA.

[0242]

[0243] Examples are provided below to facilitate a more complete understanding of toe invention. The foilawing examples illustrate toe exemplary modes of making and practicing the invention. However, toe scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

[0244] Example 1

[0245] In the experiments described herein, we tested NSS-01 therapy versus control (non-treated) groups and / or versus irinotecan (or “IRINO” or “CPT-11"), SN-38, and topotecan (or “TOPO"). SN-38 is a potent active metabolite of the prodrug irinotecan. SN-38 is the molecule responsible for Irinotecan’s therapeutic activity. Studying it in animal trials allows exploration of its pharmacological and toxicological effects without the variability introduced by the irinotecan activation process. Irinotecan is converted to SN-38 through enzymatic hydrolysis by carboxytesterases. Using SN-38 directly in experiments ensures consistent dosing and focuses on its effects without the potential confounding factor of metabolic differences. By administering SN-38, NSS can directly study its pharmacokinetics and pharmacodynamics. This can provide clearer insights into its therapeutic efficacy, and side effects. Variations in the activity of enzymes like UGT1A1, which metabolizes SN-38 into its inactive form SN-38G, can complicate irinotecan studies. The use of SN-38 bypasses the need for irinotecan’s metabolic activation and allows a more straightforward evaluation of the active drug’s effects. SN-38 is associated with the primary toxidties of irinotecan, including myelosuppression and gastrointestinal toxicity. Studying these effects in our treated (dosed) animals allows for better prediction of potential adverse effects in humans and aids in dose optimization. The experiments referenced herein (and others not included) model human response since SN-38 is the active form of irinotecan responsible for therapeutic outcomes in humans, and its use in animal trails provides a more direct model of what might occur in clinical settings, leading to better translational relevance.

[0246] Example 2

[0247] As described herein, NSS-01 can be synthesized by (a) employing aFriedlander's condensation of synthone 1 ("S1"): ((S)-4-Ethyl-4-hydroxy-7,8-dihydro- 1H-pyrano[3,4-f]indolizine- 3,6,10(4H)-trione); and synthone 2 ("S2"): (8 - amino - 5 - hydroxy - 1 ,2,3,4 -tetrohydronaphtalene -1 -one) which are dissolved in a suitable organic solvent (e.g., DMSO) in the presence of a levi’s catalyst (e.g., toluensulfonic) heated to about 60°-180° C for a period of about 10 minutes to about 24 hours (the"Classic" synthesis), or (b) employing a microwave powered ("Microwave") synthesis of S1 and S2. The NSS-01 product was then purified as described herein.

[0248] In one installment, the compounds S1 and S2 are mixed in a ratio of 1:1 (mol:mol) or 6:4 (mass:mass) into an organic solvent concentration of 10mg / ml DMSO with 40mg / ml p-toluensulfonic acid ("pTSA") added, in one embodiment, for the Classic synthesis, the reaction mixture was heated on conventional sand, mantleor oil bath to temperatures between 60° C and 180° C for between 10 minutes and 24 hours. The NSS-01 product was then subjected to purification.

[0249] In another embodiment forthe Microwave synthesis, the same reaction mixture was subjected to microwave energy (100 W to 2200 W) for 0.1 minutes to 60 minutes. The NSS-01 product was then subjected to purification as described herein.

[0250] Purification was done either by (i) washing the crude powder with a series of solvents, or by (ii) preparative HPLC on C18 column, with a gradient of 40-100% MeOH or 10-30% ACN as the mobile phase to elute the product, which was then evaporated to near dryness under a vacuum and then lyophilized to a dry powder, or by (iii) the addition of cold water, precipitated for one minute to 12 hours at temperatures from 1° Cto 100°C, then pelletized by centrifugation, and then lyophilized.

[0251] The purity and structure of the NSS-01 product were determined by analytical HPLC, 1H NMR and mass spectra, while the biological activity was confirmed through an in vitro cytotoxicity study and an in vivo anticancer activity demonstration. Purified NSS-01 was found to be essentially identical under the two heating methods described above. The purity of product as produced using the NSS synthesis protocols and purification meets or exceeds 90%.

[0252] Example 3 - Treatment of mice in cancer model (in vitro)

[0253] Experiment 1 : Kinetics of Cytotoxicity of NSS-01 and certain other camptothecins toward LS174T (Human Colon Adenocarcinoma) cells in vitro in the pretence and absence of HSA

[0254] Goal: to determine the kinetics of cytotoxicity (cytotoxicity course during time of exposure) and influence of HSA on it

[0255] Method:

[0256] LS174T cells (2x10^4 for 6-24h and 1 x10^4 for 48h and 72h) are exposed to camptothecins:Concentration (nM) for 6h-24h / Concentration (nM) exposure for 48h-72h exposureDrug / NO HSA / HSA / NO HSA / HSA NSS-01 / 100 / 500 / 25 / 250 CPT25 / 250 SN-3825 / 250 TOP0 100 / 100

[0257] All tiie concentrations were diluted 3-fold on 6 consecutive wells, in triplicate. The solutions were left 1.5h in order to reach equilibrium of the lactone / carboxy forms. 100ul were transferred to each well and then incubated for the desired time (6h, 12h, 18h, 24h, 48h and 72h). The solution containing drug was discarded and replaced with RPMI containing MTT (0.5mg / ml), incubated for 4h at 37C, at 5% CO2 and humidity. The solution was then discarded and 75ul DMSO added to each well to dissolve the formazane. The weds were then read at OD540nm in a Molecular Devices Spectramax 250 plate reader.

[0258] Result: NSS-01 possesses a longer half-life than CPT-11 SN-38, or Topotecan.

[0259] Example 4- Treatment of mice in cancer model (In vivo)

[0260] Experiment 2: Influence of HSA on the anticancer activity of certainCamptothecins In vivo

[0261] Goal: to determine the influence of circulating HSA on the antitumor effect of certain camptothecins in vivo.

[0262] Cells: LS174T cells are thawed briefly at 37C and diluted with 8ml coldRPMI, spun down 4min 600 xg and cultured in 12ml full RPMI (10% FCS, P / S) in 5% CO2.

[0263] When ~80% confluent, the cells are split into 4 flasks of 225cm2 in 60ml RPMI and cultured for 2 days;

[0264] When ~80% confluent, trypsin harvested (extended trypsinisation by Trypsin-EDTA), washed 2 x in dear RPMI and cells counted.

[0265] Dilluted to 2x10^7 / ml in RPMI (no serum nor antibiotics) and extensively re-suspended (20 x up / down by a 10ml pipet). Then kept on ice till used (less than 2h).

[0266] Tumors: NOD SCID mice of J AX origin used; the LS174T tumor cells kept on ice are re-suspended 3X by 1ml syringe up / down, (needle no.18), the needle then replaced with a no.25On day 0, mice were shaved and injected with 100ul (2x10^6 cells) into right flank subcutaneously, (no mice restraint and no anesthesia - just gentle handling).

[0267] The mice were observed daily for signs tumor formation.

[0268] The tumors were visible by day 6 on all tumor injected mice.

[0269] On day 6 the mice’s body weight and tumor size were recorded.

[0270] According to those values, the mice were stratified and subdivided into 4 therapy groups; each group had a "with HSA" and "without HSA" sub-group.

[0271] Therapy: On day 8 the mice’s body vreight and tumor sizes were recorded.

[0272] According to those values, the mice were stratified and subdivided into groups:1. Control (200ul saline i.p. and 100 ul blank vehicle i.v. 4h later)2. Control+HSA (200ul20% HSA i.p. and 10Oul blank vehicle 4h later) THERAPY: DOSE (80% Maximum Tolerated Dose ("MTD")3. OPT therapy (200ul saline i.p. and 100ul CPT 4h later) 10mg / kg4. CPT+HSA (200ul HSA i.p. and 100ul CPT 4h later) 10mg / kg5. TOPO therapy (200ul saline i.p. and lOOul TOPO 4h later) 10mg / kg6. TOPO+HSA (200ul HSA i.p. and 100ul TOPO 4h later) 10mg / kg7. CPT-11 therapy (200ul saline i.p. and 100ul CPT-11 4h later) 25mg / kg8. CPT-11+HSA (200ul HSA i.p. and 100ul CPT-11 4h later ) 25mg / kg

[0273] Tumor size and body mass measurements were followed and recorded every two days during the next 14 days.

[0274] Body mass was measured by lab scale to 3 digits.

[0275] Tumor size was measured by caliper in three dimensions (a, b and c).

[0276] The approximate tumor volume was calculated as (a x b x c) / 2.

[0277] On day 14 the mice were sacrificed by cervical dislocation.

[0278] Tumors were surgically removed, weighed and photographed for documentation.

[0279] Results: NSS-01 expressed excellent anti-tumor effect in the absence of HSA. This excellent antitumor effect was preserved in the presence of HSA, and in all cases the anti-tumor effect was superior to CPT-11, SN-38, or Topotecan.

[0280] Example 4

[0281] Experiment 3: NSS-01 therapy on LS174T tumor xenografts In NODSCID mice in the presence of HSA

[0282] Goal: to demonstrate superior antitumor effect of NSS-01 in the presence of HSA.

[0283] The experiment Is based on previous in vitro experiments which show a dear anti-cancer advantage of NSS-01 over certain other camptothecin drugs when HSA is present

[0284] Cells: LS174T cells are thawed briefly at 37C and diluted with 8ml coldRPMI, spun down 4min 600 x g and cultured in 12ml full RPMI (10% FCS, P / S) in 5% CO2.[002*5] When confluent *80% of the cells are split into 4 flasks of 225cm2 in 60mlRPMI and cultured for 2 days; when ~80% confluent trypsin harvested (extended trypsinisation by Trypsin-EDTA), washed 2 x by clear RPMI and counted. Diluted to 2x10^7 / ml in RPMI (no serum nor antibiotics) and extensively re-suspended (20 x up / down by 10ml pipet). Kept on ice till used (less than 2h).

[0284] Tumors: NOD-SCID mice used; the tumor cells kepton ice re-suspended 3X by 1ml syringe up / down (needle no.18), the needle replaced with a no.25.lnjected 100ul (2x10^6cells) in shaved flank of mice. The mice were observed daily for signs of tumor formation. The tumors were visible by day 4 on afl mice. On day 6 the mice body weight and tumor size was recorded.

[0287] According to those values, the mice were stratified and subdivided into groups:1. Control (200ul saline i.p. and 100ul empty vehicle i.v. 4h later).2. Controi+HSA (200ul 20% HSA i.p. and 100ul empty vehicle 4h later) THERAPY: DOSE (80% MTD)3. SN-38 therapy (200ul saline i.p. and 100ul SN-384h later) 10mg / kg4. SN-38+HSA (200ul HSA i.p. and 100ul SN-384h later) 10mg / kg5. NSS-01 therapy (200ul saline i.p. and 100ul NSS-01 4h later) 10mg / kg6. NSS-01 +HSA (200ul HSA i.p. and 100ul NSS-01 4h later) 10mg / kg

[0288] therapy: Onday 7, HSA was injected followed by 10mg / kg drug dose in appropriate groups. The mice were followed daily for health and tumor progression. Tumor size and body mses were measured and recorded every two days during the next 14 days, Body mass was measured by lab scale to 3 digits. Tumor size was measured by caliper in three dimensions (a, b and c). The approximate tumor volume was calculated as (a x b x c) / 2. On day 14 the mice were sacrificed by cervical dislocation. Tumors were surgically removed, weighed and photographed for documentation.

[0289] Results: As seen in FIG. 3 and FIG. 4, NSS-01 therapy demonstrated superior antitumor effect compared to SN-38 therapy, both in the presence and absence of HSA.

[0290] Example 5

[0291] Experiment 4: Anticancer effect of NSS-01 and other camptotheclns against LS174T cancer xenografts on nude mice in HSA presence

[0292] Goal: to estimate anticancer effect of NSS-01 against LS174T cancer xenografts regarding:1. HSA presence2. route of delivery (s.c., i.p., per os, i.v.)3. L / C equilibrium and bound / free drug

[0293] Cells: LS174T

[0294] inoculation: 2x10^6 / 100ul RPM11640, s.c. in left or right flank; The mice were checked daily for tumor formation. On day 8, the mice were stratified and randomly assigned to the groups.

[0295] MICE: Nude, mixed gender, 10-12 weeks old

[0296] Groups: Treatment / Dose / Route / N1. Control / nontreated / 82. NSS-01 / 10mg / kg / s.c., i.p., per os, i.v. / 6 each3. Topo / 10mg / kg / s.c., i.p., per os, i.v. / 6 each4. Irino / 20mg / kg / s.c., i.p., per os, i.v. / 6 each

[0297] Drugs: NSS-01, Topotecan and Irinotecan (CPT-11) are SUSPENDED in20% HSA solution (Baxter) and left 2h to equilibrate.

[0298] Therapy: All the mice were i.p. pre-injected with 200ul of 20% HSA solution followed by the drug injection. 12h and 24h later, additional doses of 100ul of HSA were injected i.p.

[0299] The tumors' size and body mass followed every second day for 2 weeks.At the end of the experiment, the mice were sacrificed, and tumors were removed and weighed.

[0300] Results: As seen in Images 5.0 through 10.0, NSS-01 therapy demonstrated superior antitumor effect compared to topotecan and irinotecan in every delivery method tested: subcutaneous ("s.c."), intraperitoneal ("i.p."), peroral ("per os" or "p.o."), and intravenously ("i.v.").

[0301]

[0302] Experiment s: NSS-01 therapy of LS174T xenografts on constitutivelyHSA+ hFcRn+ Rag- / - mice

[0303] Goal: to preliminarily determine:1. Does LS174T grow in the HSA+ hFcRn+ Rag- / - mice?2. Does NSS-01 have the same effect as on HSA-negative mice?

[0304] The experiment was performed by the same technique as on nude mice(Exp. 4).

[0305] The NSS-01 dose was 10mg / kg divided into two equal portions, administered i.p. and s.c, at the same time, to deliver a long-lasting lbw level in circulation.

[0306] Results:

[0307] As seen in FIG. 11 and FIG. 12:1. LS174T does grow in HSA+ hFcRn+ Rag - / - mice, with the growth kinetic comparable to LS174T growth in nude and NOD-SCID mice. HSA+ hFcRn+ Rag - / - mice are a suitable model for human cancer study.2. The anti-tumor effect of NSS-01 vs LS174T in HSA+ mice was excellent

[0308] Bramofo 7

[0300] Experiment 6: Comparison of NSS-01, SN-38, Topo and Mno activity in HSA+ Rag - / - mice

[0310] Goal: to estimate the antitumor activity of NSS-01 in HSA positive mice and compare with certain other camptothecins in vivo.

[0311] Cells: LS174T cells are thawed briefly at 37C and diluted with 8ml coldRPMI, spun down 4 minutes 600 x g and cultured in 12ml full RPMI (10% FCS, P / S) in 5% CO2.

[0312] When confluent -80% the cells are split into 3 flasks of 225cm2 in 60mlRPMI and cultured for 2 days; when *80% confluent, trypsin harvested (extended trypsinization by Trypsin-EDTA), washed 2 x by dear RPMI and counted. Diluted to 1.5x10^7 / ml in RPMI (no serum nor antibiotics) and extensively re-suspended (20 x up / down by 10ml pipet). Kept on ice till used (less than 2h).

[0313] tumors: HSA+ Rag - / - mice of GenOway origin were used; the tumor cells were kept on ice re-suspended 3X by 1ml syringe up / down (needle no.18), the needle replaced with no.25. Mice were shaved and injected 100ul (1.5x10^6 cells) into right flank subcutaneously (no mice restraint and no anesthesia - just gentle handling)-

[0314] The mice were observed daily for signs of tumor formation. The tumors were visible by day 6 on all mice.

[0315] Therapy: On day 6 the mice's body weight and tumor size were recorded.

[0316] According to those values, the mice are stratified and Subdivided into groups:1. Control (100ul saline split i.p. and s.c.). THERAPY: DOSE (80% MTD)2. NSS-01 Therapy (10mg / kg of NSS-01 nano in -100ul vehicle split i.p. and s.c.) 10mg / kg3. SN-38 Therapy (10mg / kg of SN-38 nano in ~100ul vehicle split i.p. and s.c.) 10mg / kg4. Topo Therapy (10mg / kg SN-38 in vehicle split i.p. and s.c.) 10mg / kg5. IRINO Therapy (20mg / kg Irino in vehicle split i p. and s.c.) 20mg / kg

[0317] Tumor size and body mass were measured and recorded every two days during the next 14 days. Body mass was measured by lab scale to 2 digits. Tumor size was measured by caliper in three dimensions (a, b and c). The approximate tumor volume was calculated as (a x b x c) / 2. On day 14 the mice were sacrificed by cervical dislocation. Tumors were surgically removed, weighed and photographed for documentation.

[0318] Results: As seen in Figure 13, NSS-01 therapy had a superior antitumor effect compared to SN-38 therapy, topotecan, and irinotecan, in HSA+ Rag- mice, with an average tumor volume (mm^3), post therapeutic encounter, of 64.56% less than (smaller than) the average for SN-38 and a greater superiority compared with topotecan and irinotecan. As seen in image 14.0, NSS-01 therapy had no significant loss of body weight compared with other standard of care camptothecins.

[0319]

[0320] Experiments 7 and 8: Comparison of NSS-01 and SN-38 activity Jn HSA+ Rag - / - older mice: influence of age and route of application

[0321] Goal 1 (exp. 7): to estimate the toxicity and antitumor effect of NSS-01 and SN-38, they were given s.c.+i.p. to older mice (6*8 months old).

[0322] Goal 2 (exp.8): to estimate the antitumor activity of NSS-01 and SN-38, they were orally delivered to HSA positive mice.

[0323] Cells: LSI 74T cells are thawed briefly at 37C and diluted with 8ml cold RPMI, spun down 4mln 600 x g and cultured in 12ml full RPMI (10%FCS, P / S) in 5% CO2.

[0324] When confluent -80% the cells are split into 3 flasks of 225cm2 in 60mlRPMI and cultured for 2 days; When -80% confluent, trypsin harvested (extendedtrypsinisation by Trypsin-EDTA), washed 2 x by dear RPMI and counted. Diluted to 1.8x10^7 / ml in RPMI (no serum nor antibiotics) and extensively re-suspended (20 x up / down by 10ml pipet). Kept on ice till used (less than 2h).

[0325] tumors: HSA+ Rag - / - hFcRn+ older mice of GenOway origin were used. The tumor cells were kept on ice re-suspended 3X up / down by 1ml syringe (needle no.18), the needle replaced with no. 25. Mice were Shaved and injected 100ul (1.8x10^6 cells) into right flank subcutaneously (no mice restraint and no anesthesia - just gentle handling).

[0326] The mice were observed daily for signs of tumor formation. The tumors were visible by day 6 on all mice.

[0327] Therapy: On day 6 the mice’s body weight and tumor size were recorded.

[0328] According to those values, the mice were stratified and subdivided into groups;1. Control (100ul 20% HSA 8.c.+i.p. and orally) 62. NSS-01 Therapy s.c.+i.p. (10mg / kg of NSS-01 suspended in ~100ul 20% HSA split s.c. and i.p.) 63. SN-38 Therapy s.c.+i.p. (10mg / kg of SN-38 susp. in ~100ul 20% HSA s.c.+i.p.) 64. NSS-01 Therapy (10mg / kg of NSS-01 nano in ~100ul 20% HSA orally) 65. SN-38 Therapy (10mg / kg of SN-38 nano in ~100ul 20% HSAorally)66. NSS-01 Therapy (1pmg / kg of NSS-01 nano in ~100ul 20% HSA orally, split in 2 doses 12h apart) 67. SN-38 Therapy (10mg / kg of SN-38 nano in ~100ul 20% HSA orally, split in 2 doses 12h apart) 68. NSS-01 Therapy (10mg / kg of NSS-01 nano in ~100ul 20% HSA orally, split in 4 doses 12h apart) 69. SN-38 Therapy (1 Omg / kg of SN-38 nano in ~100ul 20% HSA orally, split in 4 doses 12h apart) 6

[0329] Dose: ~80% MTD 10mg / kg (2mg / ml stock, 100ul / 20g mouse). On day s the mice body weight and tumor size were recorded, and therapy was applied. Tumorsize and body mass were measured and recorded every two days during the next 14 days. Body mass was measured by lab scale. Tumor size was measured by caliper in three dimensions (a, b and c). The approximate tumor volume was calculated as (a x b x c) / 2. On day 14 the mice were sacrificed by cervical dislocation. Tumors were surgically removed, weighed and photographed for documentation.

[0330] Results:

[0331] Experiment 7: As seen in FIG. 16, NSS-01 s.cJ.p. therapy was 60.78% more effective in antitumor activity in older mice In terms of average tumor volume, compared with SN-38 s.c.i.p. therapy, and as seen in FIG. 18, NSS-01 s.clp. therapy was 61.11% more effective than SN-38 s.c.i.p. therapy in terms of average tumor mass. Despite the demonstrated superior antitumor effect of NSS-01 (as seen in FIG.16), no significant toss of body weight was observed during NSS-01 therapy (10mg / kg dose) in HSA expressing mice (as seen in FIG. 17)

[0332] Experiment 8: As seen in FIG. 20, NSS-01 retains potent antitumor activity when applied orally or as a single or repeated dose, yet without meaningful toxicity (see Fig. 21 ). Furthermore, repeated dosing of NSS-01 at prolonged time intervals (e g., applied in 4 split doses 12 hours apart) resulted in superior antitumor activity.

[0333] Example 8

[0334] Experiments:

[0335] Goal: to estimate anticancer effect of NSS-01 against CF-Pac1-Luc(human pancreatic) cancer xenografts1. Mice: female, nude, aged (approx. 6 months)2. Primary and distant and / or metastatic tumors3. Very large tumors at startup (Therapy 1 began ~15 days after inoculation while Therapy 2 began approximately 22 days after Inoculation)4. HSA presence

[0336] Inoculation 1: 1x10^6 / 100ul RPMI 1640, s.c. in right flank (all mice)

[0337] Inoculation 2: 7 days later 1x10^6 / 100ul RPMI, intrapleural / intramuscular in right rib cage (Therapy Group 1: 12 mice)

[0338] Inoculation 3: 14 days later 1x10^6 / 10001 RPMI, subcutaneously at left shoulder (Therapy Group 2: 10 mice)

[0330] day 14, mice which received inoculation 2 (Therapy Group 1) are stratified and randomly assigned to 2 groups

[0340] day 21, mice which received inoculation 3 (Therapy Group 2) are stratified and randomly assigned to 2 groups

[0341] MICE: Nude, female, ~6 months old

[0342] At day zero of therapy (which for Therapy 1 treatment group began 15 days after Inoculation 1 and for Therapy 2 treatment group began 22 days after Inoculation 1) the mice were injected with 3 mg / mouse luciferin and imaged in vivo. As seen in FIG. 22, both the control group (on left) and the treatment group (on right) expressed luciferin modulated fluorescence (see white light markers of tumors in third row of pictures).

[0343] Therapy 1 treatment began 15 days after inoculation 1 (8 days afterInoculation 2): Duration: 4 weeks (8 doses of 5 mg / kg, 40mg / kg total dose). Therapy 2 treatment began 22 days after Inoculation 1 and 8 days after inoculation 3. Duration: 3 weeks (6 doses of 5 mg / kg, 30mg / kg total dose)

[0344] Drug: NSS-01 , SUSPENDED in saline at 1 mg / ml. Preparation: to 0.9mlSaline injected 0.1ml 10mg / ml NSS-01 in DMSO added, stirred.

[0345] Therapy method: All mice in Therapy 1 and Therapy 2 groups (treated and control / untreated) were i.p. pre-injected with 200ul of 20% HSA solution 6h before every drug injection.

[0346] The tumors size and body mass followed every third-fourth day. At the end of the experiment, tire mice were injected with ludferih (3ug / mouse) and imaged in vivo. The tumors and Organs of interest were excised and imaged in vivo. The tumors were accurately massed, photographed, samples embedded into OTC and stored at -20°C fortissue section and histopathology analysis.

[0347] Results and Discussion:

[0348] The control groups (not treated with NSS-01 ) showed extensive tumor growth over the study period (see FIG.s 25, 27, 32). In the control groups, every main tumor was accompanied by a secondary and / or metastatic tumor. As illustrated in FIG . 25, in the control groups, the mice have very large primary CF-Pac1 tumors and easily visible secondary CF-Pac1 tumors in the Therapy 2 control group. Upon surgical resection of these tumors, massive involvement of tumor mass into the chest cavity and through the rib cage protruding to the dorsal side was revealed. Metastases was visible on interior organs (lungs, liver, kidney). As illustrated in FIG. 27, a non-treated (control) Therapy 1 mouse had developed multiple metastatic and / or distant tumors, while as illustrated in FIG. 26, a Therapy 1 treatment group mouse developed no metastatic or distant tumors. The significant difference in size of excised primary tumors between treated and untreated mice can be seen in FIG.25, and no distant or metastatic secondary tumors were found in either Therapy Group. The metastatic tumors for the Therapy 1 control group could not be excised intact and photographed, but are illustrated in the images of a. control group mouse in FIG. 27.

[0349] Therapy Group 1 (5mg / kg NSS-01 dose given 2x weekly for 4 consecutive weeks): Individual tumor volumes over time for the treated group are shown in FIG. 23. As summarized in FIG. 29, the average primary tumor volume for the Therapy 1 treated group declined approximately 42.2% from 164.54 (mm3) on Day zero (0) to 95.07 (mm3) on Day 28, while the average primary tumor volume (mm3) for theuntreated / control group increased by approximately 1,275% from 162.87 mm3 to 2,239.1 mm3. Furthermore, as seen in FIG.28, of the six (6) mice in Therapy Itreated group, the tumor volume declined in four of the six mice with a maximum percentage decline of 82.2% (Mouse 5).

[0350] Therapy Group 2 (5mg / kg NSS-01 dose given 2x weekly for 3 consecutive weeks): As seen in FIG. 31, the average primary tumor volume for the treated group grew approximately 18.7% from Day zero (323.08 mm3) to Day 21 (383.36 mm3), while the average primary tumor volume (mm3) for the untreated control group increased approximately 575% from 355.78 mm3 to 2,400.77 mm3. Furthermore, as seen in FIG. 26, the tumor volume (mm3) for three of the five treated group mice declined during the 21-day therapy period with a maximum decline of 63% (mouse 3).

[0351] Imaged and excised tumors of treated, and control groups are pictured inFIG. 25: No secondary (intrapleural injected), metastatic nor distant tumors were observed in treated groups (Therapy 1 or Therapy 2). As illustrated in FIG. 29, in the NSS-01 Therapy 1 (treated) group, the average tumor volume for treated mice was approximately 23 times-smaller tian the average tumor volume for the untreated / control group, and as illustrated in FIG. 31 , for the Therapy 2 treated mice the average tumor volume was approximately 6 times smaller than the averagefortthe control group. Additionally, no sign of metastases was observed on in vivo imaging nor during surgical examination of the treated groups.

[0352] In conclusion: NSS-01 was tested versus primary tumors, distent tumors, and metastatic tumors. NSS-01 is effective versus pancreatic cancer in primary, distant, and metastatic cancer challenges. NSS-01 showed resistance to Inhibition or challenge by Human Seram Albumin. NSS-01 inhibits topoisomerase 1 and stabilizes DNA strands leading to DNA breakage and a subsequent programmed cell death (PCD) or apoptosis of the pancreatic cancer cellular makeup. NSS-01 was shown to be nontoxic to the receiver of the anti-cancer therapy as demonstrated by body weight value stability (see FIG.s 36 through 41 ) and overall lack of behavioral changes, pain signals, and food / water intake and output NSS-01 is a surprisingly potent, non-toxic, easily dose controlled, anti-pancreatic cancer entity,

[0353] Example 10

[0354] Experiment 10 Protocol for In Vitro Testing of NSS-01 Against HumanGlioma Cancer Cells, and Human Ovarian Cancer Cells

[0355] Purpose: Evaluate the cytotoxic effects of NSS-01 on human glioma and human ovarian cancer cels in vitro.

[0356] Materials and Reagents1. Cell Line:Human glioma cell line (U87-MG)Human ovarian cancer cell line (OVCAR3)2. Culture Media:RPMl-1640 (depending on cell line)Fetal bovine serum (FBS)Penidllin-streptomycin3. Reagents:NSS-01Dimethyl sulfoxide (DMSO) for NSS-01 stock preparationPhosphate-buffered saline (PBS)Trypsin-EDTA4. Assay Reagents:Cell viability assay : MTT, XTT, or CCK-8 kitApoptosis detection kit (e.g., Annexin V / PI or TUNEL assay)Propidium iodide (PI) for cel cycle analysis (s-phase action validation)5. Equipment:CO2incubatorBiosafety cabinetMicroplate reader (for absorbance or fluorescence measurements)Flow cytometer for performing apoptosis or cell cycle assaysFluorescence microscope for imaging-based assay

[0357] Procedure:

[0358] Cell Culture Preparation. Thaw and culture glioma cells in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator (37°C, 5% CO2).

[0359] Subculture cells when they reach 70-80% confluency, ensuring healthy exponential growth and lack of contamination.

[0360] Preparation of Camptothecin Stock Solution. Dissolve NSS-01 inDMSO to prepare a concentrated stock solution. Store the stock solution at -20°C (frost free only), protected from light (disconnect light). Dilute NSS-01 in toe culture medium to desired working concentration immediately before use.

[0361] Cell Seeding. Plate cells in 96-well plates (for viability assays) or 6-well plates (for apoptosis and cell cycle assays). Seed cells at a density of: - 96-well plate: 5 × 103-1 × 104cells / well in 100 μL medium. - 6-well plate: 1 × 105-2 x 105cells / well in 2 ml medium. 3.3. Allow cells to adhere overnight before treatment, and visualize to validate

[0362] Camptothecin treatment Treat cells With varying concentrations of NSS-01 (e.g., 0.1, 0.5, 1, 5, 10 μM) for 24, 48, and 72 hours. Include controls: - Untreated ceils (negative control). - DMSO-treated cells (vehicle control, same concentration as NSS-01 stocks). Optional: Include a positive control (e.g., temozolomide or another known glioma drag).

[0363] Cell Viability Assay. Add toe appropriate reagent (e.g., MTT, XTT, or CCK-8) to each well after toe treatment period, incubate for the time specified by the kit (e g., 2-4 hours). Measure absorbance at the recommended wavelength (e.g., 570 nm for MTT) using a microplate reader. Normalize absorbance values to the vehicle control to calculate percentage cell viability.

[0364] Apoptosis Assay. Harvest treated cells and wash with cold PBS. Stain with Annexin V-FITC and propidium iodide (Pl) following the manufacturer's protocol. Analyze samples using a flow cytometer to quantity early and late apoptotic populations.

[0365] Cell Cycle Analysis. Fix cells in 70% ethanol at 4°C overnight Stain with PI and RNase A for 30 minutes at room temperature. Analyze DNA content using a flow cytometer to assess cell cycle distribution.

[0366] Microscopic Observations. Observe cell morphology under a light or fluorescence microscope to detect signs of apoptosis, such as cell shrinkage, membrane blebblng, or nuclear condensation.

[0367] Data Analysis

[0368] Viability Assay:

[0369] Cell Viability (%)=Absorbance of treated cellsAbsorbance of control cells×100CeU Viability (%)=Absort>ance of control cellsAbsorbance of treated cells×100

[0370] Statistical Analysis:

[0371] Use GraphPad Prism or similar software for statistical analysis.

[0372] Compare treatment groups using ANOVA or t-tests. Report significance at p <0.05.

[0373] Results:

[0374] Viability Assay: NSS-01 therapy reduced cancer cell viability more effectively than control (non-treated) in both target cancer cell lines.

[0375] Apoptosis Assay: NSS-01 therapy showed a higher percentage of apoptotic cells (early and late apoptosis) in both target cell lines compared to control (non-treated) cells.

[0376] Cell Cycle Assay: NSS-01 therapy results in S-phase arrest versus no such effect in the non-treated cells. NSS-01 shows a potent anti-cancer effect, through, but possibly not limited to, an S-phase interaction resulting in stabilization of cellular DNA, and a resultant PCD (programmed cel death) or apoptotic event yielding a desired anti-canoer activity in both targeted cancer cell lines (human ovarian and human glioma), in vitro.

[0377] EXAMPLE 11

[0378] Goal: to measure the anticancer effect of NSS-01 versus Comparator drags against cancer (EJ-1 bladder cancer, MCF-7 breast cancer, A2780 Ovarian cancer) regarding:1. HSA presence2. Use of NSS-01 produced through Classic or Microwave Synthesis

[0379] EJ-1 Cell Une Overview• Origin: Human bladder carcinoma tissue• Morphology: EpithetiaHike Spades: Homo sapiens• Sex: Female· Disease: Bladder cancer• Growth Conditions: Typically cultured at 37°C, 5% CO2, in Eagle’s Minimum Essential Medium (EMEM) with 10% fetal bovine serum• Ha-ras mutation: Reported in EM, which is assodated with oncogenic transformationSTR profiling: Confirms EM Is genetically identical to the T24 cell line, a widely used bladder cancer models• Infinite lifespan: EM cells are immortalized and suitable for tongterm studies

[0380] MCF-7 Cell Line OverviewEstablished: 1973 by Dr. Herbert Soule at the Michigan Cancer Foundation• Source: Pleural effusion from a 69-year-old woman with metastatic breast adenocarcinoma• Name: "MCF-7" stands for Michigan Cancer Foundation -7, the seventh successful culture attempt• Subtype: Luminal A breast cancer• Receptor Status:1. ER-positive (estrogen receptor a)2. PR-positive (progesterone receptor)3. HER2-negative• Morphology: Epithelial-like; grows in monolayers with cobblestone appearance• Growth Behavior. Hormone-dependent; slow-growing with low metastatic potential

[0381] A2780 Ceil Line Overview• Source: Tumor tissue from an untreated female patient with ovarian endometrioid adenocarcinoma• Species: Homo sapiens• Sex: Female• Morphology: Epithelial; grows as a monolayer and in suspension• Disease: Ovarian cancer• STR Profile: Verified and widely referenced (Cellosaurus ID: CVCL_0134)• Mutation profile: Includes alterations in genes like ATM, PIK3CA, PTEN, and RRAS2• Microsatellite instability: MSHow phenotype· Omics data: Extensive datasets available for genomics, epigenetics, and drug screening

[0382] Experimental Protocol:

[0383] Cell Culture Reparation:

[0384] Human bladder (EJ), breast (MCF-7), and ovarian (A2780) cancer cell lines were obtained from ATCC. Cells were thawed, resuspended in RPMM640 medium supplemented with penicillin / streptomycin and 10% fetal bovine serum (FBS), cultured in flasks until approximately 80% confluence, harvested via trypsinization, and seeded at concentrations of 1x10^4 cancer cells / Well (EJ, MCF-7) and 0.75x10^4 cancer cells / well (A2780) into 96-well plates.

[0385] Drug Preparation and Exposure:

[0386] Irinotecan, SN-38, Topotecan, and NSS-01 (for "Classic" and "Microwave") were prepared from powdered form, dissolved in dimethyl sulfoxide (DMSO), and diluted to appropriate working concentrations based on molecular weights. Cells were exposed to each drug at defined initial concentrations (SN-38, NSS- 01 at 1 μM; Irinotecan, Topotecan at 10 μM) with serial dilutions (1 :4), in the presence or absence of 20 mg / ml Human Serum Albumin for 24 hours at 37°C.

[0387] Cytotoxicity Measurement

[0388] Post-treatment, cell viability was assessed via the MTT assay, the medium was aspirated, cells incubated with MTT solution (0.5 mg / ml) for 4 hours,followed by dissolution of formed forrnazan crystals in DMSO. Absorbance was measured in triplicate at 540 nm (Spectramax 250), and data were analyzed to calculate mean IC50 values using logistic regression models.

[0389] Results and Comparative Analysis:

[0390] EJ-1 Bladder Cancer Ceils (See Fig. 43,44,45):

[0391] NSS-01 (produced via our “Classic", and “Microwave" or "MW" syntheses) demonstrated superior antiprolifsrative (anticancer) potency, displaying much tower IC50 values than Irinotecan, SN-38 and Topotecan both in the presence of HSA and in its absence. In the absence of HSA, NSS-01 classic and microwave syntheses recorded mean IC50 values of approximately 1.4 nM and 2.0 nM, respectively, which represents only 23.7% and 33.9% of the lowest comparator (SN-38) value of 5.9 nM.In the presence of HSA, NSS-0T classic and microwave syntheses recorded mean IC50 values of 17.3 nM and 7.0 nM, respectively, which represent 38.6% and 15.6% of the lowest comparator (SN-38) value of 44.8 nM.

[0392] MCF-7 Bredst Cancer Cells (See Fig. 43, 46,47):

[0393] NSS-01 (produced via our “Classic*, ami “Microwave" or "MW" syntheses) demonstrated superior antiproliferative (anticancer) potency, displaying much lower IC50 values than Irinotecan, SN-38 and Topotecan both in the presence of HSA arid in its absence. In the absence of HSA, NSS-01 classic and microwave syntheses recorded mean IC50 values of approximately 33.9 nM and 30.7 nM, respectively, which represents only 75.5% and 68.4%, respectively, of the fewest comparator (SN-38) value of 44.9 nM. In the presence of HSA, NSS-01 classic arid microwave syntheses recorded mean IC50 values of 35.8 nM and 29.0 nM, respectively, which represent 78.3% and 63.5%, respectively, of the lowest comparator (SN-38) value of 45.7 nM.

[0394] A2780 Ovarian Cancer Cells (See Fig. 43, 48,49):

[0395] NSS-01 (produced via our “Classic”, and “Microwave" or "MW“ syntheses) demonstrated superior antiproliferative (anticancer) potency, displaying much tower IC50 values than Irinotecan, SN-38 and Topotecan both in the presence of HSA and in its absence. In the absence of HSA, NSS-01 classic and microwave synthesesrecoreled mean IC50 values of approximately 2.3 nM and 2.4 nM, respectively, which represents only 27.7% and 28.9%, respectively, of the lowest comparator (SN-38) value of 8.3 nM. In the presence of HSA, NSS-01 classic and microwave syntheses recorded mean IC50 values of 24.4 nM and 21.4 nM, respectively, which represent 20.7% and 18.1%, respectively, of the lowest comparator (SN-38) value of 118.1 nM.

[0396] CONCLUSION:Thecamptothecin derivative 5,6-dihydro-4H-benzo{de] quinoline-camptothecin ("NSS- 01") presents substantially enhanced cytotoxic potency against EJ-1 human bladder cancer (cell line), MCF-7 human breast cancer (cell line), and A2780 human ovarian cancer (cell line) versus Comparator camptothedns, both with and without (or, to presence and absence of) Human Serum Albumin. NSS-01's marked superiority, particularly in physiologically relevant conditions, strongly indicates potential clinical advantages over existing camptothecin-based therapies.

[0397]

[0398] Dosing Consideration and Rationale

[0399] When choosing the experimental dosage regime, three facts about NSS- 01 and camptdthecto class comparator drugs were considered:1. S-phase specific mode of action2. High cytotoxicity3. The common dose of 10mg / kg for CPT-11 and 0.75 -2.0 mg / m2 for topotecan.

[0400] Considering the above, and the results of our experimental studies, some of which are summarized in this document, we concluded that NSS-01 provides superior anti-tumor activity at a lower dose with a prolonged exposure regimen than the comparator drugs.

[0401] In one example, a 10mg / kg dose can be administered to portions of 2,5 mg / kg to 2mg / kg and delivered over time to 4 or 5 doses, respectively. A slew-release dosing schedule will allow the desired therapeutic trough levels of NSS-01 to be maintained as the target cells cycle into the S-phase, where the NSS-01 mode of actionis expressed. The NSS-01 test regimen duration, being surprisingly non-toxic, extended up to 4 wee ks .

[0402] For reference, we note certain widely available and / or FDA approved dosing guidance of certain comparator drugs for selected cancers:

[0403] Colorectal Cancer

[0404] Induding: Irinotecan

[0405] Regimens for neoadjuvant therapy for resectable metastatic disease include:

[0406] FOLFIRI: Irinotecan 180 mg / m2IV over 30-90 min on day1 plus leucovorin 400 mg / m2IV infusion to match duration of irinotecan infusion on day 1 plus 5-FU 400 mg / m2IV bolus on day 1, then 1200 mg / m2 / day for 2 days (total 2400 mg / m2over 46-48 hours) continuous infusion; repeat every 2 weeks.

[0407] FOLFiRI plus bevadzumab: Bevacizumab 5 mg / kg over 30-80 min on day 1 plus irinotecan 180 mg / m2IV over 30-90 min on day 1 plus leucovorin 400 mg / m2IV infusion to match duration of irinotecan infusion on day 1 plus 5-FU 400 mg / m2IV bolus on day 1, then 1200 mg / m2 / day for 2 days (total 2400 mg / m2over 46-48 hours) continuous infusion; repeat every 2 weeks for 4-6 cycles

[0408] FOLFIRI plus cetuximab (only for pan-RAS wild-type tumors): Cetuximab 400 mg / m2loading dose over 2 hours on day 1, then cetuximab 250 mg / m2over 1 hour weekly plus irinotecan 180 mg / m2IV over 30-90 min on day 1 plus leucovorin 400 mg / m2IV infusion to match duration of irinotecan infusion on day 1 plus 5-FU 400 mg / m2IV bolus on day 1, then 1200 mg / m2 / day for 2 days (total 2400 mg / m2over 46- 48 hours) continuous infusion; repeatevery 2 weeks for 4-6 cyclesor

[0409] FOLFOXIRI: Irinotecan 165 mg / m2over60 minutes then oxaliplatin 85 mg / m2plus leucovorin 400 mg / m2concurrently over 120 minutes then 5-FU 1600 mg / m2 / day for 2 days (total 3200 mg / m2over 48 hours); repeat every 2 weeks for 4-6 cycles-or

[0410] FOLFOXIRI plus bevacizumab: Bevacizumab 5 mg / kg IV over 30-90 min plus irinotecan 165 mg / m2over 60 minutes then oxaliplatin 85 mg / m2plus leucovorin 400 mg / m2concurrently over 120 minutes then 5-FU 1600mg / m2 / day for 2 days (total 3200 mg / m2over 48 hours); repeat every 2 weeks for 4-6 cycles

[0411] Chemotherapy for advanced or metastatic (colorectal cancer) disease

[0412] Chemotherapy for advanced or metastatic disease includes the use of multiple drugs as single agents or as combination regimens, as follows:• Patients with right-skie tumors are less likely to respond to EGFR therapy with cetuximab or panitumumab.• BRAF V600E mutation makes response to anti-EGFR therapy less likely..• Choice of initial therapy for advanced disease is based on goals of treatment, location of tumor, mutational profile, toxicity profile of the drugs, and patient's performance status.• mFOLFOX6, FOLFIRI, CapeOx, FOLFOXIRI, capecitabine, and infusional 5- FU / LV with or without targeted agents are all considered appropriate first-line agents.• For patients who are not candidates for intensive therapy, single-agent 5-FU / LV, capecitabine, irinotecan, cetuximab or panitumumab, and nivolumab or pembrotizumab, can be used in the appropriate setting.

[0413] Stage IV:

[0414] First-line chemotherapy for bevacizumab candidates includes:

[0415] FOLFIRI plus bevacizumab: Bevacizumab 5 mg / kg over 30-90 min on day 1 plus irinotecan 180 mg / m2IV over 30-90 min on day 1 plus leucovorin 400 mg / m2IV infusion to match duration of irinotecan infusion on day 1 plus 5-FU 400 mg / m2IV bolus on day 1. then 1200 mg / m2 / day for 2d (total 2400 mg / m2over 46 h) continuous infusion; repeat every 2 weeks for 4-6 cycles with reevaluation for maintenance therapy

[0416] FOLFOXIRI plus bevacizumab: Bevadzumab 5 mg / kg over 30-90 min on day 1 plus irinotecan 165 mg / m2IV over 60 mln on day 1 plus oxaliplatin 85 mg / m2IV over 2h on day 1 plus leucovorin 400 mg / m2IV infusion to match duration of irinotecan infusion on day 1 plus 5-FU 1600 mg / m2 / dayfor 2d (total 3200 mg / m2over 48 h) continuous infusion; repeat every 2 weeks.

[0417] First-line chemotherapy for patients who are not candidates for bevacizumab includes:

[0418] FOLFIRI: Irinotecan 180 mg / m2IV over 30-90 mln on day1 plus leucovorin 400 mg / m2IV infusion to match duration of irinotecan infusion on day 1 plus 5-FU 400 mg / m2IV bolus on day 1, then 1200 mg / m2 / day for 2-d (total 2400 mg / m2over 46 h) continuous infusion; repeat every 2 weeks

[0419] FOLFOXIRI: irinotecan 165 mg / m2over 60 minutes then oxaliplatin 85 mg / m2plus leucovorin 200 mg / m2concurrently over 120 minutes, then 5-FU 3200 mg / m2over 48 hours; repeat every 2 weeks

[0420] FOLFIRI plus cetuximab (only for pan-RAS and BRAF V600E wild-type tumors): Cetuximab 500 mg / m2IV over 2 hr every 2 weeks or cetuximab 409 mg / m2loading dose over 2 h on day 1, then cetuximab 250 mg / m2over 1 h weekly plus irinotecan 180 mg / m2IV over 30-90 min on day 1 plus leucovorin 400 mg / m2IV infusion to match duration of irinotecan infusion on day 1 plus 5-FU 400 mg / m2IV bolus on day 1, then 1200 mg / m2 / day for 2-d (total 2400 mg / m2over 46 h) continuous infusion; repeat every 2 weeks

[0421] FOLFIRI plus panitumumab (only for pan-RAS and BRAF V600E wild-type tumors): Panitumumab 6 mg / kg IV infusion over 1 h on day 1 plus irinotecan 180 mg / m2IV over 30-90 min on day 1 plus leucovorin 400 mg / m2IV infusion to match duration of irinotecan infusion on day 1 plus 5-FU 400 mg / m2IV bolus on day 1, then 1200 mg / m2 / day for 2 days (total 2400 mg / m2over 46 h) continuous infusion; repeat every 2 weeks

[0422] For patients with previous oxaliplatin-based therapy as first-line treatment(ie, FOLFOX, CapeOx, CapeOx plus bevacizumab, or FOLFOX plus bevacizumab), one of the following regimens including irinotecan can be used (among others):

[0423] FOLFIRI: Irinotecan 180 mg / m2IV over 30-90 min on day1 plus leucovorin 400 mg / m2IV infusion to match duration of irinotecan infusion on day 1 plus 5-FU 400 mg / m2IV bolus on day 1, then 1200 mg / m2 / day for 2 days (total 2400 mg / m2over 46 h) continuous infusion; repeat every 2 weeks

[0424] FOLFIRI + bevacizumab or ziv-aflibercept or ramucirumab: Irinotecan 180 mg / m2IV over 30-90 min on day 1 plus leucovorin 400 mg / m2IV infusion to match duration of irinotecan infusion on day 1 plus 5-FU 400 mg / m2IV bolus on day 1, then 1200 mg / m2 / day for 2 days (total 2400 mg / m2over 46 h) continuousinfusion plus bevacizumab 5 mg / kg IV on day 1 or ziv-aflibercept 4 mg / kg iv over 60 min on day 1 or ramudrumab 8 mg / kg IV over 60 mins on day 1, repeat every 2 weeks

[0425] FOLFIRI plus cetuximab or panitumumab (only for pan-RAS and BRAF V600E wild-type tumors): Cetuximab 400 mg / m2loading dose IV over 2 h on day 1, then cetuximab 250 mg / m2IV over 1 h weekly or panitumumab 6 mg / kg IV over 60 min on day 1 plus irinotecan 180 mg / m2IV over 30-90 min on day1 plus leucovorin 400 mg / m2IV infusion to match duration of irinotecan infusion on day1 plus 5-FU 400 mg / m2IV bolus on day 1, then 1200 mg / m2 / day for 2-d (total 2400 mg / m2over 46-48 h) continuous infusion; repeat every 2 weeks tor 4-6 cycles, then reevaluate

[0426] irinotecan plus cetuximab or panitumumab (only for pan-RAS and BRAF V600E wild-type tumors): Irinotecan 180 mg / m2IV over 30-90 min on day 1 plus cetuximab 400 rng / m2loading dose over 2 h on day 1, then cetuximab 250 mg / m2over 1 h weekly or panitumumab 6 mg / kg iv over 60 min on day 1; repeat every2 weeks

[0427] For patients who had irinotecan therapy as first-line treatment (ie., FOLFIR1 plus bevacizumab), the following regimens (among others) can be used:

[0428] Irinotecan plus cetuximab or panitumumab (only for pan- RAS and BRAF V600E wild-type tumors, and if anti-EGFR therapy was not used in combination with FOLFIRI): Irinotecan 180 mg / m2IV over 30-90 min on day 1 plus cetuximab 500 mg / m2IV over 2 h every 2 wk or cetuximab 400 mg / m2loading dose IV over 2 h on day 1, then cetuximab 250 mg / m2IV over 1 h weekly or panitumumab 6 mg / kg IV oyer 60 min on day 1; repeat every 2 weeks

[0429] Single agent regimen 1: Irinotecan 125 mg / m2 IV over 90 minutes on Days 1 ,8,15, and 22 then 2 week rest.

[0430] Single agent regimen 2: Irinotecan 350 mg / m2 IV over 90 minutes on Day l every3weeks

[0431] Small Cell Lung Cancer

[0432] Topotecan

[0433] indicated for SCLC sensitive disease after failure of first-line chemotherapy (for relapsed or refractory disease):

[0434] IV Infusion - 1.5mg / m2IV qDay x 5 days (total 7.5 mg / m2); repeat at 21- day cycles

[0435] Capsules -2.3mg / m2PO QD x 5 days (total 11.5 mg / m2); repeat at 21- day cycles

[0436] Cervical Cancer

[0437] Topotecan

[0438] First-line therapy for stage IV recurrent or metastatic disease protocols include:

[0439] Bevacizumab 15 mg / kg IV over 30-90 min plus paditaxel 175 mg / m2IV over 3 h on day 1 plus topotecan 0.75 mg / m2IV over 30 min on days 1 -3 every 3 weeks (category 1)

[0440] Other combinations— induding cisplatin / topotecan, carboplatin / padltaxel, and topotecan / paditaxel— can also be considered for appropriate individuals (category 2A)

[0441] Second-line therapy for stage IV recurrent or metastatic disease:

[0442] Agents that the National Comprehensive Cancer Network (NCCN) recommends as useful in second-fine therapy (category 2B) include the following (among others):Topotecan• Irinotecan• Docetaxel• 5-FU• Gemcitabine• Albumin-bound paclitaxel• CisplatinCarboplatin

[0443] Ovarian Cancer

[0444] Topotecan

[0445] Treatment recommendations for recurrent disease

[0446] Platinum-resistant recurrence:_If recurrence occurs less than 6 months after initial or subsequent complete dinical response to platinum-containingchemotherapy , the patient should be treated with one of the regimens below. There is no standard for the number of cycles of treatment given in this situation. Often treatment is changed because of progressive disease or toxicity ..Although liposomal doxorubicin is a good first choice, many other agents with similar efficacy are available; the final choice depends on individual circumstance and patient and physician preference. Some of the choices include the following:• Liposomal doxorubicin 40-50 mg / m2IV over 30 min; every 21 daysor• Gemcitabine 1000 mg / m2IV over 30 min on Days 1 and 8; every 21 days or• Topotecan 1.25-1.5 mg / m2IV over 30 min on Days 1-5; every 21 days or• Paclitaxel 80 mg / m2IV over 1 h weekly or• Docetaxel 75-100 mg / m2IV over 1 h every 21 days or• Etoposide 50 mg / m2 / day PO for 21 days every 28 days or• Nanopartide albumin-bound paclitaxel 100 mg / m2IV over 30 min given weekly (Days 1, 8, and 15) every 28 days

[0447] Alternatively , consideration may be given to bevacizumab as a single agent or in combination, as follows:• Bevacizumab 10 mg / kg IV every 14 d in combination with one of the following IV chemotherapy regimens: paditaxel, pegylated liposomal doxorubicin, or topotecan (topotecan is given weekly) or• Bevacizumab 15 mg / kg IV every 21 d in combination with topotecan (every 21 d)or• Bevacizumab 15 mg / kg IV (initially over 90 min, then over 60 min, and finally over 30 min for subsequent infusions); every 21 d until progression or• Bevacizumab 10 mg / kg IV on days 1 and 15 plus topotecan 4 mg / m2IV on Days 1, 8, and 15, every 28 days• Bevacizumab 15 mg / kg IV on Day 1 every 21 days plus olaparib 300 mg PO BID; continue olaparib until disease progression, unacceptable toxicity, or completion of 2 years of treatment Bevacizumab should be given for a total of 15 months including with chemotherapy and given as maintenance. Discontinue olaparib in patients with a complete response (no radiological evidence of disease) at 2 years.

[0448] Pancreatic Cancer

[0449] Common treatment recommendations for resectable local disease:

[0450] Stages / - / /

[0451] Neoadjuvant therapy

[0452] National Comprehensive Cancer Network (NCCN) guidelines list the following regimens as preferred for neoadjuvant therapy:• FOLFIRINOX (oxaliplatin 85 mg / m2IV, leucovorin 400 mg / m2IV, irinotecan 180 mg / m2IV, 5-fluorouracil [5-FU]) 400 mg / m2IV bolus then 2400 mg / m2IV; with or without subsequent chemoradiation• Modified FOLFIRINOX (mFOLFIRINOX; oxaliplatin 85 mg / m2, leucovorin 400 mg / m2, irinotecan 150 mg / m2, 5-FU 2.4 g / m2over 46 hours) ± subsequent dhemoradiation• Gemcitabine + albumin-bound paclitaxel ± subsequent chemoradiation• in patients with known BRCA1 / 2 or PALB2 mutations: FOLFIRINOX or mFOLFIRINOX ± subsequent chemoradiation or gemcitabine + cisplatin (2—6 cycles) ± subsequent chemoradiation

[0453] Adjuvant chemotherapy:• Gemcitabine monotherapy has been the standard of care since the CONKO-001 trial in 2008• Gemcitabine 1000 mg / m2IV over 30 min weekly for 3 wk; every 4 wk for six cycles• mFOLFIRINOX, in the Unicancer Gl PRODIGE 24 / CCTG PA.6 trial, demonstrated superior results compared with gemcitabine monotherapy (median overall survival of 54.5 versus 35 months, respectively)• mFOLFIRINOX: Every 14 days for 12 cycles

[0454] Treatment recommendations for metastatic disease

[0455] First-line treatment recommendations for advanced metastatic pancreatic cancer (stage IV):• Paclitaxel protein bound 125 mg / m2plus gemcitabine 1000 mg / m2IV over 30-40 min on days 1, 8, and 15 of each 28-day cycle• Gemcitabine 1000 mg / m2IV over 30 min weekly for 7 wk, followed by 1 wk off, then weekly for 3 wk; every 28 days or• Gemcitabine 1000 mg / m2IV over 30 min on days 1 and 15 plus cisplatin 50 mg / m2IV over 1 h on days 1 and 15; every 28 days or• Gemcitabine 1000 mg / m2IV weekly for 7 wk plus erlotinib 100 mg PO daily on days 1-56, followed by 1 wk off; then gemcitabine 1000 mg / m2IV on days 1, 8, and 15 every 28 d plus erlotinib 100 mg PO daily on days 1-28 for upto four cycles or• Gemcitabine 1000 mg / m2IV weekly for 3 wk; every 28 d; plus capecitabine 1660 mg / m2 / day weekly for 3 wk; every 28 d or• For patients with stage IV disease, median overall survival on gemcitabine-based therapy is from 5.5 to 7 mo; the non-gemcitabine-based regimen FOLFIRINOX (5-FU / leucovorin, irinotecan, and oxaliplatin) showed improved survival of 11.1 mo : Oxaliplatin 85 mg / m2IV on day 1 plus irinotecan 180 mg / m2IV on day1 plus leucovorin 400 mg / m2IV on day 1, followed by 5-FU 400 mg / m2IV bolus on day 1 and then 2400 mg / m2IV infusion over 46 h on days 1 and 15• Irinotecan liposomal 50 mg / m2IV (dose is regardless of UGT1A1*28 allele genotype), followed by oxaliplatin 60 mg / m2 IV, followed by leucovorin 400 mg / m2IV, fallowed by 5-FU 2400 mg / m2IV inftised over 46 h every 3 wk

[0456] Second-line treatment recommendations for advanced metastatic pancreatic cancer:• Capecitabine 1250 mg / m2PO BID for 14d; every 3 wkor• Capecitabine 1000 mg / m2PO BID for 14 d; every 3 wk plus erlotinib 150 mg PO daily continuously or• Irinotecan liposomal 70 mg / m2IV infused over 90 min, followed by leucovorin 400 mg / m2IV infused over 30 min, followed by 5-FU 2400 mg / m2IV infused over 46 h every 3 wkor• 5-FU 2000 mg / m2IV over 24 h tin days 1,8, 15, and 22 plus leucovorin 200 mg / m2IV over 30 min on days 1, 8, 15, and 22 plus oxaliplatin 85 mg / m2IV on days8 and 22; every 42 d

[0457] Those skilled in the art to which the present invention pertains may make modifications resulting in other embodiments employing principles of the present invention without departing from its spirit or characteristics, particularly upon considering the foregoing teachings. Accordingly, the described embodiments are to be considered in all respects only as illustrative, and not restrictive, and the scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description dr drawings. Consequently, while the present invention has been described with reference to particular embodiments, modifications of structure, sequence, materials, and the like apparent to those skiRed in the art, still fell within the scope of the invention as claimed by the applicant.While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not Bmlt the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. What is claimed is:

1. A method of treating a subject afflicted with cancer, the method comprising administering to the subject a composition comprising 5,6-dihydro-4H-benzo[de] quinoline-camptothecin or a pharmaceutically acceptable salt thereof.

2. The method of claim 1 , wherein the 5,6-dihydro-4H-benzo[de] quinoline-camptothecin prevents or reduces cancer cell proliferation and / or increases cancer cell death in the presence of HSA.

3. The method of claim 1 , wherein the cancer is a solid tumor or a liquid cancer.

4. The method of claim 1 , wherein the cancer is an adenocarcinoma or a sarcoma.

5. The method of claim 1 , wherein the cancer has metastasized.

6. The method of claim 1 , wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

7. The method of claim 1 wherein the composition is administered to the patient by infusion, oral or injection.

8. The method of claim 1 , wherein composition comprises the S isomer of 5,6-dihydro- 4H-benzo[de] quinoline-camptothecin, the R isomer of 5,6-dihydro-4H-benzo[de] quinoline-camptothecin, or both.

9. The method of claim 1 , wherein the composition is administered locally, regionally, or systemically.

10. The method of claim 1 , wherein the subject has been, is presently, or will be administered one or more additional anti-cancer therapies.

11. The method of claim 10, wherein the one or more additional anti-cancer therapy comprises surgery, chemotherapy, immunotherapy, radiotherapy, or any combination thereof.

12. A method of inhibiting the growth of one or more tumors in a subject, the method comprising administering to the subject an effective amount of a composition comprising 5,6-dihydro-4H-benzo[de] quinoline-camptothecin or a pharmaceutically acceptable salt thereof.

13. A method(s) of synthesizing 5,6-dihydro-4H-benzo[de] quinoline-camptothecin) ("NSS-01"), the method(s) comprising(a) employing Friedlander’s condensation wherein synthone 1 ("S1”): ((S)-4- Ethyl-4-hydroxy-7,8-dihydro-1 H-pyrano[3,4-f]indolizine- 3,6,10(4H)-trione); and synthone 2 ("S2”): (8 - amino - 5 - hydroxy - 1 ,2,3,4 - tetrohydronaphtalene -1 - one) are dissolved in an organic solvent in a levi’s catalyst and heated to about 60° to about 180° C for a period of 10 minutes to 24 hours, which product is then purified; or(b) a microwave based synthesis of S1 and S2; to yield 5,6-dihydro-4H-benzo[de] quinoline-camptothecin (NSS-01), which product is then purified.

14. A camptothecin derivative, 5,6-dihydro-4H-benzo[de] quinoline-camptothecin) ("NSS-01 ”), synthesized by the method(s) of claim 13.

15. A formulation for treatment of cancer, wherein the active ingredient is 5,6-dihydro- 4H-benzo[de] quinoline-camptothecin (NSS-01 ) synthesized by the method(s) of claim 13.

Images