Functionally Modified Maytansine Alkaloids and Their Compositions and Methods of Use

Abstract

The present invention provides radiosensitizer prodrugs, their formulations, and methods of use. Generally, a radiosensitizer prodrug is an analogue of a radiosensitizer parent compound having one or more S-nitrosothiol moieties. Generally, the S-N bond of the S-nitrosothiol moiety can be radiolytically cleaved during radiotherapy, releasing the parent compound and nitric oxide. One or preferably both of the parent compound and nitric oxide can promote the death of tumor cells exposed to radiotherapy. Also provided are nanoparticle formulations for delivering the prodrugs, and methods of using them in combination with radiotherapy to treat tumors and cancers.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit and priority of U.S.S.N. 62 / 931,058, filed on November 5, 2019, which is incorporated herein by reference in its entirety.

[0003] Reference to Sequence Listing

[0004] The sequence listing, submitted as a text file named "UGA_2020_078_PCT_ST25", was created on October 26, 2020, and is 2,105 bytes in size, and is incorporated herein by reference in accordance with 37 C.F.R.§1.52(e)(5). Field of the Invention

[0005] The field of the present invention generally relates to functionally modified maytansinoids and their compositions or methods of use (particularly methods for treating cancer). Background of the Invention

[0007] Radiation therapy (RT) remains a major treatment option for cancer (Tang et al., J. Exp. Clin. Canc. Res. 37:87 (2018)). Although new radiation delivery techniques have been developed (e.g., 3D conformal radiation therapy, intensity - modulated radiation therapy, and image - guided radiation therapy), the maximum radiation dose that patients can tolerate has changed little (Ramroth et al., Int. J. Radiat. Oncol., Biol., Phys. 96, 736 - 747 (2016)). To improve treatment outcomes, radiosensitizers (i.e., agents that can enhance radiation toxicity at a given physical radiation dose) are typically administered during RT. These include chemotherapeutic agents such as 5 - fluorouracil, anthracyclines, paclitaxel, and platinum, which are commonly used concurrently with RT (i.e., chemoradiation) (Ferguson et al., Drug Resist. Updates 4, 225 - 232 (2001)).

[0008] To improve bioavailability and reduce systemic toxicity, nanoparticle radiosensitizers have recently been developed and explored (Wang et al., Trends Pharmacol. Sci. 39, 24 - 48 (2018), Kuncic & Lacombe, Phys. Med. Biol. 63, 02TR01 (2018)). Different from small molecule therapeutics, nanoparticles can selectively accumulate in tumors via the enhanced permeability and retention (EPR) effect and / or ligand - receptor interactions, thereby reducing the exposure of normal tissues to toxins. For example, therapeutics such as docetaxel (Werner et al., ACS Nano 5, 8990 - 8998 (2011)), wortmannin (Karve et al., Proc. Natl. Acad. Sci. U.S.A. 109, 8230 - 8235 (2012)), and histone deacetylase inhibitors (Wang et al., Biomaterials 51, 208 - 215 (2015)) can be delivered to tumors via nanoparticle carriers to enhance RT. Polyglutamate - conjugated paclitaxel nanoparticles (also known as Paclitaxel poliglumex (Xyotax)) have been clinically tested to improve RT for gliomas (Kulhari et al., Nanomedicine 12, 1661 - 1674 (2017), Jeyapalan et al., Am. J. Clin. Oncol. 37, 444 - 449 (2014)) and RT for head and neck cancers (Hahn et al., J. Clin. Oncol. 31, 6059 - 6059 (2013)).

[0009] Despite these achievements, there remains a need for improved radiosensitizer compositions.

[0010] Accordingly, an object of the present invention is to provide improved radiosensitizer compounds, their formulations, and methods of use. SUMMARY OF THE INVENTION

[0011] Radiosensitizer prodrugs, their formulations, and methods of use are provided. Generally, a radiosensitizer prodrug is an analogue of a radiosensitizer parent compound having one or more S - nitrosothiol moieties. Generally, the S - N bond of the S - nitrosothiol moiety can be radiolytically cleaved during radiotherapy, releasing the parent compound and nitric oxide. One or preferably both of the parent compound and nitric oxide can promote the death of tumor cells exposed to radiation. In a preferred embodiment, the radiation is ionizing radiation, such as that administered as radiotherapy. The compounds can also be used as sensitizers for phototherapy and / or proton therapy.

[0012] Preferred prodrugs are compounds containing the following structural motifs:

[0013]

[0014] Wherein:

[0015] linker represents a linking group,

[0016] n is an integer from 1 to 13, including the end values,

[0017] The dashed line indicates the presence or absence of a bond, and the corresponding carbon atom has no, one, or two hydrogen atoms respectively attached according to its valence, and

[0018] "Linking group" is independently absent, a substituted amide group, an unsubstituted amide group, a substituted alkyl group, a substituted alkylene group, an unsubstituted alkylene group, a substituted aryl group, a substituted heteroaryl group, a substituted alkenyl group, a substituted alkynyl group, a substituted alkoxy group, a substituted aryloxy group, a substituted alkylthio group, a substituted arylthio group, an unsubstituted carbonyl group, a substituted carbonyl group, an unsubstituted carboxyl group, a substituted carboxyl group, an unsubstituted amino group, a substituted amino group, an unsubstituted sulfonyl group, a substituted sulfonyl group, an unsubstituted aminosulfonyl group, a substituted aminosulfonyl group, an unsubstituted phosphonyl group, a substituted phosphonyl group, a substituted polyaryl, a substituted C3-C 20 cyclic group or a substituted C3-C 20 heterocycle.

[0019] In some embodiments, the compound has the following structure:

[0020]

[0021] Wherein:

[0022] R1 is a substituted amide group, an unsubstituted amide group, a substituted alkyl group, a substituted alkylene group, an unsubstituted alkylene group, a substituted aryl group, a substituted heteroaryl group, a substituted alkenyl group, a substituted alkynyl group, a substituted alkoxy group, a substituted aryloxy group, a substituted alkylthio group, a substituted arylthio group, an unsubstituted carbonyl group, a substituted carbonyl group, an unsubstituted carboxyl group, a substituted carboxyl group, an unsubstituted amino group, a substituted amino group, an unsubstituted sulfonyl group, a substituted sulfonyl group, an unsubstituted aminosulfonyl group, a substituted aminosulfonyl group, an unsubstituted phosphonyl group, a substituted phosphonyl group, a substituted polyaryl, a substituted C3-C 20 cyclic group or a substituted C3-C 20 heterocycle, and

[0023] R2, R3, R4, R5, R6, R7, R8, R9, R 10 and R 11Independently is hydrogen, halogen (F, Br, Cl, I), substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, unsubstituted alkenyl, substituted alkenyl, unsubstituted alkynyl, substituted alkynyl, unsubstituted alkoxy, substituted alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted alkylthio, substituted alkylthio, unsubstituted arylthio, substituted arylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted amino, substituted amino, unsubstituted sulfonyl, substituted sulfonyl, unsubstituted aminosulfonyl, substituted aminosulfonyl, unsubstituted phosphonyl, substituted phosphonyl, unsubstituted polyaryl, substituted polyaryl, unsubstituted C3-C 20 cyclic group, substituted C3-C 20 cyclic group, unsubstituted C3-C 20 heterocycle or substituted C3-C 20 heterocycle, or R2 and R3 together with the carbon atom to which they are bonded form an epoxide.

[0024] In some embodiments, R1 is a substituted C1-C10 amido group, an unsubstituted C1-C10 amido group, a substituted C1-C10 alkyl group, an unsubstituted C1-C10 alkylene group, a substituted C1-C10 alkylene group, an unsubstituted C1-C10 alkylene group, a substituted aryl group, a substituted heteroaryl group, a substituted C2-C10 alkenyl group, a substituted C2-C10 alkynyl group, a substituted C1-C10 alkoxy group, a substituted aryloxy group, a substituted C1-C10 alkylthio group, a substituted arylthio group, an unsubstituted C1-C10 carbonyl group, a substituted C1-C10 carbonyl group, an unsubstituted C1-C10 carboxyl group, a substituted C1-C10 carboxyl group, an unsubstituted C1-C10 amino group, a substituted C1-C10 amino group, an unsubstituted C1-C10 sulfonyl group, a substituted C1-C10 sulfonyl group, an unsubstituted C1-C10 aminosulfonyl group, a substituted C1-C10 aminosulfonyl group, an unsubstituted C1-C10 phosphonyl group, a substituted C1-C10 phosphonyl group, a substituted polyaryl group, a substituted C3-C 10 cyclic group or substituted C3-C 10 heterocycle, preferably wherein R1 is a substituted C1-C10 amido group or an unsubstituted C1-C10 amido group.

[0025] In some embodiments, R1 has the following structure:

[0026]

[0027] wherein R 12 is a substituted C1-C5 alkylene group or an unsubstituted C1-C5 alkylene group, R 13is hydrogen, a substituted C1-C5 alkyl, or an unsubstituted C1-C5 alkyl, and R 14 is a substituted C1-C5 alkylene or an unsubstituted C1-C5 alkylene, preferably R 12 is a substituted C1-C5 alkylene (preferably -CH(CH3)-), R 12 is an unsubstituted C1-C5 alkyl (preferably -CH3), and R 14 is an unsubstituted C1-C5 alkylene (preferably -(CH2)2-).

[0028] In some embodiments, the compound has a structure selected from the following:

[0029]

[0030] In some embodiments, when present, R2, R3, R4, R5, R6, R7, R8, R9, R 10 and R 11 are independently hydrogen, hydroxy, halogen (F, Br, Cl, I), substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, unsubstituted C1-C5 alkenyl, substituted C1-C5 alkenyl, unsubstituted C1-C5 alkynyl, substituted C1-C5 alkynyl, unsubstituted C1-C5 alkoxy, substituted C1-C5 alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted C1-C5 alkylthio, substituted C1-C5 alkylthio, unsubstituted arylthio, substituted arylthio, unsubstituted C1-C5 carbonyl, substituted C1-C5 carbonyl, unsubstituted C1-C5 carboxyl, substituted C1-C5 carboxyl, unsubstituted C1-C5 amino, substituted C1-C5 amino, unsubstituted C1-C5 sulfonyl, substituted C1-C5 sulfonyl, unsubstituted C1-C5 sulfamoyl, substituted C1-C5 sulfamoyl, unsubstituted C1-C5 phosphonyl, substituted C1-C5 phosphonyl, unsubstituted polyaryl, substituted polyaryl, unsubstituted C3-C6 cyclic group, substituted C3-C6 cyclic group, unsubstituted C3-C6 heterocycle or substituted C3-C6 heterocycle.

[0031] In some embodiments, when present, R2 and R3 together with the carbon atoms to which they are attached form an epoxide.

[0032] In some embodiments, when present, R4, R5, R6, and R7 are independently hydrogen, hydroxy, halogen (F, Br, Cl, I), substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl. Preferably, R4, R5, and R6 are hydrogen, and R7 is methyl.

[0033] In some embodiments, when present, R8 is hydrogen, hydroxy, halogen (F, Br, Cl, I), substituted C1-C5 carboxyl, unsubstituted C1-C5 carboxyl, substituted C1-C5 carbonyl, or unsubstituted C1-C5 carbonyl. Preferably, R8 is hydrogen, hydroxy, substituted C1-C5 carboxyl, or unsubstituted C1-C5 carboxyl, or preferably R8 is hydrogen.

[0034] In some embodiments, when present, R9 is hydrogen, substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted C1-C5 carbonyl, or unsubstituted C1-C5 carbonyl. Preferably, R9 is unsubstituted C1-C5 alkyl, or preferably R9 is methyl.

[0035] In some embodiments, when present, R 10 is halogen (F, Cl, Br, I), substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted C1-C5 carbonyl, or unsubstituted C1-C5 carbonyl. Preferably, R 10 is halogen, or preferably R 10 is Cl.

[0036] In some embodiments, when present, R 11 is hydrogen, substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl. Preferably, R 11 is unsubstituted C1-C5 alkyl, or preferably R 11 is methyl.

[0037] In a specific embodiment, the prodrug compound has the following structure:

[0038]

[0039] Other radiosensitizer parent compounds can also be modified to include one or more S-nitrosothiol moieties. Such parent compounds include, but are not limited to, nicotinamide, metronidazole, or analogs thereof, optionally selected from misoniszole, etanidazole, and nimorazole; hypoxic cell cytotoxins, optionally selected from mitomycin-C and tirapazamine; membrane active agents, optionally selected from procaine, lidocaine, and chlorpromazine; radiosensitizing nucleosides, optionally selected from 5-fluorouracil, fluorodeoxyuridine, bromodeoxyuridine, iododeoxyuridine, hydroxyurea, gemcitabine, and fludarabine, texaphryin, optionally selected from motexafingadolinium; inhibitors of sulfhydral groups, optionally selected from N-ethylmaleimide, diamides, and diethyl maleate; chemotherapeutic agents, optionally selected from paclitaxel, docetaxel, irinotecan, and cisplatin; pentoxifylline; vinorelbine; PARP inhibitors; histone deacetylase inhibitors, and proteasome inhibitors.

[0040] In some embodiments, the prodrug is formulated with a nanoparticle delivery vehicle. The particles can be, for example, polymeric nanoparticles, liposomes, inorganic nanoparticles, or proteins.

[0041] In some embodiments, the nanoparticles are polymeric nanoparticles formed from one or more amphiphilic, hydrophobic, and / or hydrophilic polymers.

[0042] For example, in some embodiments, the particles include one or more polyester hydrophobic polymers such as poly(lactic-co-glycolic acid), poly(lactic acid), and / or poly(glycolic acid). In particular embodiments, the nanoparticles include poly(lactic-co-glycolic acid) (PLGA).

[0043] In some embodiments, the nanoparticles additionally or alternatively comprise one or more hydrophilic polymers. The hydrophilic polymer can be a polyalkylene glycol. In some embodiments, the nanoparticles comprise polyethylene glycol (PEG). In one specific embodiment, the nanoparticles are polymeric nanoparticles that comprise poly(lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG).

[0044] In some embodiments, the nanoparticles have a size or size distribution of from about 10 nm to about 300 nm or from about 20 nm to about 200 nm. Additionally or alternatively, the particles can have an average size of any size from about 10 nm to about 300 nm, or from about 20 nm to about 200 nm, or from about 50 nm to about 150 nm, or from about 50 nm to about 100 nm, or from about 50 nm to about 75 nm. Generally, the particles generally have a size or size range suitable for delivering a compound (preferably by enhanced permeability and retention) to the tumor microenvironment. In some embodiments, the nanoparticles have a targeting agent conjugated thereto. For example, in some embodiments, the targeting agent targets NTSR1, typically by binding to the type 1 neurotensin receptor (NTSR1). The targeting agent can be an NTSR1 agonist or antagonist. In some embodiments, the targeting agent is neurotensin (NTS) or a variant or derivative thereof, such as NTS mut . In other embodiments, the targeting agent is SR142948A or a derivative thereof.

[0045] Also provided are pharmaceutical compositions and nanoparticle formulations thereof comprising an effective amount of the disclosed prodrug compounds.

[0046] Also provided are methods of using the compounds, nanoparticle formulations, and pharmaceutical compositions. For example, a method of treating a subject in need thereof can include administering to the subject an effective amount of the disclosed prodrug compound or its nanoparticle formulation, which is preferably in a pharmaceutical composition. The subject can have a benign or malignant tumor. In a preferred embodiment, the subject has cancer.

[0047] Typically, the subject is a person who would benefit from a radiation-based therapy, which includes but is not limited to ionizing radiation therapy, phototherapy, or proton therapy. Thus, the method can further include administering to the subject one or more doses of ionizing radiation therapy, phototherapy, or proton therapy. Typically, a dose of ionizing, phototherapy, or proton therapy radiation is administered after (e.g., minutes, hours, or days) administering the pharmaceutical composition comprising the prodrug. For example, in an exemplary embodiment, a dose of radiation is administered 1 hour to 48 hours, or 1 hour to 24 hours, or 1 hour to 12 hours, or 1 hour to 6 hours, or 2 hours to 6 hours, or 1, 2, 3, 4, or 5 hours after administering the pharmaceutical composition. In some embodiments, 1, 2, 3, 4, or 5 rounds of radiation are administered after each single dose of the prodrug. In some embodiments, the prodrug is administered one or more times for each round of radiation. In some embodiments, a prodrug cycle precedes each radiation cycle. For example, in a particular embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more rounds of administration of the pharmaceutical composition are carried out one after the other, followed by the administration of a dose of radiation.

[0048] Typically, the compound enhances the treatment of cancer compared to administering radiation alone. In some embodiments, the cancer is a radiation-sensitive cancer. In other embodiments, the cancer is a radiation-resistant cancer. The cancer can be, for example, angiosarcoma, osteosarcoma, rhabdomyosarcoma, bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, or germ cell cancer. The cancer can be an epithelial cancer. In a specific embodiment, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is composed of cells having upregulated NTSR1. Preferably, the same dose of radiation is more effective than when administered in the absence of the prodrug compound, a lower dose of radiation has the same efficacy as a higher dose administered in the absence of the prodrug compound, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1A and 1B is a schematic diagram illustrating how DM1-NO-encapsulated PLGA-b-PEG nanoparticles (DM1-NO-NP) can accumulate in tumors via the EPR effect. In the presence of radiation and / or a reduced pH in endosomes / lysosomes, the S-N bond breaks, releasing DM1 and NO. DM1 inhibits microtubule assembly, arresting cells in the G2 / M phase, which is more radiation-sensitive. At the same time, NO can react with ROS to form free radicals such as peroxynitrite, causing DNA and lipid damage. The combined action enhances the efficacy of RT. Figure 1CIt is a bar graph which shows that in the presence of 6 Gy X-ray irradiation, the NO release using DM1-NO increased from 3.68 μM to 18.88 μM, and the NO release using DM1-NO-NP increased from 2.96 μM to 13.87 μM. Quantification was based on the Greiss assay. PBS, cell culture medium (RPBI-1640), DM1, and DM1-NP were tested as controls. Figure 1D It is a histogram showing the DLS analysis results of DM1-NO-NP in water. Figure 1E It is a graph showing the ζ potential of DM1-NO-NP in water. Figure 1F It is a bar graph illustrating the stability of DM1-NO under environmental conditions. After synthesis, the dried product of DM1-NO was kept at room temperature, and the total amount of remaining NO was quantified using the Griess assay on days 0, 7, 14, and 21. Figure 1G It is a graph illustrating the stability of DM1-NO in solution. DM1-NO (20.09 μmol) was dissolved in PBS at different pH values (5.5, 6.5, and 7.4). Time-dependent NO release was measured on a Sievers NOA280i system which evaluates NO based on the gas-phase chemiluminescence reaction between NO and ozone. Figure 1H It is a line graph showing the release curve of DM1-NO-NP, tested in PBS at different pH values by the Greiss assay at 37 °C. Figure 1I It is a histogram showing the DLS analysis results of DM1-NP. Figure 1J It is a graph showing the ζ potential of DM1-NP. Figure 1K It is a series of images of representative colony formation assays. H1299 cells were treated with 20 nM of DM1 and DM1-NO for 12 hours, followed by -X-ray irradiation (6 Gy). The resulting cells were plated for colony formation assays. Figure 1L It is a graph showing the UV-vis analysis of DM1-NO, PLGA-b-PEG, and DM1-NO-NP. Figure 1M It is a graph showing the FT-IR analysis of DM1-NO, PLGA-b-PEG, and DM1-NO-NP. Figure 1N It is a graph showing the time-dependent DLS analysis results (hydrodynamic size) of DM1-NO-NP incubated in PBS for 24 hours.

[0050] Figure 2A It is a line graph showing cell viability, tested with H1299 cells using the MTT assay at 72 hours. DM1, DM1-NO, DM1-NP, and DM1-NO-NP were studied. Figure 2BIt is a line graph showing the results of a colony formation assay, which was tested with H1299 cells. DM1+RT, DM1-NO+RT, and RT alone were tested separately. The results were fitted to a linear quadratic (LQ) model.

[0051] Figures 3A - 3D It is a bar graph showing the effect on intracellular oxidative stress. The study was conducted with H1299 cells incubated with DM1, DM1-NO, DM1-NP, DM1-NO-NP, or PBS. Figures 3A and 3B show the cytosolic and mitochondrial SOD activities: the SOD levels without X-ray irradiation (3A), and the SOD levels when the cells were first incubated with DM1, DM1-NO, DM1-NP, DM1-NO-NP, or PBS and then irradiated with X-rays (6 Gy) (3B). Figure 3C shows the change in intracellular ·OH radical levels evaluated by the MB assay. A decrease in absorption at 664 nm indicates an increase in ·OH levels. All cells were irradiated with 6 Gy. Figure 3D shows the intracellular 1 O2, which was evaluated by measuring SOSG fluorescence at 525 nm. All cells were irradiated with 6 Gy. Based on the results of at least three repeated experiments, the data are presented as mean ± standard. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant difference.

[0052] Figure 4A It is a bar graph showing the median fluorescence intensity (MFI) of DAF-FM. H1299 cells were incubated with 20 nM DM1, DM1-NO, DM1-NP, DM1-NO-NP, or PBS for 12 hours and then irradiated with 6 Gy. The cells were stained with DAF-FM (for NO) and Eth-III (for membrane-ruptured cells), and imaged by confocal microscopy. Figure 4B It is a bar graph showing the intracellular NO levels measured by the Greiss assay. H1299 cells were incubated with DM1, DM1-NO, DM1-NP, DM1-NO-N, or PBS and then irradiated with (w / RT) or without (w / o RT) 6 Gy. Figure 4C It is based on Figure 4A The imaging results of the assay described in show the MFI of Eth-III. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant difference.

[0053] Figure 5AIt is a bar graph showing the relative increase in MFI compared to the control. H1299 cells were incubated with 20 nM DM1, DM1-NO, DM1-NP, DM1-NO-NP, or PBS for 6 hours and then received 6 Gy irradiation. Peroxynitrite sensor green staining was performed to measure ONOO- in the cells. For the control, the cells received neither drug incubation nor irradiation. The cells were imaged by confocal microscopy. Figure 5B It is a bar graph showing relative fluorescence units (RFU). H1299 cells received the same treatment as described in Figure 5A and the fluorescence activity was measured on a microreader instead of a confocal microscope. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant difference.

[0054] Figure 6A It is a bar graph showing the number of foci per cell. Anti-γH2AX staining was performed 12 hours after treatment of H1299 cells with 20 nM DM1-NO-NP plus 6 Gy irradiation (DM1-NO-NP+RT). DM1-NP+RT, DM1-NO+RT, DM1+RT, RT alone, and untreated cells were studied for comparison. The cells were imaged and analyzed by Image-J. Figure 6B It is a bar graph showing the level of lipid peroxidation, which was evaluated by measuring the red / green (590 / 510 nm) fluorescence ratio using the BOBIPY lipid assay. H1299 cells were treated with 20 nM DM1-NO-NP for 12 hours, with and without 6 Gy irradiation (DM1-NO-NP+RT). DM1-NP+RT, DM1-NO+RT, DM1+RT, RT alone, and untreated cells were studied for comparison. *, P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant difference.

[0055] Figures 7A - 7E It is a graph showing the results of cell cycle analysis based on PI staining and evaluated by flow cytometry. Before analysis, H1299 cells were incubated with PBS (7A), DM1 (7B), DM1-NO (7C), DM1-NP (7D), or DM1-NO-NP (20 nM, DM1 concentration) (7E). Figure 7F It is a bar graph showing the fraction of cells in S, G0 / G1, or G2 / M phase, which is based on the analysis results described in Figures 7A - 7E *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant difference. Figure 7GIt is a bar graph showing the percentage of tubulin inhibition. Tubulin polymers were collected by centrifugation (35000×g, 1 h, at 30 °C), and the amount of tubulin precipitate was quantified by measuring the protein concentration. ***, P < 0.001. Figure 7H It is a line graph showing cell viability, which was tested with H1299 cells using 72-h MTT in the presence of RT. DM1, DM1-NO, and DM1-NO-NP were studied.

[0056] Figures 8A - 8D It shows the results of the therapy study conducted on H1299 tumor-bearing nude mice. The animals received intravenous injection of PBS, DM1, DM1-NO, DM1-NP, or DM1-NO-NP, and then received X-ray irradiation (6 Gy) 4 hours later. The animals receiving only PBS injection were studied as controls. Figure 8A It is a series of tumor photos of all treatment groups dissected on the 24th day. Figure 8B It is a graph showing the weights of excised tumor masses from all treatment groups. Figure 8C It is a tumor growth curve. Significant tumor inhibition was observed in the animals of the DM1-NO-NP + RT group. Figure 8D It is a body weight chart. No obvious body weight loss was observed in the animals of the DM1-NO-NP + RT group. Scale bar, 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant difference.

[0057] Figures 9A - 9C It shows the results of the hematological analysis for toxicity assessment. Blood was collected from balb / c mice (n = 3) that received intravenous injection of 260.8 nmol / kg (equivalent to the dose used in the therapy study) of DM1-NO-NP or DM1 or PBS on the 10th day. Figures 9A - 9C It is a bar graph showing the counts of red blood cells (RBC) and white blood cells (WBC) (9A), platelet count (9B), and plateletcrit (PCT, mean platelet volume or platelet distribution width) count (9C). ns, no significant difference. All the indicators in 9A - 9C are within the normal range.

[0058] Figures 10A - 10GShows the results of in vivo toxicity studies. Blood or tissue samples were collected from balb / c mice (n = 3) that received intravenous injection of 206.8 nmol / kg (equivalent DM1 dose) of DM1-NO-NP or DM1 or PBS on day 10. Figures 10A - 10C are bar graphs showing the results of blood biochemical analyses of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (10A), blood urea nitrogen (BUN) (10B), and creatinine (CR) (10C). Figures 10D and 10E are bar graphs showing the results of liver tissue analyses of ALT (10D) and AST (10E) levels. Figure 10F is a bar graph showing blood electrolyte levels (sodium, potassium, chloride, bicarbonate, glucose, calcium, inorganic phosphate, and magnesium). Figure 10G is a bar graph showing the levels of total protein, albumin, and lipid (cholesterol) in the blood. ns, no significant difference.

[0059] Figure 11 are PET images obtained after testing 64Cu-labeled DM1-NO-PLGA and NTS mut -DM1-NO PLGA NP in H1299 tumor-bearing mice. These images show the difference between DM1-NO-PLGA NP, which is thought to accumulate in tumors via the EPR effect, and NTS mut -DM1-NO PLGA NP, which is thought to accumulate in tumors via both the EPR and NTSR1 targeting. DETAILED DESCRIPTION OF THE INVENTION

[0061] I. DEFINITIONS

[0062] "Derivatives" and "analogues", which are used interchangeably and in relation to a given compound, refer to another compound or moiety that has structural similarity, functional similarity, or both, to the specific compound. Structural similarity can be determined using any criteria known in the art, such as the Tanimoto coefficient, which provides a quantitative measure of the similarity between two compounds based on their molecular descriptors. Preferably, the molecular descriptors are 2D properties, such as fingerprints, topological indices, and maximum common substructures, or 3D properties, such as overall shape and molecular fields. The Tanimoto coefficient ranges between 0 and 1 (inclusive) for different and identical molecule pairs. A compound can be considered a derivative or analogue of a specific compound if its Tanimoto coefficient with the specific compound is between 0.5 and 1.0 (inclusive), preferably between 0.7 and 1.0 (inclusive), and most preferably between 0.85 and 1.0 (inclusive). A compound is functionally similar to a specific compound if it induces the same effect as the specific compound. "Derivatives" or "analogues" can also refer to modifications of a compound or moiety, including but not limited to hydrolysis, reduction, or oxidation products. Hydrolysis, reduction, and oxidation reactions are known in the art.

[0063] The terms "inhibit" and "reduce" mean to decrease or lower activity or expression. This can be a complete or partial inhibition or reduction of activity or expression. The inhibition or reduction can be compared to a control or standard level. The inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

[0064] The term "nanoparticle" refers to any particle with a diameter greater than 1 nm and less than 1000 nm.

[0065] The terms "nitrosylated", "nitrosylation", "nitrosation" and related terms are used to describe structures and do not limit the structures to those made from specific starting materials or by specific synthetic routes. Unless a specific and explicit contrary indication is provided, the terms refer to structural features regardless of how the structures are formed, and the structures are not limited to those made by any particular method. The term used herein to describe a structure refers to an organic compound or moiety containing a covalently bonded nitric oxide (NO) group. When nitric oxide is bonded through its nitrogen atom to a sulfur atom in an organic compound, the organic compound is commonly referred to as an "S-nitrosothiol" and contains an "-SNO" group referred to as an "S-nitrosothiol moiety".

[0066] The term "targeting agent" refers to a compound that can direct a nanoparticle to a receptor site on a selected cell or tissue type, can be used as an attachment molecule or for coupling or attaching another molecule. The term "direct" in relation to a compound means causing the nanoparticle to preferentially attach to a selected cell or tissue type. Such a targeting agent typically binds to its receptor with high affinity and specificity.

[0067] As used herein, "treatment / treating" refers to the medical management of a subject aimed at curing, ameliorating, stabilizing or preventing a disease, pathologic condition or disorder. The term includes active treatment, i.e., treatment specifically directed toward improving a disease, pathologic condition or disorder, and also includes etiologic treatment, i.e., treatment directed toward removing the cause of the relevant disease, pathologic condition or disorder. In addition, the term also includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathologic condition or disorder; prophylactic treatment, i.e., treatment involving minimizing or partially or completely inhibiting the development of the relevant disease, pathologic condition or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy directed toward improving the relevant disease, pathologic condition or disorder. It should be understood that while treatment is aimed at curing, ameliorating, stabilizing or preventing a disease, pathologic condition or disorder, it does not actually need to result in a cure, amelioration, stabilization or prevention. Treatment effects can be measured or evaluated as described herein and known in the art in a manner applicable to the disease, pathologic condition or disorder involved. Such measurements and evaluations can be made in a qualitative and / or quantitative manner. Thus, for example, the characteristics or features of a disease, pathologic condition or disorder and / or the symptoms of a disease, pathologic condition or disorder can be reduced to any effect or to any amount.

[0068] As used herein, "substituted" refers to all permissible substituents of the compounds or functional groups described herein. In the broadest sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Exemplary substituents include, but are not limited to, halogen, hydroxy, or any other organic group having any number of carbon atoms (preferably 1-14 carbon atoms), and optionally including one or more heteroatoms (such as oxygen, sulfur or nitrogen groups) in straight-chain, branched or cyclic structural forms. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxy, aralkyl, substituted aralkyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxy, substituted carboxy, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C 20 cyclic group, substituted C3-C 20 cyclic group, heterocycle, substituted heterocycle, amino acid, poly(lactic-co-glycolic acid), peptide and polypeptide groups. Such alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxy, aralkyl, substituted aralkyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxy, substituted carboxy, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C 20 cyclic group, substituted C3-C 20 cyclic group, heterocycle, substituted heterocycle, amino acid, poly(lactic-co-glycolic acid), peptide and polypeptide groups may be further substituted.

[0069] Heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of the organic compounds described herein, which satisfy the valence of the heteroatom. It should be understood that "substituted" or "substitution" includes the implicit condition that such substitution is consistent with the permissible valences of the substituted atom and the substituents, and the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation (such as by rearrangement, cyclization, elimination, etc.).

[0070] Unless a specific and explicit contrary indication is provided, the term "substituted" refers to a structure, such as a moiety on a compound or a larger compound, regardless of how the structure is formed. The structure is not limited to a structure made by any particular method.

[0071] As used herein, "aryl" refers to a C5-C 26 membered aryl, fused aryl, fused heterocyclic or biaryl ring system. Broadly, as used herein, "aryl" includes 5-, 6-, 7-, 8-, 9-, 10-, 14-, 18- and 24-membered monocyclic aryl groups, such as benzene, naphthalene, anthracene, phenanthrene, pyrene, corannulene, coronene, etc.

[0072] "Aryl" also encompasses polycyclic ring systems having more than two cyclic rings, wherein two or more carbons are common to two adjacent rings (i.e., "fused rings"), and at least one ring is aromatic, such that one or more of the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclic.

[0073] The term "substituted aryl" refers to such an aryl in which one or more hydrogen atoms on one or more aromatic rings are replaced by one or more substituents, said substituents including but not limited to halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, carbonyl (such as ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, imino, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic, alkaryl, haloalkyl (such as CF3, -CH2-CF3, -CCl3), -CN, aryl, heteroaryl and combinations thereof.

[0074] "Heterocycle", "heterocyclic", and "heterocyclic group" are used interchangeably and refer to a cyclic group linked through a ring carbon or nitrogen atom of a monocyclic or bicyclic ring containing 3 to 10 ring atoms and preferably 5 to 6 ring atoms, consisting of carbon and 1 to 4 heteroatoms, each heteroatom selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y), where Y is absent or is H, O, C1-C 10 alkyl, phenyl, or benzyl, and optionally containing 1 to 3 double bonds and optionally substituted with one or more substituents. By definition, a heterocyclic group is different from a heteroaryl group. Examples of heterocycles include, but are not limited to, piperazinyl, piperidinyl, piperidone, 4-piperidone, dihydrofuro[2,3-b]tetrahydrofuran, morpholinyl, piperazinyl, piperidinyl, piperidone, 4-piperidone, piperonyl, pyranyl, 2H-pyrrolyl, 4H-quinolizinyl, quinuclidinyl, tetrahydrofuranyl, 6H-1,2,5-thiadiazinyl. The heterocyclic group may optionally be substituted with one or more substituents as defined above for alkyl and aryl groups.

[0075] The term "heteroaryl" refers to C5-C 26A heteroaryl, fused aryl, biaryl ring system, or a combination thereof, wherein one or more carbon atoms in one or more aromatic ring structures have been replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, oxygen, sulfur, and nitrogen. Broadly, as used herein, "heteroaryl" includes: 5-, 6-, 7-, 8-, 9-, 10-, 14-, 18-, and 24-membered monocyclic aromatic groups that may include 1 to 4 heteroatoms, such as pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, etc. Heteroaryl may also be referred to as "aryl heterocycle" or "heteroaromatic compound". "Heteroaryl" also encompasses polycyclic ring systems having more than two rings, wherein two or more carbons are common to two adjacent rings (i.e., "fused rings"), and wherein at least one ring is heteroaromatic, for example, one or more of the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocycle, or a combination thereof. Examples of heteroaryl rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, dihydroindolyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, naphthyridinyl, octahydroisoquinolinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, hydroxyindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridobenzimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thiophenyl, thiophenothiazolyl, thiophenoxazolyl, thiophenobenzimidazolyl, thiophenyl, and xanthenyl. One or more rings may be substituted as defined below for "substituted heteroaryl".

[0076] The term "substituted heteroaryl" refers to a heteroaryl in which one or more hydrogen atoms on one or more heteroaromatic rings are replaced by one or more substituents, including but not limited to halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, carbonyl (such as ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, imino, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic group, alkaryl, haloalkyl (such as CF3, -CH2-CF3, -CCl3), -CN, aryl, heteroaryl and combinations thereof.

[0077] As used herein, "alkyl" refers to saturated aliphatic groups, including straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic), alkyl-substituted cycloalkyl and cycloalkyl-substituted alkyl. In a preferred embodiment, the straight-chain or branched-chain alkyl has 30 or fewer carbon atoms in its main chain (e.g., C1-C 30 , for branched-chain is C3-C 30 ), preferably 20 or fewer carbon atoms, more preferably 15 or fewer carbon atoms, and most preferably 10 or fewer carbon atoms. Similarly, preferred cycloalkyls have 3-10 carbon atoms in their ring structure, and more preferably 5, 6 or 7 carbons in the ring structure. As used throughout the specification, examples and claims, the term "alkyl" (or "lower alkyl") is intended to include both "unsubstituted alkyl" and "substituted alkyl", the latter referring to an alkyl moiety having one or more substituents replacing the hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxy, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphite, amino, amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic group, aralkyl or aromatic or heteroaromatic moiety.

[0078] Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means as defined above, but an alkyl having 1 to 10 carbons, more preferably 1 to 6 carbon atoms, in its main chain structure. Similarly, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Throughout the application, the preferred alkyl groups are lower alkyls. In a preferred embodiment, the substituents designated as alkyl herein are lower alkyls.

[0079] "Alkyl" includes one or more substitutions on one or more carbon atoms of a hydrocarbon group and heteroalkyl. Suitable substituents include, but are not limited to: halogen, such as fluorine, chlorine, bromine or iodine; hydroxyl; -NRR', where R and R' are independently hydrogen, alkyl or aryl, and where the nitrogen atom is optionally quaternized; -SR, where R is hydrogen, alkyl or aryl; -CN; -NO2; -COOH; carboxylate; -COR, -COOR or -CON(R)2, where R is hydrogen, alkyl or aryl; azide, aralkyl, alkoxy, imino, phosphonate, phosphonite, silyl, ether, sulfonyl, sulfonamide, heterocyclic group, aromatic or heteroaromatic moiety, haloalkyl (such as -CF3, -CH2-CF3, -CCl3); -CN; -NCOCOCH2CH2; -NCOCOCHCH; -NCS; and combinations thereof.

[0080] Those skilled in the art should understand that, if appropriate, the moieties substituted on the hydrocarbon chain can themselves be substituted. For example, the substituents of a substituted alkyl can include halogen, hydroxyl, nitro, thiol, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphonite), sulfonyl (including sulfate, sulfonamide, sulfamoyl, sulfoxide and sulfonate) and silyl groups, as well as ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylate and ester), haloalkyl, -CN, etc. Cycloalkyl can be substituted in the same way.

[0081] The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups that are similar in length and possible substitution to the alkyl described above, but contain at least one double bond or triple bond, respectively.

[0082] The term "substituted alkenyl" refers to an alkenyl moiety having one or more substituents that replace one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphonite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamide, sulfonyl, heterocyclic group, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0083] The term "substituted alkynyl" refers to an alkynyl moiety having one or more substituents that replace one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0084] The term "phenyl" is well recognized in the art and refers to the aromatic moiety -C6H5, i.e., a benzene ring lacking one hydrogen atom.

[0085] The term "substituted phenyl" refers to a phenyl as defined above having one or more substituents that replace one or more hydrogen atoms on one or more carbons of the benzene ring. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0086] As used herein, "amino" and "amine" are well recognized in the art and refer to substituted and unsubstituted amines, for example, moieties that can be represented by the following general formula:

[0087]

[0088] wherein R, R' and R" each independently represent hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, -(CH2) m-R”’, or R and R’ together with the N atom to which they are attached complete a heterocycle having 3 to 14 atoms in the ring structure; R”’ represents a hydroxyl group, a substituted or unsubstituted carbonyl group, an aryl group, a cycloalkyl ring, a cycloalkenyl ring, a heterocycle or a polycycle; and m is an integer of 0 or from 1 to 8. In a preferred embodiment, only one of R and R’ can be a carbonyl group, for example R and R’ do not form an imine together with nitrogen. In a preferred embodiment, R and R’ (and optionally R”) each independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or -(CH2) m -R”’. Thus, as used herein, the term “alkylamine” refers to an amino group as defined above having a substituted or unsubstituted alkyl group attached thereto (i.e., at least one of R, R’ or R” is an alkyl group).

[0089] As used herein, “carbonyl” is well recognized in the art and includes such moieties that can be represented by the following general formula:

[0090]

[0091] wherein X is a bond or represents oxygen or sulfur, and R represents hydrogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted alkaryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group, -(CH2) m -R” or a pharmaceutically acceptable salt, R’ represents hydrogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted alkaryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group or -(CH2) m -R”; R” represents a hydroxyl group, a substituted or unsubstituted carbonyl group, an aryl group, a cycloalkyl ring, a cycloalkenyl ring, a heterocycle or a polycycle; and m is an integer of 0 or from 1 to 8. When X is oxygen and R is as defined above, this moiety is also referred to as a carboxyl group. When X is oxygen and R is hydrogen, this formula represents ‘carboxylic acid’. When X is oxygen and R’ is hydrogen, this formula represents ‘formate’. When X is oxygen and R or R’ is not hydrogen, this formula represents ‘ester’. Generally, when the oxygen atom in the above formula is replaced by a sulfur atom, this formula represents a ‘thiocarbonyl’ group. When X is sulfur and R or R’ is not hydrogen, this formula represents ‘thioester’. When X is sulfur and R is hydrogen, this formula represents ‘thiocarboxylic acid’. When X is sulfur and R’ is hydrogen, this formula represents ‘thioformate’. When X is a bond and R is not hydrogen, the above formula represents ‘ketone’. When X is a bond and R is hydrogen, the above formula represents ‘aldehyde’.

[0092] The term "substituted carbonyl" refers to a carbonyl as defined above, wherein one or more hydrogens in R, R', or the group attached to the moiety

[0093]

[0094] are independently substituted. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphonite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic group, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0095] The term "carboxy" is as defined above for formula

[0096]

[0097] and is more specifically defined by the formula -R iv COOH, where R iv is alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, alkaryl, aralkyl, aryl or heteroaryl. In a preferred embodiment, straight-chain or branched-chain alkyl, alkenyl and alkynyl have 30 or fewer carbon atoms in their main chain (e.g., C1-C 30 for straight-chain alkyl, C3-C 30 for branched-chain alkyl, C2-C 30 for straight-chain alkenyl and alkynyl, C3-C 30 for branched-chain alkenyl and alkynyl), preferably 20 or fewer carbon atoms, more preferably 15 or fewer carbon atoms, most preferably 10 or fewer carbon atoms. Similarly, preferred cycloalkyl, heterocyclic group, aryl and heteroaryl have 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

[0098] The term "substituted carboxy" refers to a carboxy as defined above, wherein R ivOne or more hydrogen atoms therein are substituted. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic group, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0099] As used herein, "heteroalkyl" refers to a straight-chain or branched-chain group containing at least one heteroatom or a carbon-containing cyclic group, or a combination thereof. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein the nitrogen, phosphorus and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized.

[0100] Examples of saturated hydrocarbon groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl and homologs and isomers such as n-pentyl, n-hexyl, n-heptyl, n-octyl. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl and 3-butynyl.

[0101] The terms "alkoxy" or "alkoxyl", "aryloxy" or "aryloxyl" generally describe compounds represented by the formula -OR v wherein R v includes, but is not limited to, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroalkyl, alkaryl, alkylheteroaryl.

[0102] As used herein, the term "alkoxy" or "alkoxyl" refers to an alkyl group as defined above having an oxygen group attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, etc. "Ether" is two hydrocarbons covalently linked by oxygen. Thus, the substituent of the alkyl group that makes the alkyl group an ether is or resembles an alkoxy group, such as can be represented by one of -O-alkyl, -O-alkenyl and -O-alkynyl. The term alkoxy also includes cycloalkyl, heterocyclic group, cycloalkenyl, heterocycloalkenyl and aralkyl having an oxygen group attached to at least one carbon atom as allowed by valence.

[0103] The term "substituted alkoxy" refers to an alkoxy group having one or more substituents that replace one or more hydrogen atoms on one or more carbons of the alkoxy backbone. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0104] The term "phenoxy" is well recognized in the art and refers to a compound of the formula -OR v wherein R v is (i.e., -O-C6H5). Those skilled in the art recognize that phenoxy is a type of aryloxy group.

[0105] The term "substituted phenoxy" refers to a phenoxy group as defined above having one or more substituents that replace one or more hydrogen atoms on one or more carbons of the benzene ring. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0106] The terms "aryloxy" and "aryloxide", which are used interchangeably herein, can be represented by -O-aryl or O-heteroaryl, where aryl and heteroaryl are as defined herein.

[0107] As used interchangeably herein, the terms "substituted aryloxy" and "substituted aryloxyl" represent -O-aryl or -O-heteroaryl having one or more substituents that replace one or more hydrogen atoms on one or more ring atoms of aryl and heteroaryl as defined herein. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphonite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0108] As used herein, the term "alkylthio" refers to an alkyl group as defined above having a sulfur group attached thereto. The "alkylthio" moiety is represented by -S-alkyl. Representative alkylthios include methylthio, ethylthio, etc. The term "alkylthio" also encompasses cycloalkyl having a sulfur group attached thereto.

[0109] The term "substituted alkylthio" refers to alkylthio having one or more substituents that replace one or more hydrogen atoms on one or more carbon atoms of the alkylthio backbone. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphonite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0110] The term "phenylthio" is well recognized in the art and refers to -S-C6H5, i.e., phenyl attached to a sulfur atom.

[0111] The term "substituted phenylthio" refers to a phenylthio as defined above having one or more substituents that replace the hydrogen on one or more carbons of the benzene ring. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic group, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0112] "Arylthio" refers to -S-aryl or -S-heteroaryl, where aryl and heteroaryl are as defined herein.

[0113] The term "substituted arylthio" represents -S-aryl or -S-heteroaryl having one or more substituents that replace the hydrogen atoms on one or more ring atoms of the aryl and heteroaryl rings as defined herein. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic group, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0114] As used herein, "aralkyl" refers to an alkyl substituted with a substituted or unsubstituted aryl or heteroaryl.

[0115] As used herein, "alkaryl" refers to an aryl (e.g., an aromatic or heteroaromatic group) substituted with a substituted or unsubstituted alkyl.

[0116] The terms "amide" or "amido" are used interchangeably and refer to "unsubstituted amido" and "substituted amido", and are represented by the following general formula:

[0117]

[0118] Wherein, E does not exist, or E is a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic group, wherein independently of E, R and R' each independently represent hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted alkaryl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH2) m -R''', or R and R' together with the N atom to which they are attached complete a heterocycle having 3 to 14 atoms in the ring structure; R''' represents hydroxyl, substituted or unsubstituted carbonyl, aryl, cycloalkyl ring, cycloalkenyl ring, heterocycle or polycycle; and m is an integer of 0 or 1 to 8. In a preferred embodiment, only one of R and R' can be carbonyl, for example, R and R' do not form an imine together with nitrogen. In a preferred embodiment, R and R' each independently represent a hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or -(CH2) m -R'''. When E is oxygen, a carbamate is formed. As understood by those of ordinary skill in the art, a carbamate cannot be linked to another chemical substance such as to form an oxygen-oxygen bond or other unstable bonds.

[0119] The term "sulfonyl group" is represented by the following formula,

[0120]

[0121] Wherein E does not exist, or E is alkyl, alkenyl, alkynyl, aralkyl, alkaryl, cycloalkyl, aryl, heteroaryl, heterocyclic group, wherein independently of E, R represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted amine, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted alkaryl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH2) m -R''', or E and R together with the S atom to which they are attached complete a heterocycle having 3 to 14 atoms in the ring structure; R''' represents hydroxyl, substituted or unsubstituted carbonyl, aryl, cycloalkyl ring, cycloalkenyl ring, heterocycle or polycycle; and m is an integer of 0 or 1 to 8. In a preferred embodiment, only one of E and R can be a substituted or unsubstituted amine to form a "sulfonamide" or "sulfonamido group". Substituted or unsubstituted amines are as defined above.

[0122] The term "substituted sulfonyl" means a sulfonyl group in which E, R, or both are independently substituted. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphonite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic group, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0123] The term "sulfonic acid" refers to a sulfonyl group as defined above, where R is hydroxy and E is absent, or E is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted alkaryl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0124] The term "sulfate" refers to a sulfonyl group as defined above, where E is absent, oxygen, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy (as defined above), and R is independently hydroxy, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy (as defined above). As understood by those of ordinary skill in the art, when E is oxygen, the sulfate cannot be linked to another chemical substance such as to form an oxygen-oxygen bond or other unstable bonds.

[0125] The term "sulfonate" refers to a sulfonyl group as defined above, where E is oxygen, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy (as defined above), and R is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted amine, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted alkaryl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH2) m -R”’, where R”’ represents hydroxy, substituted or unsubstituted carbonyl, aryl, cycloalkyl ring, cycloalkenyl ring, heterocycle or polycycle; and m is an integer from 0 or 1 to 8. As understood by those of ordinary skill in the art, when E is oxygen, the sulfonate cannot be linked to another chemical substance such as to form an oxygen-oxygen bond or other unstable bonds.

[0126] The term "sulfamoyl" refers to a sulfonamide or sulfonamide represented by the following formula

[0127]

[0128] wherein E is absent, or E is a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted alkaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic group, wherein independently of E, R and R’ each independently represent hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted alkaryl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH2) m -R”’, or R and R’ together with the N atom to which they are attached complete a heterocycle having 3 to 14 atoms in the ring structure; R”’ represents hydroxy, substituted or unsubstituted carbonyl, aryl, cycloalkyl ring, cycloalkenyl ring, heterocyclic or polycyclic; and m is 0 or an integer from 1 to 8. In a preferred embodiment, only one of R and R’ can be carbonyl, for example, R and R’ do not form an imine together with nitrogen.

[0129] The term "sulfoxide" is represented by the following formula,

[0130]

[0131] wherein E is absent, or E is alkyl, alkenyl, alkynyl, aralkyl, alkaryl, cycloalkyl, aryl, heteroaryl, heterocyclic group, wherein independently of E, R represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted amine, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted alkaryl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH2) m -R”’, or E and R together with the S atom to which they are attached complete a heterocycle having 3 to 14 atoms in the ring structure; R”’ represents hydroxy, substituted or unsubstituted carbonyl, aryl, cycloalkyl ring, cycloalkenyl ring, heterocyclic or polycyclic; and m is 0 or an integer from 1 to 8.

[0132] The term "phosphonyl" is represented by the following formula,

[0133]

[0134] wherein E is absent, or E is a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted alkaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic group, wherein independently of E, R vi and Rvii is independently hydrogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted alkaryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group, -(CH2) m -R”’, or R and R’ together with the P atom to which they are attached complete a heterocycle having 3 to 14 atoms in the ring structure; R”’ represents a hydroxyl group, a substituted or unsubstituted carbonyl group, an aryl group, a cycloalkyl ring, a cycloalkenyl ring, a heterocyclic ring or a polycyclic ring; and m is an integer of 0 or 1 to 8.

[0135] The term "substituted phosphonyl" means a phosphonyl in which E, R vi and R vii are independently substituted phosphonyls. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic group, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0136] The term "phosphonyl" defines such a phosphonyl in which E is absent, oxygen, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy (as defined above), and independently of E, R vi and R vii are independently hydroxyl, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy (as defined above). As understood by those of ordinary skill in the art, when E is oxygen, the phosphonyl cannot be linked to another chemical substance such as to form an oxygen-oxygen bond or other unstable bonds. When E, R vi and R vii are substituted, the substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thiocarboxylate), alkoxy, phosphoryl, phosphate, phosphonate, phosphite, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclic group, alkaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0137] The term "polyaryl" refers to a chemical moiety comprising two or more aryl groups, heteroaryl groups, and combinations thereof. The aryl groups, heteroaryl groups, and combinations thereof are fused or linked by single bonds, ethers, esters, carbonyls, amides, sulfonyls, sulfonamides, alkyls, azo groups, and combinations thereof.

[0138] The term "substituted polyaryl" refers to such a polyaryl in which one or more aryl groups or heteroaryl groups are substituted by one or more substituents, including but not limited to halogens, azides, alkyls, aralkyls, alkenyls, alkynyls, cycloalkyls, hydroxyls, carbonyls (such as carboxyl, alkoxycarbonyl, formyl, or acyl), silyls, ethers, esters, thiocarbonyls (such as thioesters, thioacetates, or thiocarboxylates), alkoxys, phosphoryls, phosphates, phosphonates, phosphinites, amino groups (or quaternized amino groups), amido groups, amidines, imines, cyano groups, nitro groups, azido groups, mercapto groups, alkylthios, sulfates, sulfonates, sulfamoyl groups, sulfoxides, sulfonamido groups, sulfonyls, heterocyclic groups, alkaryls, haloalkyls, -CN, aryl groups, heteroaryl groups, and combinations thereof.

[0139] The term "C3-C 20 cyclic" refers to a substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloalkynyl, substituted or unsubstituted heterocyclic group having 3 to 20 carbon atoms as permitted by geometric constraints. The cyclic structure is formed by a monocyclic or fused ring system. The substituted cycloalkyl, cycloalkenyl, cycloalkynyl, and heterocyclic groups are substituted as defined above for alkyl, alkenyl, alkynyl, and heterocyclic groups, respectively.

[0140] The terms "hydroxyl / hydroxy" are used interchangeably and are represented by -OH.

[0141] The terms "cyano" and "nitrile" are used interchangeably to refer to -CN.

[0142] The term "phosphate" refers to -O-PO3.

[0143] The terms "azide" or "azido" are used interchangeably to refer to -N3.

[0144] The term "substituted C1-C x alkyl" refers to an alkyl having 1 to x carbon atoms, wherein at least one carbon atom is substituted, where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x alkyl" refers to an unsubstituted alkyl having 1 to x carbon atoms, where "x" is an integer from 1 to 10.

[0145] The term "substituted C1-C x alkylene" refers to an alkylene having 1 to x carbon atoms, wherein at least one carbon atom is substituted, where "x" is an integer from 1 to 10. The term "unsubstituted C1-Cx "Alkylene" refers to an unsubstituted alkylene having 1 to x carbon atoms, where "x" is an integer from 1 to 10. As used herein, the term "alkylene" refers to a moiety having the formula -(CH2) a -, where "a" is an integer from 1 to 10.

[0146] The term "substituted C2-C x alkenyl" refers to an alkenyl having 2 to x carbon atoms, where at least one carbon atom is substituted, where "x" is an integer from 2 to 10. The term "unsubstituted C2-C x alkenyl" refers to an unsubstituted alkenyl having 2 to x carbon atoms, where "x" is an integer from 2 to 10.

[0147] The term "substituted C2-C x alkynyl" refers to an alkynyl having 2 to x carbon atoms, where at least one carbon atom is substituted, where "x" is an integer from 2 to 10. The term "unsubstituted C2-C x alkynyl" refers to an unsubstituted alkynyl having 2 to x carbon atoms, where "x" is an integer from 2 to 10.

[0148] The term "substituted C1-C x alkoxy" refers to an alkoxy having 1 to x carbon atoms, where at least one carbon atom is substituted, where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x alkoxy" refers to an unsubstituted alkoxy having 1 to x carbon atoms, where "x" is an integer from 1 to 10.

[0149] The term "substituted C1-C x alkylamino" refers to an alkylamino having 1 to x carbon atoms, where at least one carbon atom is substituted, where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x alkylamino" refers to an unsubstituted alkyl having 1 to x carbon atoms, where "x" is an integer from 1 to 10. The terms "alkylamine" and "alkylamino" are used interchangeably. In any alkylamino, when the nitrogen atom is substituted by one, two, or three substituents, the nitrogen atom can be referred to as a secondary, tertiary, or quaternary nitrogen atom, respectively.

[0150] The term "substituted C1-C x alkylthio" refers to an alkylthio having 1 to x carbon atoms, where at least one carbon atom is substituted, where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x alkylthio" refers to an unsubstituted alkylthio having 1 to x carbon atoms, where "x" is an integer from 1 to 10.

[0151] The term "substituted C1-Cx "Carbonyl" refers to a carbonyl group having 1 to x carbon atoms, where at least one carbon atom is substituted, and where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x "Carbonyl" refers to an unsubstituted carbonyl group having 1 to x carbon atoms, where "x" is an integer from 1 to 10.

[0152] The term "substituted C1-C x "Carboxyl" refers to a carboxyl group having 1 to x carbon atoms, where at least one carbon atom is substituted, and where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x "Carboxyl" refers to an unsubstituted carboxyl group having 1 to x carbon atoms, where "x" is an integer from 1 to 10.

[0153] The term "substituted C1-C x "Amido" refers to an amido group having 1 to x carbon atoms, where at least one carbon atom is substituted, and where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x "Amido" refers to an unsubstituted amido group having 1 to x carbon atoms, where "x" is an integer from 1 to 10.

[0154] The term "substituted C1-C x "Sulfonyl" refers to a sulfonyl group having 1 to x carbon atoms, where at least one carbon atom is substituted, and where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x "Sulfonyl" refers to an unsubstituted sulfonyl group having 1 to x carbon atoms, where "x" is an integer from 1 to 10.

[0155] The term "substituted C1-C x "Sulfonic acid" refers to a sulfonic acid having 1 to x carbon atoms, where at least one carbon atom is substituted, and where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x "Sulfonic acid" refers to an unsubstituted sulfonic acid having 1 to x carbon atoms, where "x" is an integer from 1 to 10.

[0156] The term "substituted C1-C x "Sulfamoyl" refers to a sulfamoyl group having 1 to x carbon atoms, where at least one carbon atom is substituted, and where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x "Sulfamoyl" refers to an unsubstituted sulfamoyl group having 1 to x carbon atoms, where "x" is an integer from 1 to 10.

[0157] The term "substituted C1-C x"Sulfoxide" refers to a sulfoxide having 1 to x carbon atoms, where at least one carbon atom is substituted, and where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x "Sulfoxide" refers to an unsubstituted sulfoxide having 1 to x carbon atoms, where "x" is an integer from 1 to 10.

[0158] The term "substituted C1-C x "Phosphoryl" refers to a phosphoryl having 1 to x carbon atoms, where at least one carbon atom is substituted, and where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x "Phosphoryl" refers to an unsubstituted phosphoryl having 1 to x carbon atoms, where "x" is an integer from 1 to 10.

[0159] The term "substituted C1-C x "Phosphinyl" refers to a phosphinyl having 1 to x carbon atoms, where at least one carbon atom is substituted, and where "x" is an integer from 1 to 10. The term "unsubstituted C1-C x "Phosphinyl" refers to an unsubstituted phosphinyl having 1 to x carbon atoms, where "x" is an integer from 1 to 10.

[0160] The term "substituted C0-C x "Sulfonyl" refers to a sulfonyl having 0 to x carbon atoms, where if present, at least one carbon atom is substituted, and where "x" is an integer from 0 to 10. The term "unsubstituted C0-C x "Sulfonyl" refers to an unsubstituted sulfonyl having 0 to x carbon atoms, where "x" is an integer from 0 to 10.

[0161] The term "substituted C0-C x "Sulfonic acid" refers to a sulfonic acid having 0 to x carbon atoms, where if present, at least one carbon atom is substituted, and where "x" is an integer from 0 to 10. The term "unsubstituted C0-C x "Sulfonic acid" refers to an unsubstituted sulfonic acid having 0 to x carbon atoms, where "x" is an integer from 0 to 10.

[0162] The term "substituted C0-C x "Sulfamoyl" refers to a sulfamoyl having 0 to x carbon atoms, where if present, at least one carbon atom is substituted, and where "x" is an integer from 0 to 10. The term "unsubstituted C0-C x "Sulfamoyl" refers to an unsubstituted sulfamoyl having 0 to x carbon atoms, where "x" is an integer from 0 to 10.

[0163] The term "substituted C0-C x"Sulfoxide" refers to a sulfoxide having from 0 to x carbon atoms, where at least one carbon atom is substituted, where "x" is an integer from 0 to 10. The term "unsubstituted C0-C x "Sulfoxide" refers to an unsubstituted sulfoxide having from 0 to x carbon atoms, where "x" is an integer from 0 to 10.

[0164] The term "substituted C0-C x "Phosphoryl" refers to a phosphoramidoyl having from 0 to x carbon atoms, where if present, at least one carbon atom is substituted, where "x" is an integer from 0 to 10. The term "unsubstituted C0-C x "Phosphoryl" refers to an unsubstituted phosphoryl having from 0 to x carbon atoms, where "x" is an integer from 0 to 10.

[0165] The term "substituted C0-C x "Phosphinyl" refers to a phosphinyl having from 0 to x carbon atoms, where if present, at least one carbon atom is substituted, where "x" is an integer from 0 to 10. The term "unsubstituted C0-C x "Phosphinyl" refers to an unsubstituted phosphinyl having from 0 to x carbon atoms, where "x" is an integer from 0 to 10.

[0166] The term substituted "C x "Alkyl", "C x "Alkylene", "C x "Alkenyl", "C x "Alkynyl", "C x "Alkoxy", "C x "Alkylamino", "C x "Alkylthio", "C x "Carbonyl", "C x "Carboxyl", "C x "Amido", "C x "Sulfonyl", "C x "Sulfonic acid", "C x "Sulfamoyl", "C x "Phosphoryl" and "C x "Phosphinyl" respectively refer to an alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, carbonyl, carboxyl, amido, sulfonyl, sulfonic acid, sulfamoyl, sulfoxide, phosphoryl and phosphinyl having x carbon atoms, where at least one carbon atom is substituted, where "x" is an integer from 1 to 10. The term unsubstituted "C x "Alkyl", "C x "Alkylene", "C x "Alkenyl", "C x "Alkynyl", "C x "Alkoxy", "C x "Alkylamino", "C x"alkylthio", "C x "carbonyl", "C x "carboxyl", "C x "amido", "C x "sulfonyl", "C x "sulfonic acid", "C x "sulfamoyl", "C x "phosphoryl" and "C x "phosphonoyl" respectively refer to unsubstituted alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, carbonyl, carboxyl, amido, sulfonyl, sulfonic acid, sulfamoyl, sulfoxide, phosphoryl and phosphonoyl having x carbon atoms, where "x" is an integer from 1 to 10.

[0167] The terms unsubstituted "C0 sulfonyl", "C0 sulfonic acid", "C0 sulfamoyl", "C0 phosphoryl" and "C0 phosphonoyl" respectively refer to unsubstituted alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, carbonyl, carboxyl, amido, sulfonyl, sulfonic acid, sulfamoyl, sulfoxide, phosphoryl and phosphonoyl having 0 carbon atoms.

[0168] As used herein, "halogen" refers to fluorine, chlorine, bromine or iodine.

[0169] The term "radiosensitivity" refers to the relative susceptibility of cells to the harmful effects of ionizing radiation. The higher the radiosensitivity of a cell, the less radiation is required to kill the cell. In general, cell radiosensitivity has been found to be directly proportional to the cell division rate and inversely proportional to the cell's DNA repair capacity.

[0170] The term "radiation resistance" refers to cells that do not die when exposed to clinically appropriate doses of ionizing radiation.

[0171] The term "neoplastic cell" refers to a cell that undergoes abnormal cell proliferation ("neoplasia"). The growth of neoplastic cells exceeds that of the surrounding normal tissue and is not coordinated with the growth of the surrounding normal tissue. Even after the stimulus has ceased, the growth generally persists in the same excessive manner and usually results in the formation of a tumor.

[0172] The term "tumor" or "neoplasm" refers to a mass of abnormal tissue containing neoplastic cells. Neoplasms and tumors can be benign, pre-malignant or malignant.

[0173] The term "cancer" or "malignant neoplasm" refers to cells that exhibit uncontrolled growth, invade adjacent tissues and often metastasize to other parts of the body.

[0174] The term "antineoplastic agent" refers to a composition, such as a drug or a biological agent, that can inhibit or prevent the growth, invasion and / or metastasis of cancer.

[0175] The terms "individual", "host", "subject", and "patient" are used interchangeably to refer to any individual who is the target of administration or treatment. A subject can be a vertebrate, such as a mammal. Thus, a subject can be a human or a veterinary patient.

[0176] The term "therapeutically effective" means an amount of the composition being used that is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration need only be a reduction or alteration, and not necessarily an elimination. A therapeutically effective amount of a composition for treating cancer is preferably an amount sufficient to cause tumor regression or render the tumor sensitive to radiation or chemotherapy.

[0177] The term "pharmaceutically acceptable" means a substance that is not biologically or otherwise undesirable, i.e., the substance can be administered to a subject without causing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition in which it is present. As is well known to those skilled in the art, a carrier will naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0178] The term "treatment" refers to the medical management of a patient aimed at curing, ameliorating, stabilizing, or preventing a disease, pathologic condition, or disorder. The term includes active treatment, i.e., treatment specifically directed toward the amelioration of a disease, pathologic condition, or disorder, and also includes causative treatment, i.e., treatment as against the cause of the relevant disease, pathologic condition, or disorder. In addition, the term also includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, pathologic condition, or disorder; prophylactic treatment, i.e., treatment involving minimizing or partially or completely inhibiting the development of the relevant disease, pathologic condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy directed toward the amelioration of the relevant disease, pathologic condition, or disorder.

[0179] II. Compositions

[0180] Compounds containing one or more S-nitrosothiol moieties and / or nanoparticles encapsulating such compounds are described. The compounds can render cancer cells sensitive to radiotherapy. In some forms, the compounds are maytansinoid analogs. In some forms, the compounds include compounds having the structure shown below:

[0181]

[0182] In some forms, these compounds can be encapsulated within the nanoparticles, on the surface of the nanoparticles, or both. In some forms, the compounds can be encapsulated within the nanoparticles. In some forms, the compounds can be on the surface of the nanoparticles. The compounds can be conjugated to the nanoparticles covalently or non-covalently. In some forms, the compounds can be encapsulated within the nanoparticles and non-covalently conjugated to the nanoparticles. In some forms, the compounds can be on the surface of the nanoparticles and covalently conjugated to the surface of the nanoparticles. The nanoparticles can be polymeric nanoparticles, liposomes, or inorganic nanoparticles.

[0183] In some forms, the nanoparticles are polymeric nanoparticles. In some forms, the polymeric nanoparticles contain amphiphilic copolymers. In some forms, the amphiphilic polymers contain polyesters (such as poly(hydroxy acids)) and polyalkylene oxides (such as polyethylene glycol). In some forms, the amphiphilic polymers contain poly(lactic-co-glycolic acid)-polyethylene glycol. In some forms, the nanoparticles have a size of about 50 nm to about 150 nm (such as about 78 nm). Optionally, the nanoparticles contain targeting agents.

[0184] Subsequent paragraphs include further details regarding the compounds, nanoparticles, and components that can be included in the nanoparticles.

[0185] A. Compounds

[0186] Compounds are provided for use in combination with radiotherapy. The compounds contain one or more S-nitrosothiol moieties. The compounds are prodrug compounds that are designed such that when exposed to radiation (preferably ionizing radiation) during radiotherapy, the S-N bond breaks, releasing the parent compound and nitric oxide.

[0187] Typically, the parent compound is a chemotherapeutic agent and / or a radiosensitizer, and relative to the un-nitrosated parent compound, one or more S-nitrosothiol moieties inhibit the toxicity of the prodrug form of the compound, and the nitric oxide released from the prodrug when the S-N bond breaks increases oxidative stress in cells, or a combination thereof.

[0188] In some embodiments, the parent compound and thus the compound released from nitric oxide when the S-N bond breaks is an inhibitor of microtubule polymerization.

[0189] In some forms, the compounds can include the structural motifs shown below:

[0190]

[0191] wherein linker represents a linking group, and the numbers are mainly for naming. The dashed lines between positions 11 and 12 and between positions 13 and 14 indicate that the bond may or may not be present, and according to the valence, the carbon atoms at positions 11, 12, 13, and 14 have no, one, or two hydrogen atoms respectively attached thereto. n is an integer from 1 to 13, including the end values, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. The "linking group" is independently absent, a substituted amide group, an unsubstituted amide group, a substituted alkyl group, a substituted alkylene group, an unsubstituted alkylene group, a substituted aryl group, a substituted heteroaryl group, a substituted alkenyl group, a substituted alkynyl group, a substituted alkoxy group, a substituted aryloxy group, a substituted alkylthio group, a substituted arylthio group, an unsubstituted carbonyl group, a substituted carbonyl group, an unsubstituted carboxyl group, a substituted carboxyl group, an unsubstituted amino group, a substituted amino group, an unsubstituted sulfonyl group, a substituted sulfonyl group, an unsubstituted aminosulfonyl group, a substituted aminosulfonyl group, an unsubstituted phosphonyl group, a substituted phosphonyl group, a substituted polyaryl group, a substituted C3-C 20 cyclic group or substituted C3-C 20 heterocycle. The "linking group" can also be a substituted C1-C 10 amide group, an unsubstituted C1-C 10 amide group, a substituted C1-C 10 alkyl group, an unsubstituted C1-C 10 alkylene group, a substituted C1-C 10 alkylene group, an unsubstituted C1-C 10 alkylene group, a substituted aryl group, a substituted heteroaryl group, a substituted C2-C 10 alkenyl group, a substituted C2-C 10 alkynyl group, a substituted C1-C 10 alkoxy group, a substituted aryloxy group, a substituted C1-C 10 alkylthio group, a substituted arylthio group, an unsubstituted C1-C 10 carbonyl group, a substituted C1-C 10 carbonyl group, an unsubstituted C1-C 10 carboxyl group, a substituted C1-C 10 carboxyl group, an unsubstituted C1-C 10 amino group, a substituted C1-C 10 amino group, an unsubstituted C1-C 10 sulfonyl group, a substituted C1-C 10 sulfonyl group, an unsubstituted C1-C 10 aminosulfonyl group, a substituted C1-C 10 aminosulfonyl group, an unsubstituted C1-C 10 phosphonyl group, substituting C1-C 10 phosphonyl group, a substituted polyaryl group, a substituted C3-C 10 cyclic group or substituted C3-C10 Heterocycle.

[0192] Maytansine alkaloids are macrolide compounds that can inhibit the proliferation of cancer cells at sub - nanomolar concentrations, making their efficacy 100 to 1000 times that of cisplatin (Lopus, Cancer Lett. 307, 113 - 118 (2011), Remillard et al., Science 189, 1002 - 1005 (1975)). Maytansine alkaloids kill cancer cells by inhibiting microtubule assembly (Oroudjev et al., Mol. Cancer Ther. 9, 2700 - 2713 (2010)). This anti - mitotic effect can enrich cells in the mitotic phase, which is more sensitive to RT (Lopus et al., Mol. Cancer Ther. 9, 2689 - 2699 (2010), Yenjerla et al., Methods Cell Biol. 95, 189 - 206 (2010)). However, due to a lack of specificity and unacceptable systemic toxicity, maytansine has failed as an anticancer agent in clinical trials (Moertel et al., J. Natl. Cancer Inst. 60, 93 - 96 (1978), Rosenthal et al., Cancer Treat. Rev. 64, 1115 - 1117 (1980), Lopus et al., Mol. Cancer Ther. 9, 2689 - 99 (2010)). The recent development of antibody - drug conjugates (ADCs) allows for the delivery of maytansine alkaloids (especially DM1) to cancer cells with more favorable pharmacokinetics and pharmacodynamics (Lopus, Cancer Lett. 307, 113 - 8 (2011)). Kadcyla, a DM1 - trastuzumab conjugate, can sensitize breast cancer cells to RT (Peddi & Hurvitz, Ther. Adv. Med. Oncol. 6, 202 - 209 (2014), Koshkaryev et al., Adv. Drug. Deliv. Rev. 65, 24 - 35 (2013)), confirming the potential of maytansine alkaloids as radiosensitizers when selectively delivered to tumors.

[0193] The following experiments show that S-nitrosylation of maytansine alkaloid DM1 can help inhibit toxicity and enable the drug to be delivered to tumors through the enhanced permeability and retention (EPR) effect. When the tumor is irradiated, oxidative stress increases, leading to the cleavage of the S-N bond and the release of DM1 and nitric oxide (NO). DM1 inhibits microtubule polymerization and enriches cells in the G2 / M phase, which is more radiosensitive. NO under irradiation forms highly toxic free radicals such as peroxynitrite, which also contributes to tumor suppression. These two components act additively or more than additively to enhance the effect of radiotherapy, which is confirmed in vitro by clonogenic assays and in vivo using H1299 tumor-bearing mice.

[0194] Thus, in some forms, the compound is a maytansine alkaloid or a maytansine alkaloid analogue containing one or more S-nitrosothiol moieties.

[0195] Examples of suitable maytansine alkaloid analogues include those having a modified aromatic ring and / or those having modifications at other positions. Such maytansine alkaloids are described, for example, in U.S. Patent Nos. 4,256,746, 4,294,757, 4,307,016, 4,313,946, 4,315,929, 4,322,348, 4,331,598, 4,361,650, 4,362,663, 4,364,866, 4,424,219, 4,371,533, 4,450,254, 5,475,092, 5,585,499, 5,846,545, and 6,333,410.

[0196] In some forms, the compound has the following structure:

[0197]

[0198] Wherein:

[0199] The dotted lines between positions 11 and 12 and between positions 13 and 14 indicate that the bond may or may not be present, and according to the valence, the carbon atoms at positions 11, 12, 13, and 14 have no, one, or two hydrogen atoms attached thereto, respectively.

[0200] R1 is a substituted amido group, an unsubstituted amido group, a substituted alkyl group, a substituted alkylene group, an unsubstituted alkylene group, a substituted aryl group, a substituted heteroaryl group, a substituted alkenyl group, a substituted alkynyl group, a substituted alkoxy group, a substituted aryloxy group, a substituted alkylthio group, a substituted arylthio group, an unsubstituted carbonyl group, a substituted carbonyl group, an unsubstituted carboxyl group, a substituted carboxyl group, an unsubstituted amino group, a substituted amino group, an unsubstituted sulfonyl group, a substituted sulfonyl group, an unsubstituted aminosulfonyl group, a substituted aminosulfonyl group, an unsubstituted phosphonyl group, a substituted phosphonyl group, a substituted polyaryl group, a substituted C3-C20 a cyclic group or a substituted C3-C 20 heterocycle, and

[0201] R2, R3, R4, R5, R6, R7, R8, R9, R 10 and R 11 are independently hydrogen, halogen (F, Br, Cl, I), substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, unsubstituted alkenyl, substituted alkenyl, unsubstituted alkynyl, substituted alkynyl, unsubstituted alkoxy, substituted alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted alkylthio, substituted alkylthio, unsubstituted arylthio, substituted arylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted amino, substituted amino, unsubstituted sulfonyl, substituted sulfonyl, unsubstituted aminosulfonyl, substituted aminosulfonyl, unsubstituted phosphonyl, substituted phosphonyl, unsubstituted polyaryl, substituted polyaryl, unsubstituted C3-C 20 cyclic group, substituted C3-C 20 cyclic group, unsubstituted C3-C 20 heterocycle or substituted C3-C 20 heterocycle, or R2 and R3 together with the carbon atom to which they are attached form an epoxide.

[0202] In some forms of Formula I or Formula I(a), R1 is a substituted C1-C 10 amido group, unsubstituted C1-C 10 amido group, substituted C1-C 10 alkyl, unsubstituted C1-C 10 alkylene, substituted C1-C 10 alkylene, unsubstituted C1-C10 alkylene, substituted aryl, substituted heteroaryl, substituted C2-C 10 alkenyl, substituted C2-C 10 alkynyl, substituted C1-C 10 alkoxy, substituted aryloxy, substituted C1-C 10 alkylthio, substituted arylthio, unsubstituted C1-C 10 carbonyl, substituted C1-C 10 carbonyl, unsubstituted C1-C 10 carboxyl, substituted C1-C 10 carboxyl, unsubstituted C1-C 10 amino, substituted C1-C 10 amino, unsubstituted C1-C 10 sulfonyl, substituted C1-C 10 sulfonyl, unsubstituted C1-C10 Sulfamoyl, substituted C1-C 10 Sulfamoyl, unsubstituted C1-C 10 Phosphonyl, substituted C1-C 10 Phosphonyl, substituted polyaryl, substituted C3-C 10 Cyclic group or substituted C3-C 10 Heterocycle.

[0203] In some forms of Formula I or Formula I(a), R1 is a substituted C1-C5 amide group, an unsubstituted C1-C5 amide group, a substituted C1-C5 alkyl group, a substituted C1-C5 alkylene group, an unsubstituted C1-C5 alkylene group, a substituted aryl group, a substituted heteroaryl group, a substituted C2-C5 alkenyl group, a substituted C2-C5 alkynyl group, a substituted C1-C5 alkoxy group, a substituted aryloxy group, a substituted C1-C5 alkylthio group, a substituted arylthio group, an unsubstituted C1-C5 carbonyl group, a substituted C1-C5 carbonyl group, an unsubstituted C1-C5 carboxyl group, a substituted C1-C5 carboxyl group, an unsubstituted C1-C5 amino group, a substituted C1-C5 amino group, an unsubstituted C1-C5 sulfonyl group, a substituted C1-C5 sulfonyl group, an unsubstituted C1-C5 sulfamoyl group, a substituted C1-C5 sulfamoyl group, an unsubstituted C1-C5 phosphonyl group, a substituted C1-C5 phosphonyl group, a substituted polyaryl group, a substituted C3-C6 cyclic group or a substituted C3-C6 heterocycle.

[0204] In some forms of Formula I or Formula I(a), R1 is a substituted C1-C 10 Amide group or an unsubstituted C1-C 10 Amide group.

[0205] In some forms of Formula I or Formula I(a), R1 has the following structure:

[0206]

[0207] Wherein R 12 Is a substituted C1-C5 alkylene group or an unsubstituted C1-C5 alkylene group, R 13 Is hydrogen, a substituted C1-C5 alkyl group or an unsubstituted C1-C5 alkyl group, and R 14 Is a substituted C1-C5 alkylene group or an unsubstituted C1-C5 alkylene group.

[0208] In some forms of Formula II, R 12 Is a substituted C1-C5 alkylene group (preferably -CH(CH3)-), R 12 Is an unsubstituted C1-C5 alkyl group (preferably -CH3), and R 14 Is an unsubstituted C1-C5 alkylene group (preferably -(CH2)2-).

[0209] In some forms of Formula I or Formula I(a), R2, R3, R4, R5, R6, R7, R8, R9, R 10 and R 11 are independently hydrogen, hydroxy, halogen (F, Br, Cl, I), substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, unsubstituted C1-C5 alkenyl, substituted C1-C5 alkenyl, unsubstituted C1-C5 alkynyl, substituted C1-C5 alkynyl, unsubstituted C1-C5 alkoxy, substituted C1-C5 alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted C1-C5 alkylthio, substituted C1-C5 alkylthio, unsubstituted arylthio, substituted arylthio, unsubstituted C1-C5 carbonyl, substituted C1-C5 carbonyl, unsubstituted C1-C5 carboxy, substituted C1-C5 carboxy, unsubstituted C1-C5 amino, substituted C1-C5 amino, unsubstituted C1-C5 sulfonyl, substituted C1-C5 sulfonyl, unsubstituted C1-C5 sulfamoyl, substituted C1-C5 sulfamoyl, unsubstituted C1-C5 phosphonyl, substituted C1-C5 phosphonyl, unsubstituted polyaryl, substituted polyaryl, unsubstituted C3-C6 cyclic group, substituted C3-C6 cyclic group, unsubstituted C3-C6 heterocycle or substituted C3-C6 heterocycle, or R2 and R3 together with the carbon atom to which they are attached form an epoxide.

[0210] In some forms of Formula I or Formula I(a), R2 and R3 together with the carbon atom to which they are attached form an epoxide.

[0211] In some forms of Formula I or Formula I(a), R4, R5, R6 and R7 are independently hydrogen, hydroxy, halogen (F, Br, Cl, I), substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl or unsubstituted heteroaryl. In some forms of Formula I or Formula I(a), R4, R5 and R6 are hydrogen and R7 is methyl.

[0212] In some forms of Formula I or Formula I(a), R8 is hydrogen, hydroxy, halogen (F, Br, Cl, I), substituted C1-C5 carboxy, unsubstituted C1-C5 carboxy, substituted C1-C5 carbonyl or unsubstituted C1-C5 carbonyl. In some forms of Formula I or Formula I(a), R8 is hydrogen, hydroxy, substituted C1-C5 carboxy or unsubstituted C1-C5 carboxy. In some forms of Formula I or Formula I(a), R8 is hydrogen.

[0213] In some forms of Formula I or Formula I(a), R9 is hydrogen, substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted C1-C5 carbonyl, or unsubstituted C1-C5 carbonyl. In some forms of Formula I or Formula I(a), R9 is unsubstituted C1-C5 alkyl. In some forms of Formula I or Formula I(a), R9 is methyl.

[0214] In some forms of Formula I or Formula I(a), R 10 is halogen (F, Cl, Br, I), substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted C1-C5 carbonyl, or unsubstituted C1-C5 carbonyl. In some forms of Formula I or Formula I(a), R 10 is halogen. In some forms of Formula I or Formula I(a), R 10 is Cl.

[0215] In some forms of Formula I or Formula I(a), R 11 is hydrogen, substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl. In some forms of Formula I or Formula I(a), R 11 is unsubstituted C1-C5 alkyl. In some forms of Formula I or Formula I(a), R 11 is methyl.

[0216] In some forms of Formula I or Formula I(a), the compound has the following structure:

[0217]

[0218]

[0219] wherein when present, R1, R4, R5, R6, R7, R8, R9, R 10 and R 11 are as described in any previous paragraph for some forms of Formula I or Formula I(a).

[0220] In some forms, the compound has the following structure:

[0221]

[0222] In some forms, the parent compound is another chemotherapeutic agent or radiosensitizer. Examples of known radiosensitizers include nicotinamide, metronidazole, and analogs thereof, including, for example, misonidazole, etanidazole, and nimorazole; hypoxic cell cytotoxins, such as mitomycin-C and tirapazamine; membrane-active agents, such as procaine, lidocaine, and chlorpromazine; radiosensitizing nucleosides, such as 5-fluorouracil, floxuridine, bromodeoxyuridine, iododeoxyuridine, hydroxyurea, gemcitabine, and fludarabine; texaphyrins, such as motexafin gadolinium; inhibitors of sulfhydryl groups, such as N-ethylmaleimide, diamide, and diethyl maleate; chemotherapeutic agents, such as paclitaxel, docetaxel, irinotecan, and cisplatin; pentoxifylline; vinorelbine; PARP inhibitors; histone deacetylase inhibitors; and proteasome inhibitors. See, e.g., Raviraj et al., Indian Journal of Dental Research, 25(1):83-90 (2014).

[0223] In some forms, small molecules, typically the radiosensitizers mentioned above, or maytansinoids, can be chemically modified to introduce one or more thiol groups. A thiol group can be introduced into a chemical compound using a reagent containing two or more functional groups, one of which is a thiol group. The other functional groups can be hydroxyl, carboxylic acid, amine, halide, aldehyde, ketone, etc. Depending on the reaction conducted, the thiol group can be protected by a disulfide bond that is ultimately reduced to expose the thiol group. After introduction, one or more thiol groups can react with a nitric oxide donor such as tert-butyl nitrite. S-nitrosylation can be achieved according to the protocols described in Chipinda and Simoyi, J. Phys. Chem. B 2006, 110, 5052-5061 and Pant, et al., ACS Appl. Mater. Interfaces 2017, 9, 15254-15264, the contents of which are incorporated herein by reference.

[0224] In some forms, the compound can be encapsulated and / or delivered with additional radiosensitizers (such as those mentioned above) and / or anti-cancer agents. Representative anti-cancer agents include, but are not limited to, alkylating agents (such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil and ifosfamide), antimetabolites (such as 5-fluorouracil (5-FU), gemcitabine, methotrexate, cytosine arabinoside, fludarabine and floxuridine), anti-mitotic agents (including taxanes such as paclitaxel and docetaxel, and vinca alkaloids such as vincristine, vinblastine, vinorelbine, vindesine), anthracyclines (including doxorubicin, daunorubicin, valrubicin, idarubicin and epirubicin, and actinomycins such as actinomycin D), cytotoxic antibiotics (including mitomycin, plicamycin and bleomycin), topoisomerase inhibitors (including camptothecins such as camptothecin, irinotecan and topotecan, and derivatives of epipodophyllotoxin such as amsacrine, etoposide, etoposide phosphate and teniposide), antibodies against vascular endothelial growth factor (VEGF) such as bevacizumab ( ), other anti-VEGF compounds; thalidomide ( ) and its derivatives such as lenalidomide ( ); endostatin; angiostatin; receptor tyrosine kinase (RTK) inhibitors such as sunitinib ( );tyrosine kinase inhibitors such as sorafenib( ), erlotinib( ), pazopanib, axitinib and lapatinib; transforming growth factor-α or transforming growth factor-β inhibitors, and antibodies against epidermal growth factor receptor such as panitumumab( ) and cetuximab( ).

[0225] B. Nanoparticles

[0226] The compound can be in polymeric nanoparticles, liposomes, inorganic nanoparticles or combinations thereof.

[0227] The following experiments show that nanotechnology allows a re-examination of therapeutic agents such as maytansine alkaloids, which can be effective radiosensitizers but are too toxic to be administered alone.

[0228] i. Polymeric nanoparticles

[0229] In some forms, the nanoparticles can be a substrate of a biocompatible polymer (preferably a biodegradable polymer). The polymer can be an amphiphilic, hydrophobic or hydrophilic polymer that can be hydrolyzed or enzymatically degraded in vitro or in vivo. Exemplary polymers are discussed below. Copolymers such as random, block or graft copolymers, or blends of the following polymers can also be used.

[0230] For a given polymer, the weight average molecular weight can vary, but is typically from about 1000 daltons to 1,000,000 daltons, from about 1000 daltons to about 500,000 daltons, from about 1000 daltons to about 250,000 daltons, from about 1000 daltons to about 100,000 daltons, from about 5,000 daltons to about 100,000 daltons, from about 5,000 daltons to about 75,000 daltons, from about 5,000 daltons to about 50,000 daltons or from about 5,000 daltons to about 25,000 daltons.

[0231] Compared with healthy tissues, the extracellular microenvironment of tumor tissues is generally slightly more acidic. Additionally, the lumens of endosomes and lysosomes are typically more acidic than the cytoplasm of cells. Thus, to enhance the release of payloads from nanoparticles before or after nanoparticle uptake through the process of polymer degradation, diffusion, or both, the polymer can be acid pH-responsive. In these forms, the polymer can contain ionizable groups (such as one or more amine groups) that can be ionized and cause nanoparticle swelling, or the polymer can contain chemical moieties (such as disulfides, orthoesters, acetals, ketals, hydrazones, imines, cis-aconityl compounds, esters, vinyl ethers, etc.) that cleave more rapidly in an environment with an acidic pH (such as 6.9 to 4.0) compared to an environment with a higher pH value (such as 7.2, 8, 9, or higher).

[0232] a. Amphiphilic polymer

[0233] The NP may contain one or more amphiphilic polymers, preferably biodegradable amphiphilic polymers. The amphiphilic polymer contains a hydrophobic polymer portion and a hydrophilic polymer portion. The hydrophobic polymer portion and the hydrophilic polymer portion may each independently include any hydrophobic polymer and hydrophilic polymer described in the corresponding sections below. In non-limiting examples, the hydrophobic polymer portion is a polymer formed from: polyesters such as polyhydroxy acids (such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid)), polycaprolactone, polyhydroxyalkanoates (such as poly-3-hydroxybutyrate, poly-4-hydroxybutyrate, polyhydroxyvalerate), poly(lactide-co-caprolactone); poly(anhydrides); poly(orthoesters); hydrophobic polysaccharides (such as acetalated dextran, acetylated dextran, acetylated cellulose, propionylated dextran, propionylated cellulose); and hydrophobic polyethers (such as polypropylene glycol); and copolymers thereof. The hydrophilic polymer portion may contain polymers such as polyalkylene oxides such as polypropylene glycol or polyethylene glycol (PEG); polysaccharides such as cellulose and starch; hydrophilic polypeptides such as poly-L-glutamic acid, γ-polyglutamic acid, poly-L-aspartic acid, poly-L-serine or poly-L-lysine; poly(oxyethylated polyols); poly(vinyl alcohols), such as poly(vinyl alcohol); poly(vinyl pyrrolidone); polyacrylamide or polymethacrylamide, including poly(N-hydroxyalkyl methacrylamide), such as poly(N-hydroxyethyl methacrylamide); poly(N-hydroxyalkyl methacrylate), such as poly(N-hydroxyethyl methacrylate); hydrophilic poly(hydroxy acids); and copolymers thereof. Examples of amphiphilic polymers that can be generated from this group include polyester-PEG copolymers such as poly(lactic-co-glycolic acid)-PEG (PLGA-PEG), poly(lactic acid)-PEG (PLA-PEG), poly(glycolic acid)-PEG (PGA-PEG), and polycaprolactone-PEG (PCL-PEG); hydrophobic polyether-PEGs such as polypropylene glycol-PEG (PPG-PEG), PEG-PPG-PEG, PPG-PEG-PPG; and acetylated dextran-PEG. In some forms, the amphiphilic polymer may be PLGA-PEG.

[0234] b. Hydrophobic polymer

[0235] The NP can be formed from one or more hydrophobic polymers. In some forms, the hydrophobic polymer is biodegradable. Examples of suitable hydrophobic polymers include polyesters such as polyhydroxy acids (such as poly(lactic-co-glycolic acid), poly(lactic acid), poly(glycolic acid)), polycaprolactone, polyhydroxyalkanoates (such as poly-3-hydroxybutyrate, poly-4-hydroxybutyrate, polyhydroxyvalerate), poly(lactide-co-caprolactone); poly(anhydrides); poly(orthoesters); hydrophobic polysaccharides (such as acetalated dextran, acetylated dextran, acetylated cellulose, propionylated dextran, propionylated cellulose); and copolymers thereof.

[0236] In some forms, the hydrophobic polymer includes polyesters such as polyhydroxy acids (such as poly(lactic-co-glycolic acid), poly(lactic acid), poly(glycolic acid)), polycaprolactone, polyhydroxyalkanoates (such as poly-3-hydroxybutyrate, poly-4-hydroxybutyrate, polyhydroxyvalerate), poly(lactide-co-caprolactone); poly(anhydrides); poly(orthoesters); poly(β-amino esters); and copolymers thereof.

[0237] c. Hydrophilic polymers

[0238] The NP can contain one or more hydrophilic polymers. Preferably, the hydrophilic polymer is biodegradable. Hydrophilic polymers include polyalkylene glycols such as polyethylene glycol (PEG); polysaccharides such as cellulose and starch and their derivatives; hydrophilic polypeptides such as poly-L-glutamic acid, γ-polyglutamic acid, poly-L-aspartic acid, poly-L-serine or poly-L-lysine; poly(oxyethylated polyols); poly(vinyl alcohols) such as poly(vinyl alcohol); poly(vinyl pyrrolidone); poly(N-hydroxyalkyl methacrylamides) such as poly(N-hydroxyethyl methacrylamide); poly(N-hydroxyalkyl methacrylates) such as poly(N-hydroxyethyl methacrylate); hydrophilic poly(hydroxy acids); and copolymers thereof. In some forms, the hydrophilic polymer is a polyalkylene glycol such as PEG or poloxamer.

[0239] ii. Liposomes and micelles

[0240] In some forms, the compound can be encapsulated in liposomal vesicles, lipid micelles or solid lipid nanoparticles or combinations thereof. The nanoparticles can contain one or more lipids or amphiphilic compounds. The nanoparticles are preferably made of one or more biocompatible lipids. The nanoparticles can be made of one or a mixture of different lipids that can be neutral, anionic or cationic at physiological pH (such as pH 7.4). As a non-limiting example, charged lipids can be combined with lipids that are non-ionic or uncharged at physiological pH.

[0241] In some forms, the nanoparticles can be lipid micelles. Lipid micelles for drug delivery are known in the art. For example, lipid micelles can be formed as water-in-oil emulsions with lipid surfactants. An emulsion is a blend of two immiscible phases, where surfactants are added to stabilize the dispersed droplets. Lipid micelles can be microemulsions. A microemulsion is a thermodynamically stable system composed of at least water, oil, and a lipid surfactant, resulting in a transparent and thermodynamically stable system with droplet sizes less than 1 micron, about 10 nm to about 500 nm, or about 10 nm to about 250 nm. Lipid micelles are generally useful for encapsulating hydrophobic active agents, including hydrophobic therapeutic agents, hydrophobic prophylactic agents, or hydrophobic diagnostic agents.

[0242] In some forms, the nanoparticles can be liposomes, such as liposomal vesicles. Liposomal vesicles generally contain an aqueous medium surrounded by lipids arranged in a spherical bilayer. Liposomal vesicles can be classified as small unilamellar vesicles, large unilamellar vesicles, or multilamellar vesicles. Multilamellar liposomes contain multiple concentric lipid bilayers. Liposomes can be used to encapsulate therapeutic agents, diagnostic agents, and / or prophylactic agents by entrapping hydrophilic agents within the aqueous interior or between the bilayers, or by entrapping hydrophobic agents within the bilayers.

[0243] Lipid micelles and liposomes generally have an aqueous core. The aqueous core can contain water or a mixture of water and an alcohol. Suitable alcohols include, but are not limited to, methanol, ethanol, propanol (such as isopropyl alcohol), butanol (such as n-butanol, isobutanol, sec-butanol, tert-butanol), pentanol (such as pentanol, isobutanol), hexanol (such as 1-hexanol, 2-hexanol, 3-hexanol), heptanol (such as 1-heptanol, 2-heptanol, 3-heptanol, and 4-heptanol), or octanol (such as 1-octanol), or combinations thereof.

[0244] In some forms, the nanoparticles can be solid lipid nanoparticles. Solid lipid nanoparticles are alternatives to colloidal micelles and liposomal vesicles. The size of solid lipid nanoparticles is generally submicron, i.e., about 10 nm to about 1 micron, 10 nm to about 500 nm, or 10 nm to about 250 nm. Solid lipid nanoparticles can be formed from lipids that are solid at room temperature. They are derived from oil-in-water emulsions by replacing liquid oil with solid lipid.

[0245] Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or polyethylene glycolated lipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC), including 1,2-diacyl-glycerol-3-phosphocholine; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingomyelin phospholipids such as sphingomyelin and glycosphingolipids (also known as 1-ceramidoglucosides), such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols containing a carboxylic acid group, such as cholesterol; 1,2-diacyl-sn-glycerol-3-phosphoethanolamine, including, but not limited to, 1,2-dioleoylphosphoethanolamine (DOPE), 1,2-dihexadecylphosphoethanolamine (DHPE), 1,2-distearoylphosphatidylcholine (DSPC), 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1,2-dimyristoylphosphatidylcholine (DMPC). Lipids may also include various natural (e.g., tissue-derived L-α-phosphatidyl: egg yolk, heart, brain, liver, soy) and / or synthetic (e.g., saturated and unsaturated 1,2-diacyl-sn-glycerol-3-phosphocholine, 1-acyl-2-acyl-sn-glycerol-3-phosphocholine, 1,2-diheptanoyl-SN-glycerol-3-phosphocholine) lipid derivatives.

[0246] Suitable cationic lipids include, but are not limited to, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium salts, also known as TAP lipids, such as methyl sulfate. Suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Suitable cationic lipids in liposomes include, but are not limited to, dimethyldioctadecylammonium bromide (DDAB), 1,2-diacyl-oxy-3-trimethylammonium propane, N-[1-(2,3-dioleyloxy)propyl]-N,N-dimethylamine (DODAP), 1,2-diacyl-oxy-3-dimethylammonium propane, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dialkyloxy-3-dimethylammonium propane, dioctadecylamidoglycyl spermine (DOGS), 3-[N-(N',N'-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-prop-ammonium trifluoro-acetate (DOSPA),.beta.-alanine cholesterol, cetyltrimethylammonium bromide (CTAB), diC.sub.14 -amidine, N-ferf-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine, N-(α-trimethylammonioacetyl)dilauryld-glutamate chloride (TMAG), ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride, 1,3-dioleyloxy-2-(6-carboxy-spermineamido)-propionamide (DOSPER), and N,N,N',N'-tetramethyl-,N'-bis(2-hydroxyethyl)-2,3-dioleyloxy-1,4-butanedi-ammonium iodide. In one embodiment, the cationic lipid can be a 1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazoline chloride derivative, such as 1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)-imidazoline chloride (DOTIM) and 1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazoline chloride (DPTIM).In one embodiment, the cationic lipid can be a 2,3-dialkyloxypropyl quaternary ammonium compound derivative containing a hydroxyalkyl moiety on the quaternary amine, such as 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide (DORIE-HP), 1,2-dioleoyl-oxy-propyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentyl ammonium bromide (DORIE-Hpe), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DPRIE), and 1,2-distearyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DSRIE).

[0247] Suitable solid lipids include, but are not limited to, higher saturated alcohols, higher fatty acids, sphingolipids, synthetic esters, and monoglycerides, diglycerides, and triglycerides of higher saturated fatty acids. The solid lipid can include fatty alcohols having 10-40, preferably 12-30 carbon atoms, such as cetearyl alcohol. The solid lipid can include higher fatty acids having 10-40, preferably 12-30 carbon atoms, such as stearic acid, palmitic acid, capric acid, and behenic acid. The solid lipid can include glycerides, including monoglycerides, diglycerides, and triglycerides of higher saturated fatty acids having 10-40, preferably 12-30 carbon atoms, such as glyceryl monostearate, glyceryl behenate, glyceryl palmitostearate, trilaurin, tricaprylin, trimyristin, tripalmitin, tristearin, and hydrogenated castor oil. Suitable solid lipids can include cetyl palmitate, beeswax, or cyclodextrin.

[0248] Amphiphilic compounds include, but are not limited to: phospholipids such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), di-eicosanoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), di-tricosanoylphosphatidylcholine (DTPC), and di-tetracosanoylphosphatidylcholine (DLPC), incorporated at a ratio of 0.01-60 (weight lipid / w polymer), most preferably 0.1-30 (weight lipid / w polymer). Phospholipids that can be used include, but are not limited to, phosphatidic acid, phosphatidylcholines with saturated and unsaturated lipids, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, lysophosphatidyl derivatives, cardiolipin, and β-acyl-γ-alkyl phospholipids. Examples of phospholipids include, but are not limited to: phosphatidylcholines such as dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, di-pentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), di-eicosanoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), di-tricosanoylphosphatidylcholine (DTPC), di-tetracosanoylphosphatidylcholine (DLPC); and phosphatidylethanolamines such as dioleoylphosphatidylethanolamine or 1-hexadecyl-2-palmitoyl glycerophosphoethanolamine. Synthetic phospholipids with asymmetric acyl chains (e.g., one acyl chain with 6 carbons and another acyl chain with 12 carbons) can also be used.

[0249] In some embodiments, liposomes can be coated with a water-soluble biocompatible polymer. Suitable polymers include, but are not limited to, polyoxyalkenes such as polyethylene glycol (PEG), poly(ethylene glycol)-polypropylene block copolymers such as poly(N-isopropylacrylamide) (PNIPAM), polyacrylamide (PAM), poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), poly(phosphobetaine), and polyethyleneimine (PEI). In some forms, the polymer can be a liposome coated with polyethylene glycol, collectively referred to as pegylated liposomes.

[0250] iii. Inorganic nanoparticles

[0251] In some forms, the particles can have an inorganic composition, including but not limited to minerals, including silica, silicates; sulfides (such as bismuth sulfide (Bi2S3), gold-bismuth sulfide (Au-siBi2S3)), oxides, halides, carbonates, sulfates, phosphates; iron(II) oxide, iron(III) oxide. In some forms, the particles can also be made of one or more metals, such as gold nanoparticles, silver nanoparticles, copper, platinum, palladium, ruthenium, or combinations thereof.

[0252] iv. Size

[0253] The size of the particles can vary. In some forms, the particle size is from about 5 nm to less than 1,000 nm. In some forms, the particle size is from about 10 nm to about 750 nm. In some forms, the particle size is from about 10 nm to about 500 nm. In some forms, the particle size is from about 10 nm to about 250 nm. In some forms, the particle size is from about 50 nm to about 250 nm. In some forms, the particle size is from about 50 nm to about 150 nm. In some forms, the particle size is from about 50 nm to about 100 nm. In some forms, the particle size is about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 78 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm or about 150 nm.

[0254] v. Targeting agent

[0255] Compounds containing one or more S-nitrosothiol moieties and / or nanoparticles for delivering the compounds may also comprise a targeting agent. The targeting agent can be a peptide, nucleic acid, glycoprotein, carbohydrate, lipid or small molecule that binds to one or more targets associated with an organ, tissue, cell, subcellular site or extracellular matrix.

[0256] In some forms, one or more targeting agents can be conjugated to the compound or nanoparticle, preferably covalently. The targeting agent can be covalently associated with the compound or nanoparticle directly or indirectly through a linking group. Although this document mainly discusses attaching the targeting agent to the nanoparticle, in some embodiments, the compound containing one or more S-nitrosothiol moieties is conjugated to a targeting agent such as a peptide or protein through a suitable linking group and used without a nanoparticle. Preferably, the targeting agent does not interfere with the activity of the compound and the compound does not interfere with receptor binding. It is believed that if the spacer is long enough, the interference of the drug with receptor binding will be low.

[0257] In some embodiments, conjugation of the target agent to the compound or nanoparticle is achieved by linking a thiol (-SH) (e.g., on cysteine) and an amine using a crosslinker such as sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). For example, the compound or particle can have a free amine (e.g., PLGA-b-PEG-amine), and the targeting agent can have a cysteine with -SH available for crosslinking. In other embodiments, a carboxyl-to-amine crosslinker such as EDC / NHS can be used. In a particular example, the particle has a free carboxyl (e.g., PLGA-b-PEG-COOH), and the targeting agent has a free amine. Click chemistry can also be used for conjugation. A common example is azide-alkyne conjugation via copper(I)-catalyzed or copper-free strain-promoted cycloaddition. Strain-promoted alkyne-nitrone cycloaddition is also viable. The azide and alkyne can be conjugated to the compound or nanoparticle and the targeting ligand, respectively, and click chemistry joins the two components together.

[0258] The particles can be composed of a mixture of polymers, e.g., with and without moieties for conjugating the targeting agent. For example, by reference to the description of the specific foregoing embodiments, the particles can be formed from a mixture comprising PLGA-b-PEG and PLGA-b-PEG-amine or a mixture of PLGA-b-PEG and PLGA-b-PEG-COOH. Any suitable ratio of polymers can be used and can be used to regulate the relative amount of targeting agent present on the nanoparticle surface (i.e., ligand surface density). For example, the molar ratio of the polymer having the conjugating moiety to the polymer not having the conjugating moiety can be X:Y, where X and Y are independently any integer from 1 to 100 (including the end values). In an exemplary, non-limiting embodiment, the molar ratio of the polymer having the conjugating moiety to the polymer not having the conjugating moiety is 1:10. The ratio of peptide to particle can then be calculated (Derman et al., J Biomed Sci, 22, 89 (2015)).

[0259] Preferably, the targeting agent binds to a molecule specific for tumor cells (targeting moiety) or can be expressed at a higher level on tumor cells compared to non-tumor cells.

[0260] Examples of targeting agents include: peptides such as iRGD, NGR, iNGR, RGR, LyP1; small molecules such as folic acid; aptamers, antibodies, antigen-binding fragments or fusion proteins of antibodies.

[0261] Exemplary antibodies and fragments that can be used include monoclonal and polyclonal antibodies, single-chain antibodies, single-chain variable fragments (scFv), diabodies, triabodies, tetra-bodies, disulfide-linked Fvs (sdFV), Fab’, F(ab’)2, Fv, and single-domain antibody fragments (sdAb). The antibodies can bind to targets in cancer cells or the tumor microenvironment. Exemplary cancer antigen targets are discussed below.

[0262] Examples of targeting peptides are described in U.S. Patent Nos. 6,177,542, 7,544,767, and 6,576,239; U.S. Patent Application Publication No. 20090257951; and Hoffman et al., Cancer Cell, vol. 4 (2003). Available NGR peptides include: peptides such as X2CNGRCX2 (SEQ ID NO: 1), CX2(C / X)NGR(C / X)X2C (SEQ ID NO: 2), and CNGRCX6 (SEQ ID NO: 3) (where “X” is any amino acid), which can be linear or cyclic.

[0263] Peptides that can be used for tumor targeting include, for example, iRGD, LyP-1, iNGR, and RGR peptides. iRGD has a unique target within tumors; it preferentially accumulates in the hypoxic / hypo-nutrient regions of tumors (Laakkonen et al., 2002; 2004; Karmali et al., 2009). CRGRRST (SEQ ID NO: 4) (RGR; Joyce et al., 2003) is a peptide that has been successfully used to target cytokine antibody combinations to tumors (Hamzah et al., 2008). The peptide is linear, which simplifies synthesis. NGR peptides target angiogenic vasculature, including angiogenic vasculature associated with tumors, and α v integrin and α5β1 integrin (U.S. Patent Nos. 6,576,239 and 6,177,542 and U.S. Patent Application Publication No. 20090257951).

[0264] In some forms, the targeting moiety is an antigen expressed by tumor cells. Antigen markers, such as serologically defined markers known as tumor-associated antigens, are known to be uniquely expressed by cancer cells or to be present at significantly higher levels (e.g., elevated in a statistically significant manner) in subjects with a malignancy relative to an appropriate control.

[0265] Tumor-associated antigens can include, for example, products encoded by cellular oncogenes or abnormally expressed proto-oncogenes (e.g., products encoded by the neu, ras, trk, and kit genes), or mutant forms of growth factor receptors or receptor-like cell surface molecules (e.g., the surface receptor encoded by the c-erb B gene). Other tumor-associated antigens include molecules that can be directly involved in transformation events, or molecules that may not be directly involved in carcinogenic transformation events but are expressed by tumor cells (e.g., carcinoembryonic antigen, CA-125, melanoma-associated antigens, etc.) (see, for example, U.S. Patent 6,699,475; Jager et al., Int. J. Cancer, 106:817-20 (2003); Kennedy et al., Int. Rev. Immunol., 22:141-72 (2003); Scanlan et al., Cancer Immun., 4:1 (2004)).

[0266] Genes encoding cellular tumor-associated antigens include abnormally expressed cellular oncogenes and proto-oncogenes. Generally, cellular oncogenes encode products directly related to cell transformation, and thus these antigens are particularly preferred targets for immunotherapy. An example is the oncogenic neu gene encoding a cell surface molecule involved in carcinogenic transformation. Other examples include the ras, kit, and trk genes. Products of proto-oncogenes (normal genes that mutate to form oncogenes) can be abnormally expressed (e.g., overexpressed), and such abnormal expression can be related to cell transformation. Thus, products encoded by proto-oncogenes can be targeted. Some oncogenes encode growth factor receptor molecules or growth factor receptor-like molecules expressed on the surface of tumor cells. An example is the cell surface receptor encoded by the c-erbB gene. Other tumor-associated antigens may or may not be directly involved in malignant transformation. However, these antigens are expressed by certain tumor cells and can thus provide effective targets. Some examples are carcinoembryonic antigen (CEA), CA 125 (associated with ovarian cancer), and melanoma-specific antigens.

[0267] For example, in ovarian cancer and other cancers, tumor-associated antigens can be detected in samples of readily accessible biological fluids such as serum or mucosal secretions. One such marker is CA125, a cancer-associated antigen that also flows into the bloodstream and can be detected in serum (e.g., Bast et al., N.Eng.J.Med., 309:883 (1983); Lloyd et al., Int.J.Canc., 71:842 (1997)). CA125 levels in serum and other biological fluids, as well as levels of other markers such as carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), tissue polypeptide specific antigen (TPS), sialyl-TN mucin (STN), and placental alkaline phosphatase (PLAP), have been measured in an effort to provide diagnostic and / or prognostic profiles for ovarian cancer and other cancers (e.g., Sarandakou et al., Acta Oncol., 36:755 (1997); Sarandakou et al., Eur.J.Gynaecol.Oncol., 19:73 (1998); Meier et al., Anticancer Res., 17(4B):2945 (1997); Kudoh et al., Gynecol.Obstet.Invest., 47:52 (1999)). Elevated serum CA125 can also accompany neuroblastoma (e.g., Hirokawa et al., Surg.Today, 28:349 (1998)), while elevated CEA and SCC, etc., can accompany colorectal cancer (Gebauer et al., Anticancer Res., 17(4B):2939 (1997)).

[0268] The tumor-associated antigen mesothelin (defined by reactivity with monoclonal antibody K-1) is present in most squamous cell carcinomas, including epithelial ovarian tumors, cervical tumors, and esophageal tumors, as well as in mesotheliomas (Chang et al., Cancer Res., 52:181 (1992); Chang et al., Int. J. Cancer, 50:373 (1992); Chang et al., Int. J. Cancer, 51:548 (1992); Chang et al., Proc. Natl. Acad. Sci. USA, 93:136 (1996); Chowdhury et al., Proc. Natl. Acad. Sci. USA, 95:669 (1998)). Using MAb K-1, mesothelin is detectable only as a cell-associated tumor marker and is not found in soluble form in the sera of ovarian cancer patients or in the conditioned medium of OVCAR-3 cells (Chang et al., Int. J. Cancer, 50:373 (1992)). However, structurally related human mesothelin polypeptides also include tumor-associated antigen polypeptides, such as the unique mesothelin-related antigen (MRA) polypeptide, which can be detected as a naturally occurring soluble antigen in the biological fluids from patients with malignancies (see WO 00 / 50900).

[0269] Tumor antigens can include cell surface molecules. Tumor antigens with known structures and known or described functions include the following cell surface receptors: HER1 (GenBank accession number: U48722), HER2 (Yoshino et al., J. Immunol., 152:2393 (1994); Disis et al., Canc. Res., 54:16 (1994); GenBank accession numbers X03363 and M17730), HER3 (GenBank accession numbers U29339 and M34309), HER4 (Plowman et al., Nature, 366:473 (1993); GenBank accession numbers L07868 and T64105), epidermal growth factor receptor (EGFR) (GenBank accession numbers U48722 and KO3193), vascular endothelial growth factor (GenBank NO: M32977), vascular endothelial growth factor receptor (GenBank accession numbers AF022375, 1680143, U48801 and X62568), insulin-like growth factor-I (GenBank accession numbers X00173, X56774, X56773, X06043, European Patent No. GB 2241703), insulin-like growth factor-II (GenBank accession numbers X03562, X00910, M17863 and M17862), transferrin receptor (Trowbridge and Omary, Proc. Nat. Acad. USA, 78:3039 (1981); GenBank accession numbers X01060 and M11507), estrogen receptor (GenBank accession numbers M38651, X03635, X99101, U47678 and M12674), progesterone receptor (GenBank accession numbers X51730, X69068 and M15716), follicle-stimulating hormone receptor (FSH-R) (GenBank accession numbers Z34260 and M65085), retinoic acid receptor (GenBank accession numbers L12060, M60909, X77664, X57280, X07282 and X06538), MUC-1 (Barnes et al., Proc. Nat. Acad. Sci. USA, 86:7159 (1989); GenBank accession numbers M65132 and M64928) NY-ESO-1 (GenBank accession numbers AJ003149 and U87459), NA 17-A (PCT publication number: WO 96 / 40039), Melan-A / MART-1 (Kawakami et al., Proc. Nat. Acad. Sci.USA, 91:3515 (1994); GenBank accession numbers U06654 and U06452), tyrosinase (Topalian et al., Proc. Nat. Acad. Sci. USA, 91:9461 (1994); GenBank accession number: M26729; Weber et al., J. Clin. Invest, 102:1258 (1998)), Gp-100 (Kawakami et al., Proc. Nat. Acad. Sci. USA, 91:3515 (1994); GenBank accession number: S73003, Adema et al., J. Biol. Chem., 269:20126 (1994)), MAGE (van den Bruggen et al., Science, 254:1643 (1991)); GenBank accession numbers U93163, AF064589, U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690, U10689, U10688, U10687, U10686, U10685, L18877, U10340, U10339, L18920, U03735 and M77481), BAGE (GenBank accession number: U19180; U.S. Patent Nos. 5,683,886 and 5,571,711), GAGE (GenBank accession numbers AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144, U19143 and U19142), any receptor of the CTA class (specifically including the HOM-MEL-40 antigen encoded by the SSX2 gene (GenBank accession numbers X86175, U90842, U90841 and X86174), carcinoembryonic antigen (CEA, Gold and Freedman, J. Exp. Med., 121:439 (1985); GenBank accession numbers M59710, M59255 and M29540) and PyLT (GenBank accession numbers J02289 and J02038)); p97 (melanotransferrin) (Brown et al., J. Immunol., 127:539-46 (1981); Rose et al., Proc. Natl. Acad. Sci. USA, 83:1261-61 (1986)).

[0270] Additional tumor-associated antigens include prostate-specific antigen (PSA) (U.S. Patent Nos. 6,677,157; 6,673,545); beta-human chorionic gonadotropin (β-HCG) (McManus et al., Cancer Res., 36:3476-81 (1976); Yoshimura et al., Cancer, 73:2745-52 (1994); Yamaguchi et al., Br. J. Cancer, 60:382-84 (1989): Alfthan et al., Cancer Res., 52:4628-33 (1992)); glycosyltransferase beta-1,4-N-acetylgalactosaminyltransferase (GalNAc) (Hoon et al., Int. J. Cancer, 43:857-62 (1989); Ando et al., Int. J. Cancer, 40:12-17 (1987); Tsuchida et al., J. Natl. Cancer, 78:45-54 (1987); Tsuchida et al., J. Natl. Cancer, 78:55-60 (1987)); NUC18 (Lehmann et al., Proc. Natl. Acad. Sci. USA, 86:9891-95 (1989); Lehmann et al., Cancer Res., 47:841-45 (1987)); melanoma antigen gp75 (Vijayasardahi et al., J. Exp. Med., 171:1375-80 (1990); GenBank accession number: X51455); human cytokeratin 8; high molecular weight melanoma antigen (Natali et al., Cancer, 59:55-63 (1987); keratin 19 (Datta et al., J. Clin. Oncol., 12:475-82 (1994)).

[0271] Tumor antigens of interest include antigens that are considered "cancer / testis" (CT) antigens in the art and that are immunogenic in subjects with a malignancy (Scanlan et al., Cancer Immun., 4:1 (2004)). CT antigens include at least 19 different antigen families that contain one or more members and are capable of inducing an immune response, including but not limited to: MAGEA (CT1); BAGE (CT2); MAGEB (CT3); GAGE (CT4); SSX (CT5); NY-ESO-1 (CT6); MAGEC (CT7); SYCP1 (C8); SPANXB1 (CT11.2); NA88 (CT18); CTAGE (CT21); SPA17 (CT22); OY-TES-1 (CT23); CAGE (CT26); HOM-TES-85 (CT28); HCA661 (CT30); NY-SAR-35 (CT38); FATE (CT43); and TPTE (CT44).

[0272] Additional tumor antigens that can be targeted include tumor-associated antigens or tumor-specific antigens, including but not limited to: α-actinin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, class I myosin, OS-9, ml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, Mage-A1, 2, 3, 4, 6, 10, 12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-related protein), TAAL6, TAG72, TLP, and TPS. Other tumor-associated antigens and tumor-specific antigens are known to those of skill in the art and are suitable for targeting by the disclosed fusion proteins.

[0273] In some forms, antigens associated with tumor neovasculature can also be targeted. Tumor-associated neovasculature provides an accessible route through which therapeutic agents can enter the tumor. In one embodiment, the viral protein contains a domain that specifically binds an antigen expressed by the tumor-associated neovasculature.

[0274] When compared to normal vasculature, the antigen can be specific to tumor neovasculature or can be expressed at higher levels in tumor neovasculature. Exemplary antigens overexpressed by tumor-associated neovasculature compared to normal vasculature include, but are not limited to, VEGF / KDR, Tie2, vascular cell adhesion molecule (VCAM), endoglin, and α5β3 integrin / vitronectin. Other antigens overexpressed by tumor-associated neovasculature compared to normal vasculature are known to those of skill in the art and are suitable for targeting by the disclosed fusion proteins.

[0275] Neurotensin receptor 1 (NTSR1) is upregulated in the majority of lung tumors (59.7%), but is expressed at low or undetectable levels in normal lung tissue (Alifano et al., Clinical Cancer Research, 16, 4401-4410 (2010)). NTSR1 upregulation is associated with poor 5-year overall survival, high metastatic rate, and increased neuroendocrine differentiation (Alifano et al., Clinical Cancer Research, 16, 4401-4410 (2010), Dupouy, Biochimie, 93, 1369-78 (2011)). Accordingly, in some embodiments, the disclosed compositions (e.g., nanoparticles) include a ligand for NTSR1 conjugated thereto.

[0276] NTSR1 is also upregulated in head and neck cancer, breast cancer, and colon cancer. Thus, in some embodiments, a composition that targets NTSR1 is used to treat cancers having upregulated NTSR1. In some embodiments, the subject has lung cancer (such as NSCLC), head and neck cancer, breast cancer, and / or colon cancer.

[0277] In some embodiments, the NTSR1 is a wild-type NTSR1 ligand, neurotensin (NTS), or a variant, analogue, or functional fragment thereof.

[0278] The wild-type sequence of human NTS is QLYENKPRRPYIL (SEQ ID NO: 5), UniProtKB - P30990 (NEUT_HUMAN) - amino acids 151 - 163, which is specifically incorporated herein by reference in its entirety. In some embodiments, the NTSR1 ligand comprises at least 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% sequence identity to SEQ ID No: 5. Neurotensin has significant sequence similarity in its 6 C-terminal amino acids to several other neuropeptides, including neuromedin N (which is derived from the same precursor) (see, e.g., UniProtKB - P30990 (NEUT_HUMAN)). This C-terminal region is responsible for the full biological activity, and the N-terminal portion has a regulatory role.

[0279] The neurotensin / neuromedin N precursor can also be processed to produce large 125 - 138 amino acid peptides that have a neurotensin or neuromedin N sequence at their C-terminus. These large peptides do not seem to be as effective as their smaller counterparts, but are also less sensitive to degradation and can represent endogenous persistent activators in many pathophysiological situations.

[0280] Due to peptidase degradation, the half-life of NTS in vivo is very short (Reinecke et al., Prog Histochem Cytochem, 16, 1 - 172 (1985), Wu et al., J Nucl Med, 55, 1178 - 1184 (2014)). Thus, in some embodiments, the NTSR1 ligand is an NTS analogue. Exemplary NTS analogues are NTS mut . NTS mut comprises amino acids 7 - 13 of SEQ ID NO: 5 (e.g., PRRPYIL (SEQ ID NO: 6), in which non-natural amino acids are introduced to stabilize the bonds between Arg8 - Arg9, Pro10 - Tyr11, and Tyrl1 - Ile12.

[0281] Provided below is NTS having a terminal cysteine to facilitate peptide conjugation to nanoparticles or compounds mutStructure:

[0282]

[0283] See also, e.g., Wu et al., J Nucl Med, 55(7):1178 - 84 (2014) doi:10.2967 / jnumed.114.137489, which is hereby incorporated by reference in its entirety.

[0284] Compared to wild - type NTS, NTS mut provides nanomolar affinity comparable to NTSR1 but is much more bio - stable. The experimental results presented below show that conjugating NTS mut to nanoparticles improves tumor accumulation ( Figure 11 ). Increased surface ligand density can enhance the binding affinity to NTSR1 but can also increase surface hydrophobicity that can negatively impact pharmacokinetics.

[0285] In another embodiment, the ligand is NTS 20.8 , as shown below, which has a cysteine to facilitate conjugation of the peptide to nanoparticles or compounds or their derivatives.

[0286]

[0287] See also, e.g., Yin et al., Amino Acids, 49(8):1325 - 1335. doi:10.1007 / s00726 - 017 - 2430 - 5, which is hereby incorporated by reference in its entirety.

[0288] In another embodiment, the ligand is SR142948A or its derivative, or its NTSR1 antagonist analogue (Moody, et al., Front Endocrinol, 9(2018), Kling, ACS Chem Biol, 11, 869 - 75(2016), Schaeffer, J Cardiovasc Pharmacol, 31, 545 - 50(1998).

[0289] In some embodiments, the ligand has the following structure:

[0290]

[0291] Other peptidomimetics and non-peptide receptor agonists and antagonists known in the art can be used as targeting ligands. See, e.g., Kleczkowska and Lipkowski, European Journal of Pharmacology, 716(1-3):54-60(2013), which is specifically incorporated herein by reference in its entirety.

[0292] III. Preparation Methods and Agents

[0293] A. Agents to be Delivered

[0294] Methods for preparing agents to be delivered are provided. Preferred agents to be delivered can be, for example, prodrug compounds of radiosensitizers, including microtubule polymerization inhibitors, which contain one or more S-nitrosothiol moieties. In some forms, the prodrug compounds are maytansine alkaloid analogs containing one or more S-nitrosothiol moieties. These maytansine alkaloids can be generated by chemical synthesis; isolated from higher plants, mosses, and microorganisms and then chemically modified to contain one or more S-nitrosothiol moieties. Cassady et al., Chem. Pharm. Bull., 52(1), 1-26(2004) provides various strategies for generating maytansine alkaloids, the content of which is incorporated herein by reference. Methods for chemically modifying general small molecules or maytansine alkaloids to contain one or more S-nitrosothiol moieties were discussed above.

[0295] B. Nanoparticles

[0296] Methods for preparing particles are also provided.

[0297] i. Polymeric Nanoparticles

[0298] Nanoprecipitation

[0299] In some forms, nanoparticles can be prepared by the nanoprecipitation method. In this method, a water-soluble or water-miscible organic solvent is used to dissolve the polymer and form an emulsion when mixed with an aqueous phase (preferably with moderate stirring). The organic solvent rapidly diffuses into the water, resulting in the immediate formation of nanoparticles after mixing. After the formation of the nanoparticles, the solvent can be removed under low pressure / vacuum. Nanoprecipitation can be used to encapsulate hydrophobic or hydrophilic compounds, although this method is typically used to encapsulate hydrophobic compounds.

[0300] ii. Other Methods for Forming Nanoparticles

[0301] The nanoparticles described herein can be formed using a variety of techniques known in the art. The technique to be used can depend on a variety of factors, including the polymer used to form the nanoparticles, the desired size range of the resulting nanoparticles, and the suitability of the therapeutic, diagnostic, and / or prophylactic agents to be incorporated. Suitable techniques include, but are not limited to:

[0302] a. Solvent evaporation

[0303] In this method, the polymer is dissolved in a volatile organic solvent. The drug (either soluble or dispersed as fine particles) is added to the solution, and the mixture is suspended in an aqueous solution containing a surfactant such as poly(vinyl alcohol). The resulting emulsion is stirred until most of the organic solvent has evaporated, leaving solid nanoparticles. The resulting nanoparticles are washed with water and dried overnight in a freeze dryer. Nanoparticles of different sizes and morphologies can be obtained by this method.

[0304] b. Solvent removal

[0305] In this method, the drug is dispersed or dissolved in a solution of the selected polymer in a volatile organic solvent. The mixture is suspended in an organic oil (such as silicone oil) by stirring to form an emulsion. Different from solvent evaporation, this method can be used to prepare nanoparticles from polymers with high melting points and different molecular weights. The external morphology of the spheres produced by this technique highly depends on the type of polymer used.

[0306] c. Spray drying

[0307] In this method, the polymer is dissolved in an organic solvent. A known amount of the active drug is suspended (for insoluble drugs) or co-dissolved (for soluble drugs) in the polymer solution. Then the solution or dispersion is spray dried.

[0308] d. Phase inversion

[0309] Nanospheres can be formed from polymers using the phase inversion method, in which the polymer is dissolved in a "good" solvent, fine particles of the substance to be incorporated (such as a drug) are mixed or dissolved in the polymer solution, and the mixture is poured into a strong non-solvent of the polymer to spontaneously generate polymer microspheres under favorable conditions, where the polymer is coated with the particles or the particles are dispersed in the polymer. The method can be used to produce nanoparticles in a wide size range (including, for example, from about 100 nanometers to about 10 micrometers). Substances that can be incorporated include, for example, imaging agents (such as fluorescent dyes) or bioactive molecules (such as proteins or nucleic acids). During the process, the polymer is dissolved in an organic solvent and then contacted with a non-solvent, which causes the dissolved polymer to undergo a phase inversion to form small spherical particles with a narrow size distribution, optionally incorporating antigens or other substances.

[0310] e. Microfluidics

[0311] Methods for preparing nanoparticles using microfluidics are known in the art. Suitable methods include those described in U.S. Patent Application Publication No. 2010 / 0022680 A1 to Karnik et al. Generally, a microfluidic device includes at least two channels that converge into a mixing device. The channels are typically formed by lithography, etching, imprinting, or molding of a polymer surface. Fluid sources are connected to each channel, and applying pressure to the sources causes fluid to flow in the channels. The pressure can be applied by syringe, pump, and / or gravity. The inlet streams of solutions having polymers, targeting moieties, lipids, drugs, payloads, etc. converge and mix, and the resulting mixture is combined with a polymer non-solvent solution to form nanoparticles having a desired size and density of moieties on the surface. By varying the pressure and flow rate in the inlet channels and the nature and composition of the fluid sources, nanoparticles having reproducible size and structure can be produced.

[0312] f. Self-assembly

[0313] In some forms, nanoparticles are formed by the self-assembly of amphiphilic block copolymers in aqueous solution. In an aqueous environment, the amphiphilic copolymer can spontaneously self-assemble to form nanoparticles having a hydrophobic core and a hydrophilic shell. In some forms, a solution containing the amphiphilic polymer is mixed with another solution containing a therapeutic, diagnostic, and / or prophylactic agent for encapsulation. In some forms, the amphiphilic polymer and the therapeutic, diagnostic, and / or prophylactic agent to be delivered are dissolved in a suitable solvent, such as tetrahydrofuran, DMSO, or dichloromethane. Preferably, the therapeutic agent is a compound described herein. The concentration of the amphiphilic polymer and the therapeutic, diagnostic, and / or prophylactic agent in the solvent can vary as needed. After forming a solution containing the amphiphilic polymer and the therapeutic, diagnostic, and / or prophylactic agent, the solution can be continuously added to an aqueous solution to induce nanoparticle formation (micellization). The nanoparticle suspension can be stirred at room temperature and then dialyzed, placed in an ultrafiltration centrifuge tube, and centrifuged to obtain the nanoparticles.

[0314] iii. Liposomal nanoparticles

[0315] Suitable methods, materials, and lipids for preparing liposomes are known in the art. Liposomal delivery vehicles are commercially available from a variety of sources. Commercially available liposomal formulations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO - BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany), and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.) and other liposomes developed according to standard procedures in the art are well known. For example, liposomes can be prepared by hydration of modified lipid films (Szoka et al., Annual review of biophysics and bioengineering, 9:467 - 508 (1980).

[0316] iv. Inorganic nanoparticles

[0317] The inorganic nanoparticles described herein can be manufactured using methods recognized in the art, such as gas - phase synthesis, liquid - phase synthesis, solid - phase synthesis, or combinations thereof. Some methods are described in Tsuzuki, Int. J. Nanotechnol. 2009, 6(5 / 6), 567 - 578, the content of which is incorporated herein by reference.

[0318] The disclosed compounds can be bound to the surface of inorganic nanoparticles by covalent or non - covalent interactions. For example, in some embodiments, the disclosed compounds are bound to the surface of inorganic nanoparticles by electrostatic interactions or hydrophobic - hydrophobic interactions. For inorganic nanoparticles having a porous structure, the compounds can also be entrapped within the pores of the particles.

[0319] IV. Pharmaceutical compositions

[0320] Pharmaceutical compositions comprising the disclosed compounds with or without a particle - based delivery platform are provided. The pharmaceutical compositions can be used, for example, for administration by parenteral (e.g., intramuscular, intraperitoneal, intravenous (IV), or subcutaneous) injection or infusion.

[0321] In some embodiments, the pharmaceutical composition is a unit dose containing an effective amount of the disclosed composition. In some embodiments, the unit dose is a unit dosage form for intravenous injection. In some embodiments, the unit dose is a unit dosage form for intratumoral injection.

[0322] In some embodiments, the composition is administered systemically, for example, by intravenous or intraperitoneal administration, in an amount effective to deliver the composition to target cells.

[0323] In certain embodiments, the composition is administered locally, such as by subcutaneous injection, or directly into the site to be treated. In some embodiments, the composition is injected or otherwise directly administered to one or more tumors. Generally, local injection results in an increased local concentration of the composition, which is greater than the concentration achievable by systemic administration. In some embodiments, the composition is locally delivered to the appropriate cells by using a catheter or syringe. Other ways of locally delivering such a composition to cells include using an infusion pump (e.g., from Alza Corporation, Palo Alto, Calif.) or incorporating the composition into a polymeric implant (see, e.g., P. Johnson and J.G. Lloyd-Jones, eds., Drug Delivery Systems (Chichester, England: Ellis Horwood Ltd., 1987), which can provide a sustained release of particles into the immediate area of the implant).

[0324] The compounds and their particle-based formulations can be provided directly to cells, such as by contacting them with the cells, or indirectly, such as by the action of any biological process. For example, the compounds and their particle-based formulations can be formulated in a physiologically acceptable carrier or vehicle and injected into the tissue or fluid surrounding the cells.

[0325] A. Formulations for Parenteral Administration

[0326] In a preferred embodiment, the composition is administered by parenteral injection in the form of an aqueous solution.

[0327] The formulation can be in the form of a suspension or an emulsion. Generally, there is provided a pharmaceutical composition comprising an effective amount of the disclosed compound and optionally comprising a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. Such compositions can include diluents such as sterile water, various buffer contents (e.g., Tris-HCl, acetate, phosphate), buffered saline for pH and ionic strength; and optionally, additives such as detergents and solubilizers (e.g., 20, 80, also known as polysorbate 20 or 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thimerosal, benzyl alcohol) and fillers (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil and corn oil), gelatin, and injectable organic esters (such as ethyl oleate). The formulation can be lyophilized and re-dissolved / re-suspended immediately before use. The formulation can be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating a bactericide into the composition, by irradiating the composition, or by heating the composition.

[0328] B. Other formulations

[0329] The disclosed compounds may also be administered topically, either alone or in particulate formulations. Topical administration may include application to the lung, nose, mouth (sublingual, buccal), vagina, or rectal mucosa. In some embodiments, the composition is administered in combination with a transdermal or mucosal transport component.

[0330] A wide range of mechanical devices designed for the pulmonary delivery of therapeutic products may be used, including but not limited to nebulizers, metered-dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are Nebulizer (Mallinckrodt Inc., St. Louis, Mo.); II Nebulizer (Marquest Medical Products, Englewood, Colo.); Metered-dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and Powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes, and Mannkind all have inhalable insulin powder formulations that have been approved or are in clinical trials, and the technology can be applied to the formulations described herein.

[0331] Oral formulations may be in the form of chewing gum, gel strips, tablets, capsules, or lozenges. Oral formulations may include excipients or other modifications to the particles that may confer intestinal protection or enhanced delivery through the gastrointestinal tract (including the intestinal epithelium and mucosa) (see Samstein et al., Biomaterials, 29(6):703-8 (2008).

[0332] Transdermal formulations may also be prepared. These are typically ointments, lotions, sprays, or patches, all of which can be prepared using standard techniques. Transdermal formulations may include penetration enhancers.

[0333] V. Methods of Use

[0334] A. Methods of Treatment

[0335] The method of use is provided. The following experiments demonstrate the use of an exemplary nanoparticle-based maytansine alkaloid radiosensitizer prodrug (DM1-NO) in the treatment of cancer. Due to nanoparticle (PLGA) encapsulation and S-nitrosylation, the toxicity of DM1 is inhibited, thereby allowing systemic delivery of the therapeutic agent to the tumor via the EPR effect. Subsequent irradiation increases oxidative stress in the tumor, leading to the cleavage of the S-N bond and the release of both DM1 and NO, both of which are potent radiosensitizers( Figure 1A ). Specifically, NO reacts with reactive oxygen species (ROS) to form free radicals, such as peroxynitrite, which can effectively oxidize lipid, lipoprotein, and DNA molecules (Bloodsworth et al., Arterioscler., Thromb., Vasc. Biol. 20, 1707-1715 (2000)). On the other hand, DM1 causes mitotic arrest and cell enrichment in the more radiosensitive G2 / M phase.

[0336] A nanoparticle radiosensitizer that can sensitize NSCLC cells to RT while causing minimal systemic toxicity is highly desirable. In the following experiments, the radiosensitizing effect of DM1-NO was first evaluated in vitro by a colony formation assay and then in vivo in a rodent NSCLC tumor model. NSCLC accounts for 85% of all lung cancer cases, and 234,030 people were diagnosed in the United States alone in 2018 (Jemal et al., Ca-Cancer J. Clin. 60, 277-300 (2010)). RT is the standard treatment for most patients with locally advanced or regional disease, and for stage I patients, it is a viable alternative to lobectomy and lymph node dissection (Baker et al., Radiat. Oncol. 11, 115 (2016)).

[0337] Accordingly, a method of treating a subject is provided. The method generally comprises administering to a subject in need thereof an effective amount of the disclosed compound having one or more S-nitrosothiol moieties. In a preferred embodiment, a particle-based delivery platform is used to deliver the composition to the subject.

[0338] In the following experiments, 200 μl of free drug (DM1, DM1-NO) or drug-loaded nanoparticles (DM1-NP, DM1-NO-NP) were administered to mice by intravenous delivery in PBS at a dose of 260.8 nmol / kg (or approximately 0.2 mg / kg). As further studies are conducted, information regarding appropriate dose levels for treating various conditions in various patients will emerge, and one of ordinary skill in the art will be able to determine the appropriate dose considering the recipient's treatment background, age, and general health. The selected dose depends on the desired therapeutic effect, the route of administration, and the duration of the desired treatment.

[0339] Generally, a dose level of 0.001 to 10 mg / kg body weight, such as 0.1 mg / kg to 1 mg / kg body weight, is administered to a mammal. Generally, for topical administration, the dose can be lower than for systemic administration. The dose can be a daily dose, or any other dosing regimen consistent with the disclosed methods. The timing of administration of the composition will also depend on the formulation and / or route of administration used. The compound can be administered once a day, but can also be administered two, three, or four times a day, or once every other day, or once or twice a week. For example, one or more treatments can be administered to a subject at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, days, weeks, or months.

[0340] The subject can have one or more malignant or non-malignant tumors. In some embodiments, the subject has cancer.

[0341] Generally, the composition is administered to the subject in combination with radiotherapy. Although mainly discussed herein with reference to ionizing radiotherapy, it is believed that the disclosed compounds can also be used as sensitizers for phototherapy and / or proton therapy. Accordingly, in the methods disclosed herein, substitution of ionizing radiation with phototherapy or proton therapy is specifically contemplated and disclosed.

[0342] In some embodiments, a compound having one or more S-nitrosothiol moieties is administered in an effective amount relative to the administration of radiation alone and / or the administration of a non-S-nitrosylated compound alone or in combination with radiation to enhance the treatment of a tumor or cancer. In some embodiments, the administration of a compound having one or more S-nitrosothiol moieties has reduced and / or inhibited systemic toxicity compared to the administration of a non-S-nitrosylated compound. In a preferred embodiment, the compound is an S-nitrosylated maytansine alkaloid compound, such as DM1-NO.

[0343] In a preferred embodiment, the radiation is ionizing radiation. Ionizing radiation therapy (also known as radiotherapy and RT) is the medical use of ionizing radiation as part of cancer treatment to control malignant cells. Ionizing radiation is generally defined as radiation having sufficient energy to release electrons from atomic orbitals, thereby causing the atom to become charged or ionized. Ionizing radiation can be administered to a subject in need thereof as part of radiotherapy for treating cancer. Examples of radiotherapy include, but are not limited to, external beam radiotherapy (EBRT or XRT) or teletherapy, brachytherapy or sealed source radiotherapy, and systemic radioisotope therapy or unsealed source radiotherapy. Radiotherapy can be administered to a subject externally (i.e., in vitro), or internally such as by brachytherapy (which typically utilizes a sealed radiation source placed in the treatment area) to the subject, and / or by systemic administration of a radioisotope by infusion or oral ingestion to the subject. Radiotherapy can include the temporary or permanent placement of a radiation source on or within a subject. Another example of radiotherapy is particle therapy, which typically includes external beam radiotherapy, where the particles are protons or heavier ions.

[0344] Radiotherapy works by damaging the DNA of dividing cells, such as cancer cells. This DNA damage is caused by one of two types of energy (photons or charged particles). This damage is either direct or indirect. Indirect ionization is the result of the ionization of water, forming free radicals, particularly hydroxyl radicals, which then damage the DNA. For example, most of the radiation effects caused by photon therapy are through the production of free radicals. One of the major limitations of photon radiotherapy is that solid tumor cells become hypoxic, and tumor cells in a hypoxic environment can be two to three times more resistant to radiation damage than those in a normal oxygen environment.

[0345] Direct damage to cancer cell DNA occurs through high LET (linear energy transfer) charged particles such as protons, boron, carbon, or neon ions. This damage is independent of tumor oxygenation because these particles act mainly through direct energy transfer, which typically results in double-strand DNA breaks. Due to their relatively large mass, protons and other charged particles have little lateral scatter in tissue; the beam does not spread much, focusing on the tumor shape and delivering a small dose of side effects to surrounding tissue.

[0346] The amount of radiation used in photon radiotherapy is measured in grays (Gy) and varies depending on the type and stage of the cancer being treated. For curative cases, typical doses for solid epithelial tumors are in the range of 60 to 80 Gy, while lymphomas are treated with 20 to 40 Gy. Postoperative (adjuvant) doses are typically about 45 - 60 Gy, divided into fractions of 1.8 - 2 Gy (for breast cancer, head and neck cancer). Many other factors are considered by radiation oncologists when selecting the dose, including whether the patient is receiving chemotherapy, the patient's comorbidities, whether radiotherapy is used before or after surgery, and the success of the surgery.

[0347] The response of cancer to radiation is described by its radiosensitivity. Highly radiosensitive cancer cells are rapidly killed by a moderate dose of radiation. These include leukemia, most lymphomas, and germ cell tumors. Most epithelial cancers are only moderately radiosensitive and require significantly higher doses of radiation (60 - 70 Gy) to achieve cure. Certain types of cancer are significantly radioresistant, meaning that the dose required to effect a cure is much higher than what can be safely delivered in clinical practice. Renal cell carcinoma and melanoma are generally considered radioresistant.

[0348] In some embodiments, the compositions and methods reduce the radiation dose required to induce a curative or prophylactic effect. For example, the disclosed compounds can increase the radiosensitivity of cancer. An effective dose of radiation therapy can be toxic to certain cancers. In some embodiments, the compounds reduce the required effective dose of radiation needed to treat cancer, thereby reducing the toxicity of the effective dose of radiation.

[0349] In other embodiments, the disclosed compounds can be used in combination with normal doses of drugs or radiation to enhance efficacy. For example, the compounds can be used to enhance radiation therapy for radioresistant cancers.

[0350] The response of tumors to radiation therapy is also related to their size. For complex reasons, very large tumors respond less well to radiation than smaller tumors or microscopic disease. Various strategies are used to overcome this effect. The most common technique is surgical resection prior to radiation therapy. This is most common in the treatment of breast cancer with wide local excision or mastectomy prior to adjuvant radiation therapy. Another approach is to shrink the tumor with neoadjuvant chemotherapy prior to definitive radiation therapy. In some embodiments, the disclosed methods allow for the treatment of tumors larger than can be treated with normal doses of radiation.

[0351] A third technique is to enhance the radiosensitivity of cancer by administering certain drugs during radiation therapy. The disclosed compositions can serve this third function. In these embodiments, the compounds increase the sensitivity of cells to radiation therapy, such as by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%. In addition, the compounds can be combined with one or more additional radiosensitizers. Examples of known radiosensitizers include cisplatin, gemcitabine, 5 - fluorouracil, pentoxifylline, vinorelbine, PARP inhibitors, histone deacetylase inhibitors, and proteasome inhibitors, as well as other radiosensitizers mentioned elsewhere herein.

[0352] Radiation therapy can be administered to a subject in combination with surgery, chemotherapy, hormone therapy, immunotherapy, or a combination thereof. For example, intraoperative radiation therapy or (IORT) is delivered immediately after surgical resection of cancer. This method has been used for breast cancer (targeted intraoperative radiation therapy or TARGIT), brain tumors, and rectal cancer.

[0353] Radiation therapy also has a variety of applications in non-malignant conditions, such as the treatment of trigeminal neuralgia, severe thyroid eye disease, pterygium, pigmented villonodular synovitis, prevention of keloid growth, and prevention of heterotopic ossification. Thus, in some embodiments, the compositions and methods are used to increase radiosensitivity to non-malignant conditions.

[0354] In other embodiments, the composition is administered to a subject in combination with photodynamic therapy (PDT), wherein the prodrug serves as a photosensitizer. When the prodrug is exposed to light of a specific wavelength, the S-N bond is cleaved, and the parent drug compound and NO are released. One or more additional photosensitizers can also be used.

[0355] In other embodiments, the composition is administered to a subject in combination with proton therapy, wherein the prodrug serves as a sensitizer. When the prodrug is exposed to proton radiation, the S-N bond is cleaved, and the parent drug compound and NO are released.

[0356] Typically, the prodrug composition is administered, for example, minutes, hours, or days before radiation therapy, for example. For example, in an exemplary embodiment, a dose of radiation is administered 1 hour to 48 hours, or 1 hour to 24 hours, or 1 hour to 12 hours, or 1 hour to 6 hours, or 2 hours to 6 hours, or 1, 2, 3, 4, or 5 hours after administration of the pharmaceutical composition. In some embodiments, 1, 2, 3, 4, or 5 rounds of radiation are administered after each single dose of the prodrug. In some embodiments, the prodrug is administered one or more times for each round of radiation. In some embodiments, a prodrug cycle precedes each radiation cycle. For example, in a particular embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more rounds of administration of the pharmaceutical composition are carried out one after the other, followed by the administration of a dose of radiation.

[0357] In some embodiments, the disclosed compositions and methods are more effective, less toxic, or a combination thereof relative to a particular concurrent or sequential chemoradiation therapy (CRT). In some embodiments, the chemotherapeutic component of the CRT is a platinum-based doublet, optionally administered concurrently with radiation. In a more specific embodiment, the disclosed compositions and methods are more effective, less toxic, or a combination thereof relative to a cisplatin + etoposide CRT regimen.

[0358] B. Cancer to be Treated

[0359] The compositions and methods described herein can be used to treat subjects suffering from benign or malignant tumors by delaying or inhibiting tumor growth, reducing the growth or size of tumors, inhibiting or reducing tumor metastasis, and / or inhibiting or reducing symptoms associated with tumor development or growth in a subject. Treatment can also be used to reduce the hyperproliferation of non-cancerous tissues, such as endometriosis, restenosis, and scar formation (fibrosis).

[0360] Malignant tumors that can be treated are classified according to the embryonic origin of the tissue from which the tumor arises. Carcinomas are tumors that originate from endodermal or ectodermal tissues, such as the epithelial layers of the skin or internal organs and glands. The disclosed compositions are particularly effective in treating carcinomas. Sarcomas, which are less common, originate from mesodermal connective tissues, such as bone, fat, and cartilage. Leukemias and lymphomas are malignancies of hematopoietic cells in the bone marrow. Leukemias proliferate as single cells, while lymphomas tend to grow as tumor masses. Malignant tumors can occur in many organs or tissues of the body to form cancers.

[0361] Types of cancers that can be treated with the provided compositions and methods include, but are not limited to, cancers of the bone, bladder, brain, breast, cervix, colorectal, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, and uterus, such as angiosarcoma, such as multiple myeloma, adenocarcinoma, and sarcoma. In some embodiments, the disclosed compositions are used to treat multiple cancer types simultaneously. The compositions can also be used to treat metastases or tumors at multiple sites.

[0362] In some embodiments, the cancer is highly radiosensitive, moderately radiosensitive, or radioresistant (i.e., low radiosensitivity cancer). Highly radiosensitive cancer cells are rapidly killed by a moderate dose of radiation. Tissues rich in actively dividing cells generally exhibit high sensitivity to radiation, while those with few such cells have low radiosensitivity (Hayabuchi, JMAJ, 47(2): 79-83 (2004)). For example, the gonads (such as the testes and ovaries), lymphoid tissue, fetal tissue, and fetal-like embryonal cell tissue are highly radiosensitive. Tissues with low radiosensitivity include adult bone, adipose tissue, muscle, and large blood vessels. Since the radiosensitivity of a tumor reflects the sensitivity of its origin tissue, malignant lymphomas originating from lymphoid tissue and seminomas originating from the testes are highly sensitive to radiation. In contrast, osteosarcoma and liposarcoma exhibit low radiosensitivity.

[0363] Epithelial tumors and cancers are considered to have moderate radiosensitivity. Such cancers may require significantly higher doses of radiation (60 - 70 Gy) to achieve cure. Among these tumors, undifferentiated carcinoma and small cell carcinoma have relatively high radiosensitivity, followed by squamous cell carcinoma. Adenocarcinoma generally has lower radiosensitivity than other types of epithelial tumors. In view of this, head and neck cancer, esophageal cancer, cervical cancer, and skin cancer (where squamous cell carcinoma is common) appear to be good indications for radiotherapy.

[0364] However, even in esophageal squamous cell carcinoma, some are highly radiosensitive while others are not. Radiosensitivity can depend not only on the histological type of the tumor but also on other factors such as the oxygen concentration in the tumor and the mitotic cycle of the tumor cells.

[0365] Renal cell carcinoma and melanoma are generally considered radioresistant.

[0366] In particular embodiments, the cancer is lung cancer, such as non - small cell lung cancer (NSCLC). In other embodiments, the cancer is head and neck cancer, breast cancer, or colon cancer.

[0367] In some embodiments, particularly those in which the nanoparticles are characterized by an NTSR1 targeting signal, the cancer has up - regulated NTSR1. In some embodiments, cancers with up - regulated NTSR1 are lung cancer (such as non - small cell lung cancer (NSCLC)), head and neck cancer, breast cancer, or colon cancer.

[0368] The disclosed compositions and methods can be further understood by the following numbered paragraphs.

[0369] 1. A compound, the compound comprising the following structural motif:

[0370]

[0371] Wherein:

[0372] linker represents a linking group,

[0373] n is an integer from 1 to 13, including the end values.

[0374] The dashed line represents the presence or absence of a bond, and the corresponding carbon atom has no, one, or two respective attached hydrogen atoms according to its valence, and

[0375] "Linking group" is independently non-existent, substituted amide group, unsubstituted amide group, substituted alkyl group, substituted alkylene group, unsubstituted alkylene group, substituted aryl group, substituted heteroaryl group, substituted alkenyl group, substituted alkynyl group, substituted alkoxy group, substituted aryloxy group, substituted alkylthio group, substituted arylthio group, unsubstituted carbonyl group, substituted carbonyl group, unsubstituted carboxyl group, substituted carboxyl group, unsubstituted amino group, substituted amino group, unsubstituted sulfonyl group, substituted sulfonyl group, unsubstituted aminosulfonyl group, substituted aminosulfonyl group, unsubstituted phosphonyl group, substituted phosphonyl group, substituted polyaryl group, substituted C3-C 20 cyclic group or substituted C3-C 20 heterocycle.

[0376] 2. The compound according to paragraph 1, wherein the compound has the following structure:

[0377]

[0378] Wherein:

[0379] R1 is a substituted amide group, unsubstituted amide group, substituted alkyl group, substituted alkylene group, unsubstituted alkylene group, substituted aryl group, substituted heteroaryl group, substituted alkenyl group, substituted alkynyl group, substituted alkoxy group, substituted aryloxy group, substituted alkylthio group, substituted arylthio group, unsubstituted carbonyl group, substituted carbonyl group, unsubstituted carboxyl group, substituted carboxyl group, unsubstituted amino group, substituted amino group, unsubstituted sulfonyl group, substituted sulfonyl group, unsubstituted aminosulfonyl group, substituted aminosulfonyl group, unsubstituted phosphonyl group, substituted phosphonyl group, substituted polyaryl group, substituted C3-C 20 cyclic group or substituted C3-C 20 heterocycle, and

[0380] R2, R3, R4, R5, R6, R7, R8, R9, R 10 and R 11 are independently hydrogen, halogen (F, Br, Cl, I), substituted alkyl group, unsubstituted alkyl group, substituted aryl group, unsubstituted aryl group, substituted heteroaryl group, unsubstituted heteroaryl group, unsubstituted alkenyl group, substituted alkenyl group, unsubstituted alkynyl group, substituted alkynyl group, unsubstituted alkoxy group, substituted alkoxy group, unsubstituted aryloxy group, substituted aryloxy group, unsubstituted alkylthio group, substituted alkylthio group, unsubstituted arylthio group, substituted arylthio group, unsubstituted carbonyl group, substituted carbonyl group, unsubstituted carboxyl group, substituted carboxyl group, unsubstituted amino group, substituted amino group, unsubstituted sulfonyl group, substituted sulfonyl group, unsubstituted aminosulfonyl group, substituted aminosulfonyl group, unsubstituted phosphonyl group, substituted phosphonyl group, unsubstituted polyaryl group, substituted polyaryl group, unsubstituted C3-C 20 cyclic group, substituted C3-C 20A cyclic group, an unsubstituted C3-C 20 heterocycle or a substituted C3-C 20 heterocycle, or R2 and R3 together with the carbon atom to which they are attached form an epoxide.

[0381] 3. For the compound according to paragraph 2, R1 is a substituted C1-C 10 amido group, an unsubstituted C1-C 10 amido group, a substituted C1-C 10 alkyl group, an unsubstituted C1-C 10 alkylene group, a substituted C1-C 10 alkylene group, an unsubstituted C1-C 10 alkylene group, a substituted aryl group, a substituted heteroaryl group, a substituted C2-C 10 alkenyl group, a substituted C2-C 10 alkynyl group, a substituted C1-C 10 alkoxy group, a substituted aryloxy group, a substituted C1-C 10 alkylthio group, a substituted arylthio group, an unsubstituted C1-C 10 carbonyl group, a substituted C1-C 10 carbonyl group, an unsubstituted C1-C 10 carboxyl group, a substituted C1-C 10 carboxyl group, an unsubstituted C1-C 10 amino group, a substituted C1-C 10 amino group, an unsubstituted C1-C 10 sulfonyl group, a substituted C1-C 10 sulfonyl group, an unsubstituted C1-C 10 sulfamoyl group, a substituted C1-C 10 sulfamoyl group, an unsubstituted C1-C 10 phosphonyl group, a substituted C1-C 10 phosphonyl group, a substituted polyaryl group, a substituted C3-C 10 cyclic group or a substituted C3-C 10 heterocycle, preferably wherein R1 is a substituted C1-C 10 amido group or an unsubstituted C1-C 10 amido group.

[0382] 4. For the compound according to paragraph 2 or 3, wherein R1 has the following structure:

[0383]

[0384] wherein R 12 is a substituted C1-C5 alkylene group or an unsubstituted C1-C5 alkylene group, R 13 is hydrogen, a substituted C1-C5 alkyl group or an unsubstituted C1-C5 alkyl group, and R14 is a substituted C1-C5 alkylene or an unsubstituted C1-C5 alkylene, preferably R 12 is a substituted C1-C5 alkylene (preferably -CH(CH3)-), R 12 is an unsubstituted C1-C5 alkyl (preferably -CH3), and R 14 is an unsubstituted C1-C5 alkylene (preferably -(CH2)2-).

[0385] 5. A compound according to any one of paragraphs 2 to 4, said compound having a structure selected from the following:

[0386]

[0387]

[0388] 6. A compound according to any one of paragraphs 2 to 5, wherein when present, R2, R3, R4, R5, R6, R7, R8, R9, R 10 and R 11 are independently hydrogen, hydroxy, halogen (F, Br, Cl, I), substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, unsubstituted C1-C5 alkenyl, substituted C1-C5 alkenyl, unsubstituted C1-C5 alkynyl, substituted C1-C5 alkynyl, unsubstituted C1-C5 alkoxy, substituted C1-C5 alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted C1-C5 alkylthio, substituted C1-C5 alkylthio, unsubstituted arylthio, substituted arylthio, unsubstituted C1-C5 carbonyl, substituted C1-C5 carbonyl, unsubstituted C1-C5 carboxyl, substituted C1-C5 carboxyl, unsubstituted C1-C5 amino, substituted C1-C5 amino, unsubstituted C1-C5 sulfonyl, substituted C1-C5 sulfonyl, unsubstituted C1-C5 sulfamoyl, substituted C1-C5 sulfamoyl, unsubstituted C1-C5 phosphonyl, substituted C1-C5 phosphonyl, unsubstituted polyaryl, substituted polyaryl, unsubstituted C3-C6 cyclic group, substituted C3-C6 cyclic group, unsubstituted C3-C6 heterocycle or substituted C3-C6 heterocycle.

[0389] 7. A compound according to any one of paragraphs 2 to 6, wherein when present, R2 and R3 together with the carbon atoms to which they are attached form an epoxide.

[0390] 8. A compound according to any one of paragraphs 2 to 7, wherein when present, R4, R5, R6 and R7 are independently hydrogen, hydroxy, halogen (F, Br, Cl, I), substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl or unsubstituted heteroaryl, preferably R4, R5 and R6 are hydrogen and R7 is methyl.

[0391] 9. A compound according to any one of paragraphs 2 to 8, wherein when present, R8 is hydrogen, hydroxy, halogen (F, Br, Cl, I), substituted C1-C5 carboxyl, unsubstituted C1-C5 carboxyl, substituted C1-C5 carbonyl or unsubstituted C1-C5 carbonyl, preferably R8 is hydrogen, hydroxy, substituted C1-C5 carboxyl or unsubstituted C1-C5 carboxyl, or preferably R8 is hydrogen.

[0392] 10. A compound according to any one of paragraphs 2 to 9, wherein when present, R9 is hydrogen, substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted C1-C5 carbonyl or unsubstituted C1-C5 carbonyl, preferably R9 is unsubstituted C1-C5 alkyl, or preferably R9 is methyl.

[0393] 11. A compound according to any one of paragraphs 2 to 10, wherein when present, R 10 is halogen (F, Cl, Br, I), substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted C1-C5 carbonyl or unsubstituted C1-C5 carbonyl, preferably R 10 is halogen, or preferably R 10 is Cl.

[0394] 12. A compound according to any one of paragraphs 2 to 11, wherein when present, R 11 is hydrogen, substituted C1-C5 alkyl, unsubstituted C1-C5 alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl or unsubstituted heteroaryl, preferably R 11 is unsubstituted C1-C5 alkyl, or preferably R 11 is methyl.

[0395] 13. A compound according to any one of paragraphs 2 to 12, the compound having the following structure:

[0396]

[0397] 14. A compound, the compound comprising an analogue of a radiosensitizer parent compound comprising one or more S-nitrosothiol moieties, wherein the S-N bond of the one or more S-nitrosothiol moieties is radiolytically cleaved, preferably ionizing radiolytically, during radiotherapy and releases the parent compound and nitric oxide.

[0398] 15. The compound according to paragraph 14, wherein the parent compound is nicotinamide, metronidazole or an analogue thereof, optionally selected from misonidazole, etanidazole and nimorazole; a hypoxic cell cytotoxic agent, optionally selected from mitomycin-C and tirapazamine; a membrane active agent, optionally selected from procaine, lidocaine and chlorpromazine; a radiosensitizing nucleoside, optionally selected from 5-fluorouracil, floxuridine, bromodeoxyuridine, iododeoxyuridine, hydroxyurea, gemcitabine and fludarabine, techsarine, optionally selected from gadolinium motexafin; an inhibitor of thiol groups, optionally selected from N-ethylmaleimide, diamide and diethyl maleate; a chemotherapeutic agent, optionally selected from paclitaxel, docetaxel, irinotecan and cisplatin; pentoxifylline; vinorelbine; a PARP inhibitor; a histone deacetylase inhibitor and a proteasome inhibitor.

[0399] 16. A nanoparticle, the nanoparticle comprising a compound according to any one of paragraphs 1-15.

[0400] 17. The nanoparticle according to paragraph 16, wherein the nanoparticle is a polymeric nanoparticle, a liposome, an inorganic nanoparticle.

[0401] 18. The nanoparticle according to paragraph 17, wherein the nanoparticle is a polymeric nanoparticle comprising one or more amphiphilic, hydrophobic and / or hydrophilic polymers.

[0402] 19. The nanoparticle according to any one of paragraphs 16 to 18, wherein the nanoparticle comprises one or more hydrophobic polymers.

[0403] 20. The nanoparticle according to paragraph 19, wherein one or more of the hydrophobic polymers is a polyester.

[0404] 21. The nanoparticle according to paragraph 20, wherein the polyester or polyesters are selected from poly(lactic-co-glycolic acid), poly(lactic acid), poly(glycolic acid).

[0405] 22. The nanoparticle according to any one of paragraphs 16 to 21, wherein the nanoparticle comprises poly(lactic-co-glycolic acid) (PLGA).

[0406] 23. The nanoparticle according to any one of paragraphs 16 to 22, wherein the nanoparticle comprises one or more hydrophilic polymers.

[0407] 24. The nanoparticle according to paragraph 23, wherein one or more of the hydrophilic polymers are polyalkylene glycols.

[0408] 25. The nanoparticle according to any one of paragraphs 16 to 24, wherein the nanoparticle comprises polyethylene glycol (PEG).

[0409] 26. The nanoparticle according to any one of paragraphs 16 to 25, wherein the nanoparticle is a polymeric nanoparticle comprising poly(lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG).

[0410] 27. The nanoparticle according to any one of paragraphs 16 to 26, wherein the nanoparticle has a size suitable for delivering the compound to the tumor microenvironment by enhanced permeability and retention.

[0411] 28. The nanoparticle according to any one of paragraphs 16 to 27, wherein the nanoparticle has a size of about 10 nm to about 300 nm.

[0412] 29. The nanoparticle according to any one of paragraphs 16 to 28, wherein the nanoparticle comprises a targeting agent conjugated thereto.

[0413] 30. The nanoparticle according to paragraph 29, wherein the targeting agent targets NTSR1.

[0414] 31. The nanoparticle according to paragraph 30, wherein the targeting agent is an agonist or antagonist of NTSR1.

[0415] 32. The nanoparticle according to paragraph 31, wherein the targeting agent is NTS or a variant thereof.

[0416] 33. The nanoparticle according to paragraph 32, wherein the targeting agent is NTS mut 。

[0417] 34. The nanoparticle according to paragraph 31, wherein the targeting agent is SR142948A or NTS 20.8 。

[0418] 35. A pharmaceutical composition comprising an effective amount of the compound according to any one of paragraphs 1 - 15.

[0419] 36. A pharmaceutical composition comprising an effective amount of the nanoparticle according to any one of paragraphs 16 - 34.

[0420] 37. A method of treating a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition according to paragraph 35.

[0421] 38. A method of treating a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition according to paragraph 36.

[0422] 39. The method according to paragraphs 37 and 38, wherein the subject has cancer.

[0423] 40. The method according to any one of paragraphs 37 to 39, the method comprising administering to the subject one or more doses of radiotherapy, optionally wherein the radiotherapy is ionizing radiotherapy, phototherapy or proton therapy.

[0424] 41. The method according to paragraph 40, wherein the compound enhances the treatment of the cancer compared to administering the radiotherapy alone.

[0425] 42. The method according to any one of paragraphs 39 to 41, wherein the cancer is a radiosensitive cancer.

[0426] 43. The method according to any one of paragraphs 39 to 41, wherein the cancer is a radioresistant cancer.

[0427] 44. The method according to any one of paragraphs 39 to 43, wherein the cancer is a vascular cancer, bone cancer, muscle cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, uterine cancer or germ cell cancer.

[0428] 45. The method according to any one of paragraphs 39 to 43, wherein the cancer is an epithelial cancer.

[0429] 46. The method according to any one of paragraphs 39 to 43, wherein the cancer is non-small cell lung cancer (NSCLC).

[0430] 47. The method according to any one of paragraphs 40 to 46, wherein the same dose of radiotherapy is more effective than when administered in the absence of the pharmaceutical composition, a lower dose of radiotherapy has the same efficacy as a higher dose administered in the absence of the pharmaceutical composition, or a combination thereof.

[0431] 48. The method according to any one of paragraphs 37 to 46, wherein a dose of radiotherapy is administered after administering the pharmaceutical composition.

[0432] 49. The method according to item 48, wherein the dose of radiotherapy is administered 1 to 48 hours, or 1 to 24 hours, or 1 to 12 hours, or 1 to 6 hours, or 2 to 6 hours or 1, 2, 3, 4 or 5 hours after administering the pharmaceutical composition.

[0433] 50. The method according to item 48 or 49, the method comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds of administration of the pharmaceutical composition, followed by administration of the dose of radiation.

[0434] 51. The method according to any one of paragraphs 40 to 50, wherein the radiation is ionizing radiation.

[0435] 52. The method according to any one of paragraphs 39 to 51, wherein the cancer comprises cells with upregulated NTSR1. Examples

[0436] Example 1: DM1 can be nitrosylated and formulated in the form of a particle delivery system

[0437] Materials and Methods

[0438] Materials:

[0439] Unless otherwise specified, all required chemicals were used without further purification. 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), (2-hydroxyethyl), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tert-butyl nitrate, polyethylene glycol (PEG, molecular weight 2000), DMAP (dimethylaminopyridine), DCC (N,N'-dicyclohexylcarbodiimide), hydrogen peroxide solution (30 wt%, in H2O), dichloromethane (DCM), methylene blue (MB), dimethyl sulfoxide (DMSO) and other chemicals were purchased from Sigma.

[0440] Carboxyl-terminated PLGA (poly(D,L-lactide-co-glycolide) (dL / g, 0.15 to 0.25)) was obtained from Lactel (Birmingham, AL, USA). Mertansine (DM1) was purchased from MeedKoo Bioscience Inc. Phosphate-buffered saline (PBS, pH 7.4, containing 138 mM NaCl, 2.7 mM KCl, and 10 mM sodium phosphate) was used for all in vitro experiments. RPMI 1640 medium containing L-glutamine (HyClone, GE Bioscience, USA) and trypsin-EDTA was purchased from Corning (Manassas, VA 20109). Antibiotic penicillin-streptomycin (Peh-Strep, MediaTech, USA) and fetal bovine serum (FBS) were purchased from Gibco-Life Technologies (Grand Island, NY 14072). H1299 non-small lung cancer cells (NSLC) were originally obtained from the American Type Culture Collection (ATCC). Free thiol assay and superoxide dismutase (SOD) assay kits were purchased from Cayman Chemical (USA). Griess reagent was obtained from Promega (USA). Nitric oxide dye DMF-FM was purchased from Thermo Scientific.

[0441] Synthesis of DM1-NO prodrug (S-nitrosylation of DM1):

[0442] S-nitrosylation of DM1 was achieved by following the reported protocol without modification (Chipinda et al., J. Phys. Chem. B, 110, 5052 - 5061 (2006), Pant et al., ACS Appl. Mater. Interfaces, 9, 15254 - 15264 (2017)). Briefly, DM1 was dissolved in DMSO, and tert-butyl nitrite was added dropwise to the reaction mixture at a molar ratio of 5:1 under gentle stirring. The resulting mixture was stirred in the dark for 45 minutes using a magnetic stirrer. Thereafter, the reaction vessel was placed in an ice bath to precipitate the DM1-NO drug. The resulting compound was filtered, rinsed with anhydrous DMSO, purified through a HyperSep C18 column, and stored at -20 °C before use.

[0443] Characterization of DM1-NO prodrug:

[0444] By high-resolution electrospray ionization mass spectrometry (HRMS-ESI) and 1The new compound (2) was characterized by H-NMR spectroscopy. Mass spectrometry was performed to confirm the structure by loading the sample into the HRMS-ESI chamber through an ionization source. The calculated theoretical isotopic distributions of compound (1) (molecular formula (C 35 H 48 ClN3O 10 S = 737.29)) and compound (2) (molecular formula (C 34 H 48 ClN4O 11 S = 766.29)) were carried out. For compound (1), the main peak was observed at 760.29, and for compound (2), the main peak was observed at 789.254, which was in good agreement with the theoretical calculated m / z value of [M+Na] + .

[0445]

[0446]

[0447] Synthesis and characterization of PLGA-b-PEG:

[0448] The synthesis of PLGA-b-PEG followed the published protocol with slight modifications (as follows) (Cheng et al., Biomaterials, 28, 869-876 (2007)).

[0449]

[0450] Briefly, PEG (2.29 g, 0.684 mmol), PLGA-COOH (1.0 g, 0.170 mmol), and 4-dimethylaminopyridine (0.023 g, 0.187 mmol) were mixed in 30 mL of anhydrous dichloromethane CH2Cl2. Next, with stirring, a 10 mL CH2Cl2 solution of N,N'-dicyclohexylcarbodiimide (DCC, 0.141 g or 0.684 mmol) was added dropwise to the reaction mixture at 0 °C. The mixture was warmed to room temperature and stirred overnight. The insoluble dicyclohexylurea was filtered out. The crude product was precipitated by adding 50 mL of 50:50 diethyl ether and methanol to the mixture. The mixture was centrifuged at 4 °C for 15 minutes. The purification step was repeated 4-5 times. The resulting white pellets were dried under high vacuum to obtain the polymer product. The yield was 68-73%. The 1 1H-NMR (CHCl3-d) data showed: δ 5.20 [m, (OCHCH3C(O)], 4.82 [m, (OCH2C(O))], 3.63 [s, (OCH2)], 1.57 [m, (CH3CH)].

[0451] Synthesis and Characterization of Nanoparticles:

[0452] Briefly, the polymer was dissolved in DMSO to a final polymer concentration of 5 mg / mL and thoroughly mixed with 1.5 mg / mL of the drug (30% concentration relative to the polymer concentration). The mixture was then added dropwise to nanopure water (Millipore) under vigorous stirring. The nanoparticles (NP) were allowed to self-assemble for 2 hours at room temperature under continuous stirring. The NP were washed four times with nanopore water using an Amicon Ultra-15 centrifugal filter device (Millipore, Billerica, MA, USA) with a molecular weight cut-off of 100 kDa. After purification, the NP solution was resuspended in PBS (1X) and stored at 4 °C until further use.

[0453] The physical properties of the NP (e.g., size distribution and ζ potential) were characterized using a dynamic light scattering (DLS) instrument (Malvern Zetasizer Nano S90). The morphology of the NP was determined using transmission electron microscopy (TEM) (FEI Tecnai20, 200 kV). The cumulative drug release of the nanoparticles studied at pH 5.0, 6.5, and 7.4 was evaluated by fluorescence thiol assay ( Figure 1H ).

[0454] Quantification of Drug and Nitric Oxide Release:

[0455] The amount of drug loaded into the polymer NP was quantified by fluorescence thiol assay (Winther et al., Biochim. Biophys. Acta, 1840, 838 - 846 (2014)). According to the manufacturer's protocol (Cayman Chemical, Ann Arbor, MI, USA), the sensitivity of this method is 400 times that of the colorimetric method (Ellman's reagent). The corresponding amount of drug loaded into the NP was quantitatively measured by comparison with a standard curve. An NP solution without loaded drug (empty-NP) was used as a negative control. The drug loading capacity of this drug in the NP was found to be approximately 3.8%, and the encapsulation efficiency was approximately 43%. Both of these values are comparable to those of PLGA NP loaded with other drugs (Tian et al., J. Mater. Chem. B, 5(30), 6049 - 6057 (2017)).

[0456] The NO concentration was evaluated to determine the amount of DM1-NO prodrug loaded into DM1-NO-NP. NO undergoes a series of reactions with several molecules in solution. Therefore, to quantify the total amount of NO, the total nitrite (NO2-) and nitrate (NO3-) concentrations in the NP solution pool were measured using a nitrate / nitrite colorimetric assay (Cayman Chemical) according to the supplier's protocol.

[0457] Results

[0458] DM1-NO was synthesized by the reaction of DM1 with tert-butyl nitrite in anhydrous DMSO. See the reaction scheme for the synthesis of DM1-NO shown below.

[0459]

[0460] The resulting compound was purified on a flash column. 1H-NMR analysis found that the peak at δ = 6.0 ppm disappeared after the reaction, indicating the successful nitrosation of the DM1 thiol group. High-resolution electrospray ionization mass spectrometry found a main peak at 789.2548, which was consistent with the calculated m / z of [M+Na]+. The DM1-NO product was analyzed on a Sievers NOA 280i system, which measures NO levels based on the gas-phase chemiluminescence reaction between NO and ozone. According to the analysis, the yield of DM1-NO was 86%.

[0461] DM1-NO is stable in powder form under ambient conditions. According to Griess analysis, less than 10% of the compound degraded after storage at room temperature for 2 weeks ( Figure 1F ). DM1-NO gradually decomposes in PBS at 37 °C ( Figure 1G ), and the degradation is accelerated at low pH (e.g., pH 6.5 or 5.5, Figure 1G , Table 1) and under X-ray irradiation ( Figure 1C ).

[0462] Table 1: NO release corresponding to Figure 1G

[0463]

[0464] For example, when 6 Gy of X-ray irradiation was applied to a solution containing DM1-NO, the NO release increased by 5.14-fold ( Figure 1C ). This enhanced degradation is attributed to the ·OH radicals generated by radiolysis of water (Azzam et al., Cancer Lett. 327, 48 - 60 (2012)), which promote the oxidation of DM1-NO.

[0465] ​DM1-NO was loaded onto PLGA-b-PEG nanoparticles by nanoprecipitation. The drug loading and encapsulation efficiency were 3.8% and 43%, respectively. Transmission electron microscopy and dynamic light scattering (DLS, Figure 1D ) revealed that the resulting DM1-NO PLGA-b-PEG nanoparticles (hereinafter referred to as DM1-NO-NP) had an average size of approximately 78 nm in water. The polydispersity index or PDI was 0.18 ± 0.01, indicating a narrow size distribution and good aqueous dispersibility. DM1-NO-NP remained stable in PBS with no significant size change within 24 hours ( Figure 1N ). The nanoparticle surface was negatively charged (ζ potential -29.39 mV, Figure 1E ), which was attributed to multiple surface hydroxyl groups.

[0466] DM1-NO-NP was loaded onto a dialysis device and the compound release was evaluated in PBS at 37 °C. Due to the hydrophobicity of S-nitrosothiols under physiological conditions, DM1-NO was released very slowly from the nanoparticles at pH 7.4, taking more than 15 hours to release approximately 50% of the payload ( Figure 1H ). At lower pH, the release was significantly accelerated. For example, at pH 6.5, the t1 / 2 decreased to 12 hours and at pH 5.5 to 10 hours ( Figure 1H ).

[0467] Example 2: Nitrosation and particle formulation independently reduce the toxicity of DM1.

[0468] Materials and Methods

[0469] Cell culture:

[0470] H1299 cells were obtained from ATCC (Manassas, VA, USA). The cells were cultured in RPMI-1640 (Gibco, Invitrogen, Carlsbad, CA, USA) growth medium supplemented with 10% fetal bovine serum (MediaTech, Manassas, VA, USA), 2 mM L-glutamine, 100 U / mL penicillin (MediaTech, USA), and 100 μg / mL streptomycin (MediaTech). The cells were maintained in a humid atmosphere containing 5% CO2 and the temperature was kept at 37 °C.

[0471] Cytotoxicity measurement (MTT assay):

[0472] An Infinite M200 microplate reader (BioTek’s Synergy TMMx, USA), and the viability of H1299 cells after different treatments was measured by bromide (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Approximately 3×10 3 cells / well were seeded into 96-well plates and cultured overnight until the cells were completely adhered to the bottom of the plates. The cells were treated with different treatment groups for 72 hours, including free drugs (DM1 and DM1-NO) and drug-loaded nanoparticles (DM1-NP and DM1-NO-NP). The final drug concentration of each treatment group in the growth medium was 0.8 - 500 nM. Thereafter, MTT reagent (5 mg / mL in 20 μL PBS) was added to each well. The cells were incubated at 37 °C for 4 hours. Then the medium in each well was removed and replaced with 100 μL DMSO. The plates were gently shaken for 5 minutes (instrument settings) to dissolve the formazan crystals, and the absorbance at 570 nm was recorded by a microplate reader. Each experimental condition was run in triplicate, and the data were shown as mean plus standard deviation (±SD).

[0473] Results

[0474] The MTT cell viability assay was performed using H1299 cells, which is a human NSCLC cell line. DM1 was very effective in inhibiting cell proliferation, with an IC50 value of 19.8 nM( Figure 2A , Table 2).

[0475] Table 2: IC50 values of DM1, DM1-NO, DM1-NP, and DM1-NO-NP, based on Figure 2A the results.

[0476]

[0477] For comparison, the IC50 of DM1-NO-NP was 98.2 nM. For comparison, PLGA-b-PEG nanoparticles encapsulated with DM1 (abbreviated as DM1-NP, Figures 1I - 1J ) were prepared and their toxicity was evaluated. Compared with the corresponding free drugs, the toxicity of DM1-NO-NP and DM1-NP was reduced( Figure 2A , Table 2), which was attributed to the controlled drug release. Between DM1-NO-NP and DM1-NP, DM1-NO-NP showed lower toxicity (IC 50 98.2 versus 52.7 nM, Figure 2B ), which was due to the S-nitrosylation of DM1.

[0478] Example 3: The toxicity of nitrosylated DM1 is activated by radiation.

[0479] Materials and methods

[0480] Colony formation assay:

[0481] Approximately 3×10 5 H1299 cells were plated in a series of 25 mm culture dishes in 5 mL of complete RPMI-1640 medium and the cells were incubated at 37 °C for 24 h. Once the cells had completely adhered to the bottom of the plate, the medium was replaced with fresh RPMI-1640 containing a final concentration of 20 nM of DM1 or DM1-NO. After incubation for an additional 12 h, the cell cultures were irradiated with different doses of X-rays (320 kV). Immediately after X-ray irradiation, the cell monolayer was trypsinized using 0.05% trypsin / EDTA (Gibco, Life Technologies, USA). Single cells were collected and plated onto 25 mm culture dishes in 5 mL of complete medium ( Figure 1K ). Then each dish was incubated for 21 days to form colonies. After that, the medium was removed and the existing colonies were fixed and stained with 0.5% crystal violet in 70% methanol. The plates were rinsed thoroughly and carefully and dried at room temperature. After drying, the colonies were manually counted under a microscope. When observed under a microscope, colonies of 21-50 cells were called surviving colonies. The survival rate of these colonies was normalized to the plating efficiency of untreated control cells. The mean and standard deviation of the surviving colonies in each treatment group were calculated.

[0482] ·OH, 1 Detection of O2 and NO free radicals:

[0483] Methylene blue (MB) (Sigma-Aldrich, USA) and Singlet Oxygen Sensor Green (SOSG) (Invitrogen, USA) were used to evaluate the production of ·OH and 1 O2, respectively. Briefly, 1 mL of aqueous solution containing MB (100 μL, 0.5 mM) or SOSG (100 μL, 10 μM) of different treatment groups (20 nM) was irradiated with 6 Gy. Immediately after irradiation, the absorption of MB at 664 nm and the fluorescence intensity of SOSG (excitation / emission: 504 / 525 nm) were measured. Unirradiated samples and empty NPs were studied as controls.

[0484] Superoxide dismutase (SOD) assay:

[0485] Approximately 3×10 4Cells were seeded at [[number]] cells / well into each well of a six-well plate and cultured overnight. Cells treated with free drugs (DM1 and DM1-NO) and drug-loaded nanoparticles (DM1-NP and DM1-NO-NP) were given the same concentration of drug (20 nM) and irradiated with 6 Gy of radiation. The plates were then incubated for 24 h. After treatment, cells were rinsed with PBS and cell pellets were collected using a cell scraper. Cells were homogenized using an ultrasonic device in ice-cold 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPS) buffer (pH 7.2, containing 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, also known as EGTA; MilliporeSigma, USA), 210 mL of mannitol, and 70 mM sucrose. After homogenization, a pellet was formed by centrifugation at 15,000×g for 15 min at 4 °C. The supernatant was collected and assayed immediately to evaluate cytosolic SOD levels by comparison with a standard curve. To measure mitochondrial SOD levels, the pellet was resuspended in ice-cold PBS and samples were measured accordingly.

[0486] Lipid peroxidation assay:

[0487] The BODIPY 581 / 591 probe (ThermoFisher Scientific, USA) was used to measure lipid peroxidation levels. Briefly, H1299 cells were incubated with DM1-NO-NP (20 nM) for [[time]] h. After replenishing the medium, X-rays (6 Gy) were applied. For controls, cells incubated with PBS, DM1, DM1-NO, and DM1-NP or not receiving radiation were studied. The BODIPY 581 / 591 probe (10 μM) was added to the cells and incubated for 30 min, and the cells were washed with ice-cold PBS. Thereafter, both red (590 nm) and green (510 nm) emissions were recorded and the ratio was calculated (BODIPY undergoes a fluorescence shift from red to green upon oxidation).

[0488] Peroxynitrite:

[0489] Peroxynitrite levels were measured using a peroxynitrite green sensor (AAT Bioquest, Sunnyvale, CA, USA), which produces a green fluorescent compound upon reaction with peroxynitrite. Briefly, approximately 5×10 3Cells were seeded in Costar black-wall / clear-bottom 96-well plates (Corning, ME-USA) and incubated with 20 nM of free drugs (DM1 and DM1-NO) or drug-loaded nanoparticles (DM1-NP and DM1-NO-NP) for 6 h, and then irradiated with 6 Gy of X-rays. After that, 10 μL of peroxynitrite green sensor solution was added to each well and incubated with the cells for 1 h. The fluorescence signal was measured on a microplate reader using FITC filter: (excitation at 490 nm, emission at 530 nm and cut-off at 515 nm). For confocal microscopy imaging, cells were seeded in CLSM special culture dishes and treated in a similar manner as described above. Images were taken on a Zeiss LSM 710 system using FITC filter.

[0490] Intracellular ROS / NO measurement:

[0491] H1299 cells (2×10 4 cells) were seeded on Nunc Lab Tek Chamber Slides (ThermoFisher Scientific, USA) and incubated with 20 nM of free drugs (DM1 and DM1-NO) or drug-loaded nanoparticles (DM1-NP and DM1-NO-NP) for 12 h. Then the cells were irradiated with X-rays (6 Gy). Serum-free RPMI-1640 medium containing (100 μL, 5 μM) 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM, ThermoFisher Scientific, USA) and ethidium homodimer III (100 μL, 10 μM) (EthD-III, Biotium, USA, Cat.#s 40050) was added to each well and allowed to incubate for 30 - 35 min. Then the NO released from the drug would react with DAF-FM to produce green fluorescence. Meanwhile, EthD-III (a DNA dye impermeable to intact plasma membranes) translocates to dead cells and binds to nuclear DNA to emit red fluorescence. To remove free dye molecules, the cells were washed three times with serum-free medium and imaged under a confocal laser microscope (Zeiss, LSM 710, USA) using the following settings. DAF-FM (FITC), ex / em: nm; EthD-III (Rhodomine (red)), ex / em:.

[0492] γ-H2AX assay:

[0493] 3×10 4Cells were seeded at each well and cultured overnight, and further incubated with 20 nM of free drugs (DM1 and DM1-NO) or drug-loaded nanoparticles (DM1-NP and DM1-NO-NP) for 12 hours. Then the cells were irradiated with 6-Gy and incubated for another hour. Then the cells were fixed in ice-cold 50% CH3OH and 50% (CH3)2CO for 20 minutes. After fixation, the cells were permeabilized with 0.5% Triton-X-100 in PBS (1X), and then blocked with 0.2% skim milk (Difco TM SkimMilk, BD, VWR, USA), 0.1% Triton X-100 (Millipore, Sigma USA) and 5% goat serum (normal goat serum, ab7481) in PBS. Then the cells were stained with anti-phosphorylated H2A.X (Ser139) antibody, conjugated anti-γ-H2AX antibody Alexa Fluor-647 (Millipore, Sigma USA USA) to detect phosphorylated histone H2A.X. The cells were washed 3 times with ice-cold PBS (1X), and the coverslips were mounted with mounting medium containing DAPI (Fluoro-Gel II, with Dapi, Electron Microscopy Sciences, USA) to counterstain the nuclei. γ-H2AX foci were counted by Image-J. The average number of foci / cell was calculated based on at least 50 cells per sample. The experimental data represent the mean of three independent experiments.

[0494] In vitro cell cycle arrest:

[0495] H1299 cells were seeded at a density of 2×10 5 cells / well on 6-well plates and cultured overnight. Then the cells were incubated with 20 nM of free drugs (DM1 and DM1-NO) or drug-loaded nanoparticles (DM1-NP and DM1-NO-NP) for 24 hours, then harvested and washed with ice-cold phosphate buffered saline (PBS), and fixed overnight at 4 °C with cold 70% (v / v) ethanol. The resulting cells were resuspended in PBS (1X) buffer containing a final concentration of 20 μg / mL RNase A and 20 μg / mL propidium iodide (eBioscience, Invitrogen, USA) for 15 minutes. The cell cycle profiles were determined using flow cytometry (HyperCyAn, Beckman USA), and analyzed using CellQuest software.

[0496] Tubulin inhibition assay:

[0497] The assay was performed according to the manufacturer's protocol (PurSolutions, USA). Briefly, β-tubulin (>97% purity) was suspended in G-PEM buffer (pH 6.9, containing 80 mM PIPES, 2 mM MgCl2, 0.5 mM EDTA and 1.0 mM GTP) at a final concentration of 1.0 mg / mL. The tubulin solution was then incubated with separate G-PEM buffer (control) and free drugs (DM1 and DM1-NO) or drug-loaded nanoparticles (DM1-NP and DM1-NO-NP). The drug concentrations ranged from 0 - 20 μmol / L and the incubation was carried out at 30 °C for 45 minutes. For the sedimentation assay, the formed polymers were centrifuged (35000×g, 1 h, 30 °C) and the pellet was further depolymerized in PEM buffer at 0 °C in 1 mM GTP and the protein concentration was determined. The sedimentation assay for each compound was performed at least twice. For TEM analysis, the samples were fixed in 0.2% glutaraldehyde (Millipore sigma, USA) and stained with 0.5% uranyl acetate (Millipore sigma, USA) to visualize the microtubule morphology. Images were obtained on a transmission electron microscope (Hitachi HT7800) TEM) operating at 120 KV.

[0498] Results

[0499] The potential of DM1-NO as a radiosensitizer was evaluated by colony formation assay. Briefly, DM1 or DM1-NO was incubated with H1299 cells, the cells were irradiated with X-rays (0 - 10 Gy), and the resulting cells were seeded onto culture dishes ( Figure 1K ). Colonies consisting of more than 50 cells after 3 weeks were counted and the data were fitted to the linear quadratic model (Table 3).

[0500] Table 3: α, β and D 10 values, based on Figure 2B the fitting results.

[0501]

[0502] DM1 showed a D10 (radiation dose for 10% partial survival) of 4.89 Gy, compared to 5.40 Gy for RT alone (Table 3). The D10 of DM1-NO was even lower, at 4.39 Gy, and the dose modification factor (DMF10) at 10% partial survival was 1.23. The fact that DM1-NO exhibited lower toxicity but higher dose modification than DM1 supports the hypothesis that DM1-NO under radiation releases DM1 and NO, which together sensitize cancer cells to RT. The MTT results also confirmed the enhanced radiosensitization of DM1-NO and DM1-NO-NP relative to DM1 ( Figure 2A , Table 2, Figure 7H , Table 4).

[0503] Table 4: Figure 7H IC of the viability data shown in 50 value.

[0504]

[0505] To better understand the radiosensitization, intracellular oxidative stress was evaluated after RT. When cells were incubated with DM1-NO, the levels of cytoplasmic and mitochondrial superoxide dismutase (SOD) increased and further elevated upon the application of radiation (6 Gy) (Figures 3A - 3B). For example, compared to DM1+RT, the DM1-NO+RT treatment increased cytoplasmic SOD activity by 70.08% and mitochondrial SOD activity by 24.36%. Similar levels of SOD elevation were observed using DM1-NO-NP (Figure 3A). Intracellular 1 O2 and ·OH levels were also measured using singlet oxygen sensor green (SOSG) and methylene blue (MB) staining, respectively. Both SOSG fluorescence activity (Figure 3C) and MB bleaching (Figure 3D, shown as absolute value change) were significantly elevated under DM1-NO-NP+RT, again confirming the increased oxidative stress (Ewing, Radiat.Res. 94, 171 - 189 (1983), Riley, Iht.J.Radiat.Biol. 65, 27 - 33 (1994)).

[0506] Changes in intracellular NO levels were evaluated using DAF-FM, a fluorescent sensor for NO. Confocal fluorescence microscopy found that the positive DAF-FM staining level of DM1-NO-NP+RT was higher than that of DM1-NP+RT. The fluorescence intensity increased 2.52-fold (for DM1-NO-NP+RT) and 1.66-fold (for DM1-NP+RT) relative to unirradiated cells ( Figure 4A)。The Griess assay observed similar results, finding that the NO release of DM1-NO-NP+RT increased by 4.36-fold and that of DM1-NP+RT increased by 1.83-fold ( Figure 4B )。Free NO will react with intracellular ROS to form reactive nitrogen species (RNS), such as peroxynitrite (Pacher et al., Physiol. Rev. 87, 315 - 424 (2007)), which are highly toxic (Fraszczak et al., J. Immunol. 184, 1876 - 1884 (2010), Korkmaz et al., Interdiscip. Toxicol. 2, 219 - 228 (2009)). In fact, peroxynitrite sensor green staining found that the intracellular peroxynitrite level was significantly increased under DM1-NO-NP+RT ( Figure 5A )。Fluorescence analysis showed that the green activity of the peroxynitrite sensor using DM1-NO-NP+RT was 2.17-fold higher than that using RT alone ( Figure 5B )。

[0507] The elevated ROS and RNS levels cause extensive damage to intracellular components. γH2AX staining found more positive staining for DM1-NO-NP+RT, with the number of foci increasing by 4.14-fold relative to RT alone ( Figure 6A )。The BODIPY lipid probe assay found that the 581 / 591 nm ratio decreased by 56.47%, indicating a significant enhancement of lipid peroxidation ( Figure 6B )。Notably, the DNA and lipid damage of DM1-NO-NP+RT was much more significant than that of DM1-NP+RT, highlighting the role of NO and its RNS derivatives in therapy. These results also correlated well with the MTT ( Figure 2A , Table 2 - 3) and Eth III staining results ( Figure 4C ), confirming that the elevated oxidative stress (mostly attributed to the NO released by DM1-NO) is the cause of radiosensitization.

[0508] On the other hand, DM1 released from DM1-NO-NP will interfere with tubulin polymerization (Lopus et al., Mol. Cancer Ther. 9, 2689-99 (2010), Bhattacharyya & Wolff, FEBS Lett. 75, 159-162 (1977)). This was investigated by incubating β-tubulin (1 mg / mL) in the presence of DM1, DM1-NO, DM1-NO-NP or PBS for 1 h and then examining fiber formation by TEM. In the absence of drug, β-tubulin self-assembled into long fibers. In comparison, all compounds or nanoparticles containing DM1 effectively inhibited fiber formation. Notably, at high concentrations (e.g., 20 μM DM1), β-tubulin began to form aggregates due to non-specific interactions between β-tubulin molecules (Bhattacharyya & Wolff, FEBS Lett. 75, 159-162 (1977)). Microtubule polymerization was quantitatively evaluated by sedimentation ( Figure 7G ). Compared with the PBS control, DM1-NO-NP inhibited polymer formation by 65.7%, 90.6% and 99.7% at 5, 10 and 20 μM (DM1 concentration), respectively.

[0509] Microtubule formation is a key step in cell mitosis. Inhibiting this process will enrich cells in the G2 / M phase (Ng et al., Cancer Res. 60, 5451-5455 (2000)). This was analyzed by flow cytometry using propidium iodide (PI) cell staining ( Figures 7A - 7E ). When H1299 cells were incubated with DM1, the proportion of cells in the G2 / M phase increased sharply from 17.5% to 85.3% ( Figure 7F ). A similar level of G2 / M arrest (83.47%) was observed using DM1-NO-NP. Mitotic arrest is also thought to contribute to the dose-modulating effect of DM1-NO-NP.

[0510] Example 4: Nitrosylated DM1 formulated in particles accumulates in tumors and kills cancer cells in response to radiation.

[0511] Materials and Methods

[0512] Tumor model establishment and therapy research:

[0513] All animal experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee (IACUC). Six-week-old female athymic nude mice (Charles River Laboratories, USA) were housed in a protective unit dedicated to immunodeficient animals. To establish a tumor model, H1299 cells (2×10 5Cells) were suspended in Matrigel solution and subcutaneously injected into mice using a 28-gauge needle. All mice were randomly divided into six groups (n = 5). When the average tumor volume reached 150 mm 3 ³, 200 μL of free drug (DM1, DM1-NO) or drug-loaded NPs (DM1-NP, DM1-NO-NP) were intravenously injected into mice at a dose of 260.8 nmol / kg in PBS. Mice treated with 200 μL of PBS were studied as controls. All other mice received X-ray irradiation (6 Gy, 320 kV) of the tumor 4 h after drug / nanoparticle injection, with the rest of the body shielded by lead. Tumor size and body weight were examined every 3 days using digital calipers, and the tumor volume was estimated as (length) × (width) 2 / 2. After 24 days, the mice were euthanized, and the tumors were excised and dissected into sections for hematoxylin and eosin (H&E) staining.

[0514] Toxicity study (AST / ALT):

[0515] Six-week-old female albino BALB / c mice were purchased from Charles River Laboratories, USA. The animals were divided into a control group and an experimental group (n = 3) and marked to allow individual identification. DM1-NO-NP was intravenously administered at a similar dose (260.8 nmol / kg) as used in the treatment study, and the mice were examined for 10 days to observe abnormal signs of toxicity, such as changes in behavior, dizziness, respiratory distress, or mortality, until the end of the study. To further evaluate liver function, the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, and blood chemistry analysis was performed using 20 μL of serum samples.

[0516] The following evaluations were performed on each treated animal: white blood cells (WBC), red blood cells (RBC), and platelets (PLT), plateletcrit (PCT, mean platelet volume or platelet distribution width), and the corresponding electrolyte panel (sodium, potassium, chloride, bicarbonate, sugar (glucose), calcium, inorganic phosphate, and magnesium, lipids (cholesterol)), albumin, and the total protein level was also analyzed.

[0517] Histological analysis:

[0518] Gross pathological changes in the major organs (including brain, heart, lung, liver, kidney, spleen) were collected and immediately transferred to a fixative (10% buffered formalin) and embedded in paraffin for further histopathological evaluation. Sections with a thickness of approximately 5 μm were prepared, stained with hematoxylin and eosin (H&E), and histopathological changes were examined using a microscope with different magnifications and objectives.

[0519] Statistical analysis:

[0520] In all experiments, means and standard errors were calculated from at least three replicates. Student's t-test was used to determine statistical significance between groups, where P < 0.05 was considered to indicate a statistically significant difference between two groups.

[0521] Results

[0522] In vivo therapy studies were performed using nude mice bearing H1299 tumors. When the tumor size reached 150 mm 3 , DM1-NO-NP (260.8 nmol / kg, equivalent to 0.2 mg DM1 / kg) was intravenously (i.v.) injected into the animals (n = 5). X-ray irradiation (6 Gy) was applied to the tumors, with the rest of the animal's body shielded by lead. A single irradiation of a similar dose is commonly used in preclinical small animal studies (Biglin et al., Radiat. Oncol. 14, 134 (2019)). For comparison, DM1, DM1-NO, and DM1-NP at the same DM1 concentration, as well as PBS, were also tested. All animals were euthanized after 24 days, and their tumors and major organs were harvested. DM1-NO-NP + RT showed the most effective tumor suppression among all treatment groups ( Figures 8A - 8C ). On day 24, the average tumor size was 219.4 mm 3 , and the weight was 0.096 g. In comparison, the tumor size and weight of the RT-only group were 1826.4 mm 3 and 0.931 g, respectively. This represents a 9.64-fold increase in the tumor inhibition rate (TIR) of DM1-NO-NP + RT relative to RT alone ( Figure 8B , 8C ). Notably, the tumor suppression of DM1-NO-NP + RT was also much greater than that of DM1-NO + RT and DM1-NP + RT (tumor volumes on day 24 were 759.33 and 589.03 mm 3 ; Figure 8B ). This indicates that both nanoparticle drug delivery and NO release contribute to the superior radiosensitization of DM1-NO-NP + RT. Meanwhile, the body weights of the animals treated with DM1-NO-NP + RT did not decrease significantly ( Figure 8D) nor signs of toxicity, indicating good tolerability. Pathologists performed blinded examinations of harvested tumor and major organ samples. For the PBS control, the tumors had approximately 50% coagulative necrosis, with only few TUNEL-positive cells at the periphery of these necrotic foci. The remaining tumor cells were large and appeared viable, with many distinct mitotic figures. For the animals in the RT group, there was more coagulative necrosis (approximately 70%), while the remaining tumor cells had degenerative changes (rounding, shrinkage, cytoplasmic eosinophilia). Compared to the other groups, the tumors in the DM1-NO-NP+RT group had more positive TUNEL staining, indicating enhanced therapeutic efficacy. No signs of toxicity were found in normal tissues such as the brain, lung, and kidney in all treatment groups.

[0523] To better understand potential adverse effects, toxicity studies were conducted in separate animals. Briefly, DM1-NO-NP was injected into normal balb / c mice at a dose of (260.8 nmol / kg). For comparison, only saline or DM1 at the same concentration was injected. Blood samples were collected after 10 days and hematological tests were performed. Complete blood count (CBC) showed that the levels of white blood cells (WBC), red blood cells (RBC), platelets (PLT), and plateletcrit (PCT) were all within the normal range and comparable to the PBS control ( Figures 9A - 9C ). The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and creatinine (CR) were also examined (Figure S8a-c). The ALT level of DM1-NO-NP increased compared to the saline control (Figure 10A), but the difference was not significant (P = 0.29). All other parameters were within the normal range (Otto et al., J. Am. Assoc. Lab. Anim. Sci. 55, 375-386 (2016)), indicating little impact of the treatment on liver and kidney functions. The ALT and AST levels were also analyzed in liver tissues, and no significant differences were found between the DM1-NO-NP+RT and PBS control groups (Figure 10D, 10E). Other markers including sodium, potassium, chloride, bicarbonate, magnesium, calcium, glucose, inorganic phosphate, and magnesium, cholesterol, albumin, and total protein levels were all within the normal range (Figure 10G, 10G). Overall, these results confirmed the low toxicity of DM1-NO-NP at the tested dose.

[0524] The above experiment illustrates the preparation of DM1-NO (a nitrosylated maytansine analogue) and its encapsulation into PLGA-b-PEG nanoparticles for tumor delivery and sensitization of cancer cells to RT. Although DM1 has the potential as a radiosensitizer, it has rarely been studied in the context of RT due to its high systemic toxicity. This problem is addressed by using nanoparticle delivery and nitrosylation, which inhibits the toxicity of the drug in the absence of radiation. Once delivered to the tumor, external irradiation disrupts the S-N bond and releases DM1 as an anti-mitotic agent. EPR-based drug delivery and the fact that radiation is confined to the tumor region provide high selectivity for this approach. Although the current study was conducted in an NSCLC model, the nanoparticles have great potential for treating other cancer types, including but not limited to colorectal cancer, brain cancer, and breast cancer.

[0525] The function of nitrosylation is at least twofold. In addition to reducing the toxicity of DM1 in the absence of radiation, it also enhances the sensitivity of cancer cells to radiotherapy. Specifically, DM1-NO is released from the nanoparticles in the tumor and degrades upon irradiation to release the highly reactive free radical NO. The latter can react with the abundant ROS in the irradiated tumor to form RNS, such as peroxynitrite. Through complementary radiosensitization mechanisms, DM1 and NO act synergistically to improve RT outcomes. Upon irradiation, both the release of NO and its reaction with ROS are promoted, which can be clinically delivered to the tumor again in a conformal manner.

[0526] The above experiments utilized PLGA-b-PEG nanoparticles, which are a well-established nanoplatform. Studies have shown good DM1-NO loading and controlled release of the drug. Meanwhile, DM1-NO, as a small molecule, can potentially be loaded onto other nanoparticle-based platforms, including those based on polymers, liposomes, or micelles. DM1-NO can also be loaded onto inorganic nanoparticles, including those with high-Z elements. High-Z nanoparticles are promising radiosensitizers because they have a large absorption cross-section for high-energy photons and may increase energy deposition in tumors (Song et al., Adv. Mater. 29, 1700996 (2017)). For example, Bi2S3 and Au-Bi2S3 nanoparticles have shown good dose modification factors (Wang et al., ACS Nano 13, 5947-5958 (2019), Nosrati et al., Sci. Eng. 5, 4416-4424 (2019)). Gd-conjugated silica nanoparticles and hafnium oxide nanoparticles are being tested clinically to enhance radiotherapy. This combination can further improve treatment outcomes. In addition to acting as a radiosensitizer, NO can also have other effects on the tumor microenvironment (Salimian, Trends Cancer 3, 659-672 (2017)). For example, NO can dilate blood vessels (Zhao et al., J. Pharmacol. Sci. 129, 83-94 (2015)), potentially making tumors more accessible to nanoparticles.

[0527] Example 5: Conjugation of NTS mut with nanoparticles improved tumor accumulation

[0528] Materials and Methods

[0529] DM1-NO-loaded PLGA nanoparticles were prepared by nanoprecipitation. Briefly, PLGA-COOH was dissolved in DMSO to a final polymer concentration of 5 mg / mL and mixed with 1.5 mg / mL of DMI-NO. The mixture was added dropwise to pure water under vigorous stirring to generate nanoparticles. After purification, the NP solution was resuspended in PBS (1X). The surface carboxyl groups were activated by EDC / NHS, and then the nanoparticles were reacted with NTS mut and p-NH2-Bn-DOTA in a 5:1 molar ratio for conjugation. The resulting nanoparticles were purified and labeled with 64 Cu for PET imaging. Only DM1-NO-encapsulated PLGA nanoparticles conjugated with NTS mut were tested for comparison. H1299 tumor-bearing mice were intravenously injected with these nanoparticles (∼100 μCi / mouse), and PET imaging was performed at 1, 4, and 24 hours.

[0530] Cys-NTS mut Chemical structure of:

[0531]

[0532] Results

[0533] DM1-NO-PLGANP accumulates in tumors through the EPR effect, while NTS mut -DM1-NO PLGA NP accumulates in tumors through both the EPR and NTSR1 targeting. See Figure 11 .

[0534] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention pertains. Publications and the materials cited therein are specifically incorporated herein by reference.

[0535] Those skilled in the art will recognize, or be able to ascertain using only routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A compound having a structure selected from the following: wherein when present, R4, R5, R6 and R8 are independently hydrogen or unsubstituted C1-C5 alkyl, R7, R9, and R 11 are independently hydrogen or unsubstituted C1-C5 alkyl, R 10 is F, Br, Cl or I, and R1 has the following structure: ， Formula II wherein R 12 is an unsubstituted C1-C5 alkylene group, R 13 is hydrogen or an unsubstituted C1-C5 alkyl group, and R 14 is an unsubstituted C1-C5 alkylene group.

2. The compound according to claim 1, having the following structure: 。 3. The compound according to claim 1, wherein R1 has the following structure: ， Formula II wherein R 12 is -CH(CH3)-, R 13 is -CH3, and R 14 is -(CH2)2-.

4. The compound according to claim 1, wherein when present, R4, R5 and R6 are hydrogen, and R7 is methyl.

5. The compound according to claim 1, wherein when present, R8 is hydrogen.

6. The compound according to claim 1, wherein when present, R9 is methyl.

7. The compound according to claim 1, wherein when present, R 10 is Cl.

8. The compound according to claim 1, wherein when present, R 11 is methyl.

9. A nanoparticle comprising the compound according to any one of claims 1 - 8.

10. The nanoparticle according to claim 9, wherein the nanoparticle is a polymeric nanoparticle, liposome, inorganic nanoparticle.

11. The nanoparticle according to claim 10, wherein the nanoparticle is a polymeric nanoparticle comprising one or more amphiphilic, hydrophobic and / or hydrophilic polymers.

12. The nanoparticle according to claim 11, wherein the nanoparticle comprises one or more hydrophobic polymers.

13. The nanoparticle according to claim 12, wherein one or more of the hydrophobic polymers are polyesters.

14. The nanoparticle according to claim 13, wherein one polyester or a plurality of polyesters are selected from poly(lactic - co - glycolic acid), poly(lactic acid), poly(glycolic acid).

15. The nanoparticle according to claim 14, wherein the nanoparticle comprises poly(lactic - co - glycolic acid) (PLGA).

16. The nanoparticle according to claim 11, wherein the nanoparticle comprises one or more hydrophilic polymers.

17. The nanoparticle according to claim 16, wherein one or more of the hydrophilic polymers are polyalkylene glycols.

18. The nanoparticle according to claim 17, wherein the nanoparticle comprises polyethylene glycol (PEG).

19. The nanoparticle according to claim 18, wherein the nanoparticle is a polymeric nanoparticle comprising poly(lactide - co - glycolide) - block - poly(ethylene glycol) (PLGA - b - PEG).

20. The nanoparticle according to claim 9, having a size suitable for delivering the compound to the tumor microenvironment by enhanced permeability and retention.

21. The nanoparticle according to claim 20, wherein the nanoparticle has a size of 10 nm to 300 nm.

22. The nanoparticle according to claim 9, further comprising a targeting agent conjugated thereto.

23. The nanoparticle according to claim 22, wherein the targeting agent targets NTSR1.

24. The nanoparticle according to claim 23, wherein the targeting agent is an agonist or antagonist of NTSR1.

25. The nanoparticle according to claim 24, wherein the targeting agent is NTS or a variant thereof.

26. The nanoparticle according to claim 25, wherein the targeting agent is Cys-NTS having the following structure mut :[[]]END]] 。 27. The nanoparticle according to claim 24, wherein the targeting agent is SR142948A or Cys-NTS having the following structure 20.8 :[[]]END]] 。 28. The nanoparticle according to claim 14, wherein the nanoparticle further comprises a targeting agent targeting NTSR1, and wherein the compound is 。 29. The nanoparticle according to claim 28, wherein the nanoparticle is a polymeric nanoparticle comprising poly(lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG).

30. The nanoparticle according to claim 29, wherein the targeting agent is Cys-NTS having the following structure mut : 。 31. A pharmaceutical composition comprising an effective amount of the compound according to any one of claims 1-8.

32. A pharmaceutical composition comprising an effective amount of the nanoparticle according to any one of claims 9-30.

33. Use of the pharmaceutical composition according to claim 32 for the preparation of a pharmaceutical product for treating a disease or disorder in a subject, wherein the disease or disorder involves radiosensitive cells and the subject has cancer.

34. Use of the pharmaceutical composition according to claim 33, further comprising administering to the subject one or more doses of radiotherapy, wherein the radiotherapy is ionizing radiotherapy, phototherapy or proton therapy.

35. Use of the pharmaceutical composition according to claim 34, wherein the compound enhances the treatment of the cancer compared to administering the radiotherapy alone.

36. Use of the pharmaceutical composition according to claim 35, wherein the cancer is a radiosensitive cancer.

37. Use of the pharmaceutical composition according to claim 35, wherein the cancer is a radioresistant cancer.

38. Use of the pharmaceutical composition according to claim 35, wherein the cancer is a vascular cancer, bone cancer, muscle cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, uterine cancer or germ cell cancer.

39. Use of the pharmaceutical composition according to claim 35, wherein the cancer is an epithelial cancer.

40. Use of the pharmaceutical composition according to claim 35, wherein the cancer is non-small cell lung cancer (NSCLC).

41. Use of the pharmaceutical composition according to claim 35, wherein the same dose of radiotherapy is more effective when administered in the presence of the pharmaceutical composition than when administered in the absence thereof, and a lower dose of radiotherapy has the same efficacy as a higher dose when administered in the absence of the pharmaceutical composition.

42. Use of the pharmaceutical composition according to claim 35, wherein a dose of radiotherapy is administered after administering the pharmaceutical composition.

43. Use of the pharmaceutical composition according to claim 42, wherein the dose of radiotherapy is administered 1 to 48 hours after administering the pharmaceutical composition.

44. Use of the pharmaceutical composition according to claim 43, the method comprising administering 1 or more rounds of the pharmaceutical composition, followed by administering the dose of radiotherapy.

45. Use of the pharmaceutical composition according to claim 35, wherein the radiotherapy is ionizing radiotherapy.

46. Use of the pharmaceutical composition according to claim 35, wherein the cancer comprises cells with upregulated NTSR1.

47. Use of the pharmaceutical composition according to claim 36, wherein the nanoparticles comprise a targeting agent targeting NTSR1.

48. Use of the pharmaceutical composition according to claim 47, wherein the compound is 。 49. Use of the pharmaceutical composition according to claim 48, wherein the nanoparticles are polymeric nanoparticles comprising poly(lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG).

50. Use of the pharmaceutical composition according to claim 49, wherein the targeting agent is Cys-NTS having the following structure mut : 。

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