pH-responsive compositions, formulations, and methods for tumor imaging

Abstract

Described herein are formulations, methods, and pH-responsive compositions useful for the detection of primary and metastatic tumor tissue.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 937,141, filed November 18, 2019, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with government support awarded under CA217528 by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Approximately 1.7 million new cancer cases are expected to be diagnosed, and approximately 610,000 Americans are expected to die from cancer in 2019. Effective imaging agents are needed to detect primary and metastatic tumor tissue.

[0004] Treatment guidelines for all stages of solid cancer prominently include surgical removal of the primary tumor and at-risk or involved lymph nodes. Despite biological and anatomical differences between these tumor types, the status of postoperative margins is one of the most important prognostic factors for local tumor control and, therefore, the chance of disease recurrence or tumor metastasis.

[0005] Surgical resection of solid tumors is a balance between oncological effectiveness and removal of normal tissue, thereby minimizing functional morbidity, as well as cosmetic outcomes. This also applies to lymph node dissection, which is often performed simultaneously with removal of the primary tumor for diagnostic and therapeutic purposes. The presence or absence of lymph node metastasis is the most important determinant of survival for gastrointestinal cancer, breast cancer, and many other solid cancers. Physical examination or imaging modalities used for staging successfully detect enlarged or abnormal nodes and aid in surgical treatment planning, but for a high percentage of patients, lymph node metastasis is present at levels too small to be detected by current methods, resulting in understaging. Because occult lymph node metastasis is common, selective local node dissection and histological examination are standard of care for many solid tumors, especially in locally advanced cases. This leads to overtreatment with a significant potential for treatment-related morbidity.

[0006] Optical imaging strategies, based on cellular imaging, natural autofluorescence, and Raman scattering, are rapidly being adapted to image tissues intraoperatively. Optical imaging offers the potential for real-time feedback during surgery, and a variety of camera systems are readily available that provide a wide view of the surgical field. One strategy to overcome the complexities encountered during surgery due to the diversity of oncogenic genotypes and histological phenotypes is to target metabolic vulnerabilities ubiquitous in cancer. Aerobic glycolysis, known as the Warburg effect, in which cancer cells preferentially take up glucose and convert it to lactate, occurs in all solid tumors and represents one such target. Summary of the Invention [Problem to be solved by the invention]

[0007] In some cases, the compositions presented herein utilize pH as a common biomarker for solid cancers, where, after cellular uptake, the ubiquitous pH difference between cancerous and normal tissues provides a sensitive and specific fluorescent response, thereby enabling the detection of tumor tissue, tumor margins, and metastatic tumors, including lymph node and peritoneal metastases.

[0008] In some cases, the compounds described herein are useful imaging agents for the detection of primary and metastatic tumor tissue (including lymph nodes). Real-time fluorescence imaging during surgery assists surgeons in delineating tumor tissue versus normal tissue and detecting metastatic lymph nodes, with the goal of achieving negative margins and complete tumor removal. Clinical benefits from improved surgical outcomes include, for example, reduced tumor recurrence and reoperation rates, avoidance of unnecessary surgeries, preservation of function, cosmesis, and informing patient treatment plans.

[0009] In certain embodiments, a compound of formula (II):

[0010] [ka] (wherein n=90 to 140, x=50 to 200, y=0 to 3, z=0 to 3, and X 1 is a halogen, -OH, or -C(O)OH) or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0011] In some embodiments, X 1 is a halogen. In some embodiments, X 1 is -Br. In some embodiments, n is 100 to 120. In some embodiments, n is 113. In some embodiments, x is 60 to 150. In some embodiments, y is 0.5 to 1.5. In some embodiments, y is 0. In some embodiments, z is 1.5 to 2.5. In some embodiments, z is 0.

[0012] In certain embodiments, provided herein are micelles comprising one or more block copolymers of formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0013] In certain embodiments, provided herein are pH-responsive compositions comprising a pH transition point and an emission spectrum. In some embodiments, the pH transition point is between 4.8 and 5.5. In some embodiments, the pH transition point is about 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. In some embodiments, the emission spectrum is between 700 and 900 nm. In some embodiments, the composition has a pH transition range (ΔpH 10-90% In some embodiments, the pH transition range is less than 0.25 pH units. In some embodiments, the pH transition range is less than 0.15 pH units. In some embodiments, the composition has a fluorescence activation ratio greater than 25. In some embodiments, the composition has a fluorescence activation ratio greater than 50.

[0014] In certain embodiments, provided herein are imaging agents comprising one or more block copolymers having the structure of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the imaging agent comprises poly(ethylene oxide)-b-poly(dibutylaminoethyl methacrylate-r-aminoethyl methyl acrylate hydrochloride) copolymer indocyanine green and an acetic acid conjugate.

[0015] In certain embodiments, there is provided a pharmaceutical composition comprising a micelle, wherein the micelle comprises 1) a compound of formula (II):

[0016] [ka] (wherein n is 90 to 140, x is 50 to 200, y is 0 to 3, z is 0 to 3, and X 1 is a halogen, -OH, or -C(O)OH) or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and 2) a stabilizer.

[0017] In some embodiments, the stabilizing agent is a cryoprotectant. In some embodiments, the stabilizing agent is a sugar, a sugar derivative, a surfactant, or a salt. In some embodiments, the stabilizing agent is a monosaccharide, a disaccharide, a trisaccharide, a water-soluble polysaccharide, or a sugar alcohol, or a combination thereof. In some embodiments, the stabilizing agent is fructose, galactose, glucose, lactose, sucrose, trehalose, maltose, mannitol, sorbitol, ribose, dextrin, cyclodextrin, maltodextrin, raffinose, or xylose, or a combination thereof. In some embodiments, the stabilizing agent is trehalose.

[0018] In some embodiments, the pharmaceutical composition comprises about 0.5% to about 25% w / v, about 1% to about 20% w / v, about 5% to about 15% w / v, about 6% to about 13% w / v, about 7% to about 12% w / v, or about 8% to about 11% w / v of a stabilizer. In certain embodiments, the pharmaceutical composition comprises about 5% w / v, about 6% w / v, about 7% w / v, about 8% w / v, about 9% w / v, about 10% w / v, about 11% w / v, about 12% w / v, about 13% w / v, about 14% w / v, or about 15% w / v of a stabilizer.

[0019] In some embodiments, the pharmaceutical composition further comprises a liquid or aqueous carrier, hi some embodiments, the liquid carrier is selected from sterile water, saline, D5W, or lactated Ringer's solution.

[0020] In some embodiments, the pharmaceutical composition comprises about 1.0 mg / mL to about 5.0 mg / mL of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 0.1 mg / kg to about 3 mg / kg, or about 0.1 to about 1.2 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 1 mg / kg, 2 mg / kg, 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, or about 7 mg / kg of the block copolymer of Formula (II). In some embodiments, the composition comprises about 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 0.8 mg / kg, 1 mg / kg, 1.2 mg / kg, 1.4 mg / kg, 1.6 mg / kg, 1.8 mg / kg, 2 mg / kg, 2.5 mg / kg, or 3 mg / kg of the block copolymer of Formula (II).

[0021] In another embodiment, about 3 mg / mL of Formula (II):

[0022] [ka] (wherein n is 90 to 140, x is 60 to 150, y is 0 to 3, z is 0 to 3, and X 1 is Br) or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and about 10% w / v trehalose in water. In some embodiments, the pharmaceutical composition is formulated for oral, intramuscular, subcutaneous, intratumoral, or intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intravenous (IV) administration.

[0023] In another aspect, provided herein are methods for imaging the pH of an intracellular or extracellular environment, the method comprising: (a) contacting the environment with a pharmaceutical composition of the present disclosure; and (b) detecting one or more optical signals from the environment, wherein the detected optical signals indicate that a micelle has reached its pH transition point and dissociated. In some embodiments, the optical signals are fluorescent signals. In some embodiments, the intracellular environment is imaged, and the cell is contacted with the pH-responsive composition under conditions appropriate to cause uptake of the pH-responsive composition. In some embodiments, the intracellular environment is part of a cell. In some embodiments, the extracellular environment is that of a tumor or vasculature cell. In some embodiments, the extracellular environment is intravascular or extravascular. In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is cancerous, and the cancer is breast, colorectal, bladder, esophageal, head and neck (HNSSC), lung, brain, prostate, ovarian, or skin (including melanoma and sarcoma) cancer.

[0024] In another aspect, provided herein are methods of removing a tumor in a patient, the method comprising: (a) detecting one or more optical signals from a tumor or a sample thereof obtained from a patient administered an effective dose of a pharmaceutical composition described herein, wherein the detected optical signals indicate the presence of a tumor; and (b) removing the tumor via surgery. In some embodiments, the optical signals indicate tumor margins. In some embodiments, the tumor is at least 90%, 95%, or 99% removed. In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, esophageal cancer, brain cancer, pancreatic cancer, skin cancer, melanoma, sarcoma, pleural metastasis, kidney cancer, lymph node cancer, cervical cancer, or colorectal cancer. In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), esophageal cancer, colorectal cancer, ovarian cancer, or prostate cancer.

[0025] In some embodiments, the pharmaceutical compositions disclosed herein are administered before surgery. In some embodiments, the pharmaceutical compositions are administered before imaging of the tumor or lymph nodes. In some embodiments, the pharmaceutical compositions disclosed herein are administered before patient management for clinical outcomes. In some embodiments, the pharmaceutical compositions are administered at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 24 hours, at least 28 hours, at least 32 hours, at least 80 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, or at least 2 weeks before surgery. In some embodiments, the pharmaceutical compositions are administered about 1 hour to about 32 hours, about 2 hours to about 32 hours, 16 hours to about 32 hours, about 20 hours to about 28 hours, about 1 hour to about 5 hours, or about 3 hours to about 9 hours before surgery. In some embodiments, the pharmaceutical compositions are administered as an injection or infusion. In some embodiments, the pharmaceutical composition is administered as a single dose or multiple doses.

[0026] In another aspect, provided herein is a method of treating cancer, the method comprising: (a) detecting one or more optical signals in a cancer patient in need thereof who has been administered an effective dose of a pharmaceutical composition described herein, wherein the detected optical signals indicate the presence of a cancerous tumor. In some embodiments, the method further comprises imaging a body cavity of the cancer patient, or imaging the cancerous tumor or a slice or sample thereof (e.g., fresh or formalin-fixed), optionally by back-table fluorescence-guided imaging after removal from the patient.

[0027] In another aspect, provided herein is a method of minimizing cancer recurrence for at least five years, the method comprising: (a) detecting one or more optical signals in a cancer patient in need thereof who has been administered an effective dose of a pharmaceutical composition disclosed herein, wherein the detected optical signals indicate the presence of a cancerous tumor, and the presence of the tumor indicates cancer recurrence; and (b) treating the cancer to minimize recurrence if the one or more optical signals are detected. In some embodiments, the method further comprises removing the tumor. In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, esophageal cancer, colorectal cancer, brain cancer, or skin cancer. In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), esophageal cancer, pleural metastasis, kidney cancer, lymph node cancer, cervical cancer, pancreatic cancer, or colorectal cancer. In some embodiments, the pharmaceutical composition is administered at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 24 hours, at least 28 hours, at least 32 hours, at least 80 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, or at least 2 weeks before imaging the patient. In some embodiments, the pharmaceutical composition is administered about 1 hour to about 32 hours, about 2 hours to about 32 hours, 16 hours to about 32 hours, about 20 hours to about 28 hours, about 1 hour to about 5 hours, or about 3 hours to about 9 hours before imaging the patient. In some embodiments, the pharmaceutical composition is administered as an injection or infusion. In some embodiments, the pharmaceutical composition is administered as a single dose or multiple doses. In some embodiments, the method further comprising imaging the cancer patient includes an intraoperative or endoscopic camera. In some embodiments, the patient in need thereof is a human patient. In some embodiments, a patient in need thereof is a canine, feline, bovine, equine, porcine, or lavatory patient.

[0028] Other objects, features, and advantages of the block copolymers, methods, and compositions described herein will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating particular embodiments, are given by way of example only, and that various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

[0029] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0030] Various aspects of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0031] [Figure 1A] Figure 1 shows mean plasma concentrations versus time in Phase 1a following a single intravenous dose of a pharmaceutical composition containing 0.1, 0.3, 0.5, 0.8, or 1.2 mg / kg of Compound 1. Figure 2 shows mean plasma concentrations (LOG) versus time. [Figure 1B] 1 shows mean plasma concentration versus time in Phase 1a following a single intravenous dose of a pharmaceutical composition containing 0.1, 0.3, 0.5, 0.8, or 1.2 mg / kg of Compound 1. 2 shows linear mean plasma concentration versus time. [Figure 2] The correlation between the mean plasma concentration of the pharmaceutical composition at 10 minutes (C10m) and dose for Compound 1 is disclosed. [Figure 3] The correlation between mean AUC 0-24hr and dose for Compound 1 is disclosed. [Figure 4A]1 shows the plasma concentration versus time of Phase 1b subjects (patients) after a single intravenous dose of a pharmaceutical composition containing 1.2 mg / kg of Compound 1. The mean plasma concentration dose for patients is shown for the plasma concentration (Log) versus time. [Figure 4B] 1 shows plasma concentrations versus time in Phase 1b subjects (patients) following a single intravenous dose of a pharmaceutical composition containing 1.2 mg / kg of Compound 1. 2 shows plasma concentrations (linear) versus time in patients. [Figure 5A] 1 shows the mean plasma concentrations versus time for Phase 1a and Phase 1b following a single intravenous administration of a pharmaceutical composition containing 0.1, 0.3, 0.5, 0.8, or 1.2 mg / kg of Compound 1. The mean plasma concentrations (Log) versus time for Phase 1a and Phase 1b are shown by dose. [Figure 5B] 1 shows mean plasma concentrations versus time for Phase 1a and Phase 1b following a single intravenous administration of a pharmaceutical composition containing 0.1, 0.3, 0.5, 0.8, or 1.2 mg / kg of Compound 1. Mean plasma concentrations (linear) for Phase 1a and Phase 1b versus time are shown by dose. [Figure 6] The (±SD) mean plasma concentrations of Compound 1 vs. dose at 10 minutes for Phase 1a and Phase 1b are shown. [Figure 7] The mean (±SD) AUC 0-24 hr versus dose for Phase 1a and Phase 1b is shown. [Figure 8A] Figure 1 shows the mean plasma concentrations of Compound 1 by tumor type. Figure 2 shows the mean plasma concentrations (Log) versus time for Phase 1a (1.2 mg / kg) and Phase 1b by tumor type. [Figure 8B] Figure 1 shows the mean plasma concentrations of Compound 1 by tumor type. Figure 2 shows the mean plasma concentrations (linear) versus time for Phase 1a (1.2 mg / kg) and Phase 1b by tumor type. [Figure 8C] 1 shows the mean plasma concentrations of Compound 1 by tumor type. 2 shows the plasma concentration (Log) versus time for Phase 1a (1.2 mg / kg) and Phase 1b patients in breast cancer. [Figure 8D] 1 shows the mean plasma concentrations of Compound 1 by tumor type. 2 shows the plasma concentration (Log) versus time for Phase 1a (1.2 mg / kg) and Phase 1b patients in colorectal cancer tumors. [Figure 8E]1 shows the mean plasma concentrations of Compound 1 by tumor type. 2 shows the plasma concentration (Log) versus time for Phase 1a (1.2 mg / kg) and Phase 1b patients in esophageal cancer tumors. [Figure 8F] 1 shows the mean plasma concentrations of Compound 1 by tumor type. 2 shows the individual plasma concentrations (Log) versus time for Phase 1a (1.2 mg / kg) and Phase 1b in head and neck (HNSCC) tumors. [Figure 8G] 1 shows the mean plasma concentrations of Compound 1 by tumor type. 2 shows plasma concentrations (linear) versus time for Phase 1a (1.2 mg / kg) and Phase 1b patients in breast cancer tumors. [Figure 8H] 1 shows the mean plasma concentrations of Compound 1 by tumor type. 2 shows plasma concentrations (linear) versus time for Phase 1a (1.2 mg / kg) and Phase 1b patients in colorectal cancer tumors. [Figure 8I] 1 shows the mean plasma concentrations of Compound 1 by tumor type. 2 shows plasma concentrations (linear) versus time for Phase 1a (1.2 mg / kg) and Phase 1b patients in esophageal cancer tumors. [Figure 8J] 1 shows the mean plasma concentrations of Compound 1 by tumor type. 2 shows plasma concentrations (linear) versus time for Phase 1a (1.2 mg / kg) and Phase 1b patients in HNSCC tumors. [Figure 9A] 1 shows intraoperative images imaged using a NOVADAQ SPY Elite camera from three patients dosed with 0.5 mg / kg of Compound 1. The left column shows white light images, and the right column shows fluorescent images. [Figure 9B] 1 shows intraoperative images imaged using a NOVADAQ SPY Elite camera from three patients dosed with 1.2 mg / kg of Compound 1. The left column shows white light images, and the right column shows fluorescent images. [Figure 10A] 1 shows images taken using a LI-COR Pearl camera of post-operative specimens from three patients dosed with Compound 1 at 0.5 mg / kg. [Figure 10B] 1 shows images taken using a LI-COR Pearl camera of post-operative specimens from three patients dosed with Compound 1 at 1.2 mg / kg. [Figure 11A]The fluorescence intensity contrast ratio of contrast to noise (CNR) is shown. [Figure 11B] Tumor-to-background (TBR) fluorescence intensity contrast ratio is shown. [Figure 12A] The mean postoperative fluorescence intensity versus dose of tumor and normal tissues from histologically confirmed samples (formalin-fixed (FF) or fresh) is shown. [Figure 12B] Shown are post-operative mean fluorescence intensities versus initial plasma concentrations in tumor and normal tissues for histologically confirmed samples (formalin-fixed (FF) or fresh). [Figure 13A] CNR fluorescence ratios calculated using postoperative mean fluorescence intensities from histologically confirmed tumor and normal regions of pathologist-selected breadloaf slices (formalin-fixed (FF) or fresh) for all 15 patients at five dose levels are shown. [Figure 13B] Shown are TBR fluorescence ratios calculated using postoperative mean fluorescence intensities from histologically confirmed tumor and normal regions of pathologist-selected breadloaf slices (formalin-fixed (FF) or fresh) for all 15 patients at five dose levels. [Figure 14] The study design is shown. Intravenous administration of Compound 1 was administered 24 hours (±8 hours) before surgery. A 10-day safety assessment (laboratory, PK, ECG) followed, and adverse events were monitored up to day 17 (a). During surgery, intraoperative images were obtained before incision of the surgical cavity and after resection (b). Immediately after resection, samples were imaged for the presence of positive surgical margins (c). During all standard pathology processing phases, fluorescent images were obtained (d, e), and H / E slices were correlated with standard histopathology slices (f-h). ECG electrocardiogram; H / E hematoxylin-eosin; SOC standard of care. [Figure 15-1]Fluorescence images of different tumor tissue slices are shown: head and neck squamous cell carcinoma of the tongue (a-f); breast cancer (g-l); esophageal cancer (m-r); and colorectal cancer (s-x). Tumors are delineated with solid black lines in the H / E slices (c, i, o, u). The mean fluorescence intensity (MFI) of tumor and nontumor tissue slices per tumor type is shown (y). Dots represent the MFI of a single tissue slice (approximately three slices per subject) from the 1.2 mg / kg cohort. HNSCC, 7 subjects, P < 0.0001; BC, 5 subjects, P = 0.0001; EC, 3 subjects, P = 0.0010; and Wilcox test, two-sided. CRC, 3 subjects. Statistics were not performed because only three data points were available. [Figure 15-2] Fluorescence images of different tumor tissue slices are shown: head and neck squamous cell carcinoma of the tongue (a-f); breast cancer (g-l); esophageal cancer (m-r); and colorectal cancer (s-x). Tumors are delineated with solid black lines in the H / E slices (c, i, o, u). The mean fluorescence intensity (MFI) of tumor and nontumor tissue slices per tumor type is shown (y). Dots represent the MFI of a single tissue slice (approximately three slices per subject) from the 1.2 mg / kg cohort. HNSCC, 7 subjects, P < 0.0001; BC, 5 subjects, P = 0.0001; EC, 3 subjects, P = 0.0010; and Wilcox test, two-sided. CRC, 3 subjects. Statistics were not performed because only three data points were available. [Figure 15-3]Fluorescence images of different tumor tissue slices are shown: head and neck squamous cell carcinoma of the tongue (a-f); breast cancer (g-l); esophageal cancer (m-r); and colorectal cancer (s-x). Tumors are delineated with solid black lines in the H / E slices (c, i, o, u). The mean fluorescence intensity (MFI) of tumor and nontumor tissue slices per tumor type is shown (y). Dots represent the MFI of a single tissue slice (approximately three slices per subject) from the 1.2 mg / kg cohort. HNSCC, 7 subjects, P < 0.0001; BC, 5 subjects, P = 0.0001; EC, 3 subjects, P = 0.0010; and Wilcox test, two-sided. CRC, 3 subjects. Statistics were not performed because only three data points were available. [Figure 16] Fluorescence results of Compound 1 using postoperative tissue samples from different tumor types are shown. Images show a representative example of head and neck squamous cell carcinoma of the tongue from a subject with negative surgical margins. In vivo and ex vivo visualization of intratumor fluorescence (a, c, g, i) is shown, with no fluorescent signal present within the surgical cavity or at the surgical resection site (b, h, d, j). Correlation of the fluorescent signal on tissue slices with histology images (e, k, f) shows tumor-negative 6.4 mm surgical margins. Representative examples of breast cancer surgery (i.e., lumpectomy) with tumor-positive surgical margins are shown (l, m, n, o). Fluorescence was detected at the abdominal surgical margins both in vivo and immediately after resection (r, s, t, u), corresponding to fluorescence localization on tissue slices (p, v) and final histopathology (q). The tumor is delineated as a solid black line on the H / E slices (f, q). H / E hematoxylin-eosin, SOC standard of care. [Figure 17-1]Clinically relevant images for HNSCC and BC are shown. (a-c) show peritoneal metastases (PM) detected intraoperatively. (d-f) show additional tumor lesions detected within the surgical cavity after resection of head and neck squamous cell carcinoma (HNSCC) of the mandible. (g-i) show false-positive fluorescent lesions from salivary gland tissue. (j-o) show additional satellite metastases of the primary tumor lesions detected in two BC subjects and confirmed upon final histopathological examination. (p-r) show additional primary tumor lesions detected in fresh tissue slices from BC subjects, representing triple-negative breast cancer not detected pre- or intraoperatively. (c, f, l, o, r) show tumors delineated as solid black lines in H / E slides. (i) shows the false-positives did not contain viable tumor tissue. [Figure 17-2] Clinically relevant images for HNSCC and BC are shown. (a-c) show peritoneal metastases (PM) detected intraoperatively. (d-f) show additional tumor lesions detected within the surgical cavity after resection of head and neck squamous cell carcinoma (HNSCC) of the mandible. (g-i) show false-positive fluorescent lesions from salivary gland tissue. (j-o) show additional satellite metastases of the primary tumor lesions detected in two BC subjects and confirmed upon final histopathological examination. (p-r) show additional primary tumor lesions detected in fresh tissue slices from BC subjects, representing triple-negative breast cancer not detected pre- or intraoperatively. (c, f, l, o, r) show tumors delineated as solid black lines in H / E slides. (i) shows the false-positives did not contain viable tumor tissue. [Figure 18A] Fluorescence microscopy is described to confirm tumor-specific activation of Compound 1. Fluorescence microscopy performed ex vivo after spraying Compound 1 onto tissue sections of freshly frozen HNSCC specimens immediately after resection is shown. DAPI was applied to Compound 1 for nuclear staining (a) and for fluorescence visualization (b). A sharp delineation of fluorescence between tumor and stromal tissue was observed (c) and correlated with the corresponding histopathological tissue section stained with hematoxylin and eosin (d). [Figure 18B]A fluorescence microscopy method is described to confirm tumor-specific activation of Compound 1. The pH-dependent activation of Compound 1 in human plasma is shown. Addition of increasing amounts of Compound 1 to human plasma did not result in any increase in fluorescence. When the test was repeated using HCl to provide protons to the plasma, the addition of increasing amounts of intact Compound 1 resulted in an increase in fluorescence, suggesting that acidosis activates Compound 1 and thus the fluorescence in a dose-dependent manner. RFU: relative fluorescence units. [Figure 19A] Correlation of fluorescence surgical margin assessment with final histopathology results. Intraoperative assessment of surgical margins during fluorescence-guided surgery can be performed either by intraoperative fluorescence imaging of the surgical cavity at the back table or by fluorescence imaging of the resected specimen. Final histopathology is correlated with fluorescence images for a breast cancer subject (a) and a head and neck squamous cell carcinoma subject (b). [Figure 19B] Correlation of fluorescence surgical margin assessment with final histopathology results. Intraoperative assessment of surgical margins during fluorescence-guided surgery can be performed either by intraoperative fluorescence imaging of the surgical cavity at the back table or by fluorescence imaging of the resected specimen. Final histopathology is correlated with fluorescence images for a breast cancer subject (a) and a head and neck squamous cell carcinoma subject (b). [Figure 20A] Dose-independent mean fluorescence intensity separation between tumor and non-tumor tissues is described. Mean fluorescence intensity (MFI) of tumor and non-tumor tissues from the 0.1 mg / kg cohort, P=0.0005, Wilcoxon test, two-tailed. Dots represent MFI of single tissue slices. [Figure 20B] Dose-independent mean fluorescence intensity separation between tumor and non-tumor tissues is described. Mean fluorescence intensity (MFI) of tumor and non-tumor tissues from the 0.3 mg / kg cohort, P=0.0078, Wilcoxon test, two-tailed. Dots represent MFI of single tissue slices. [Figure 20C]Dose-independent mean fluorescence intensity separation between tumor and non-tumor tissues is described. Mean fluorescence intensity (MFI) of tumor and non-tumor tissues from the 0.5 mg / kg cohort, P=0.0020, Wilcoxon test, two-tailed. Dots represent MFI of single tissue slices. [Figure 20D] Dose-independent mean fluorescence intensity separation between tumor and non-tumor tissues is described. Mean fluorescence intensity (MFI) of tumor and non-tumor tissues from the 0.8 mg / kg cohort, P=0.0078, Wilcoxon test, two-tailed. Dots represent MFI of single tissue slices. [Figure 20E] Dose-independent mean fluorescence intensity separation between tumor and non-tumor tissues is described. Mean fluorescence intensity (MFI) of tumor and non-tumor tissues from the 1.2 mg / kg cohort, P<0.0001, Wilcoxon test, two-tailed. Dots represent MFI of single tissue slices. [Figure 20F] Receiver operator characteristic curves were based on calculated MFI values ​​for tumor and normal tissue from the 1.2 mg / kg dose cohort, P<0.0001; area under the curve 0.9875, n=59, 95% confidence interval using the Wilson / Brown method. ROC receiver operator curve, area under the AUC curve. **P≤0.01; ***P≤0.001; ****P≤0.0001. [Figure 21] 1 shows in vivo imaging using Compound 1 fluorescence. Representative examples of in vivo imaging data using Compound 1 fluorescence. A large tongue cancer with a necrotic ulcer in the center was visualized in vivo using Compound 1 (a). A cancer located in the right mandible / floor of the mouth was visualized in vivo using Compound 1 (b). A large tongue cancer with a necrotic ulcer in the center was visualized using Compound 1 (c). A colorectal cancer with a large peritoneal metastasis was visualized in vivo using Compound 1 (d). [Figure 22] Fluorescence imaging of breast cancer and HNSCC tumors 3-9 hours and 1-5 hours after dosing with Compound 1 is shown. Images are shown using SPY Elite and VisionSense cameras. [Figure 23] Using a Da Vinci Firefly camera with updated software and hardware, we demonstrate intraoperative fluorescence of Compound 1 in prostate cancer through a thin prostate capsule. No fluorescence was detected in the surgical wound bed, consistent with negative margins confirmed via pathology examination. [Figure 24] Using a VisionSense camera, we demonstrate Compound 1 fluorescence in ovarian cancer (uterocervical recurrence). In vivo imaging was performed 6±3 hours after dosing with 3 mg / kg Compound 1 before resection. [Figure 25] Fluorescence of Compound 1 on a breadloaf slide (BLS) tissue sample corresponding to the tumor area confirmed by pathology is shown. [Figure 26] Using a 3-5 hour dosing schedule timing, we demonstrate that Compound 1 fluorescence was verified in all visible BC and HNSCC tumors using a SPY Elite camera. [Figure 27] 1 shows a mast cell tumor resected from a canine patient. Representative white light (left) and fluorescent images (right) of a mast cell tumor resected from a canine patient after administration of Compound 1. [Figure 28A] Representative images from a soft tissue sarcoma are shown. A white light image of a mast cell tumor is evident in (A). [Figure 28B] Representative images from a soft tissue sarcoma are shown, which can also be easily observed intraoperatively using the custom-built NIR camera described above before resection (B). [Figure 28C] Representative images from a soft tissue sarcoma are shown. A white light photograph of the resected tumor with tissue margins is shown (C). [Figure 28D] Representative images from a soft tissue sarcoma are shown. The corresponding fluorescent image of the resected tumor imaged with LI-COR Pearl, when overlaid with the white light image, shows that the fluorescence colocalizes with the white light anatomical structures (D). Malignancy of the resected tissue was confirmed by histopathological examination. [Figure 29A]Representative images from a canine patient with osteosarcoma are shown. White light photographs of the lesion in the amputated leg are shown; the green and black dotted lines indicate cross-sections of the locations of normal and cancerous tissue, respectively. [Figure 29B] Representative images from a canine patient with osteosarcoma are shown, showing NIR tumor images taken using a Hamamatsu PDE NIR camera. [Figure 29C] Representative images from a canine patient with osteosarcoma are shown, showing white light photographs of cross sections of normal tissue (left, minor) and cancerous tissue (right, major) as described in (A). [Figure 29D] Representative images from a canine patient with osteosarcoma are shown. (C) NIR images of cross sections of the same normal (non-fluorescent) and cancerous tissue (fluorescent tissue) are shown. [Figure 30] Representative images are shown from a canine patient with soft tissue sarcoma. A white light image of the excised soft tissue sarcoma with margins is shown on the left, and a fluorescent image of the tumor tissue (overlaid with white light) is shown on the right. Histopathology confirmed malignancy of the excised tissue. [Figure 31] Images are shown from a canine patient with primary soft tissue auricular sarcoma. White light images of the soft tissue auricular sarcoma are shown in the upper left and lower left panels. NIR images of the ear taken after amputation using a Hamamatsu PDE show the tumor fluorescing through the skin (lower middle panel). The ear was also imaged using the LI-COR system after a core needle biopsy (lower right and inset, respectively) was performed, showing residual fluorescence. Histopathological analysis of the punch biopsy confirmed the malignancy of the tissue. [Figure 32] Images are shown from a canine patient with a primary soft tissue sarcoma and lymph nodes affected by peripheral tumors. The white light image in the top left panel shows the primary soft tissue sarcoma. During surgical removal of this mass, the popliteal lymph node was noted to be enlarged (top right panel), which was removed and imaged using LI-COR (middle panel). Fluorescence imaging showed that the sectioned lymph node was affected, which was confirmed by histopathology. DETAILED DESCRIPTION OF THE INVENTION

[0032] Some embodiments provided herein describe micelle-based fluorescent imaging agents. In some embodiments, the micelles comprise a diblock copolymer of polyethylene glycol (PEG) and dibuthylamino-substituted polymethyl methacrylate (PMMA) covalently conjugated to indocyanine green (ICG) via NHS chemistry on 2-aminoethyl methacrylate hydrochloride monomers. In some embodiments, the PEG comprises the shell or surface of the stable micelles. In some embodiments, the micelles are <100 nm in size.

[0033] I. Compound In some embodiments, a compound of formula (II):

[0034] [ka] (In the formula, X 1 is a halogen, —OH, or —C(O)OH; n is 90 to 140, x is between 50 and 200; y is 0 to 3, z is 0 to 3) or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0035] In some embodiments, the block copolymer of Formula (II) is a compound. In some embodiments, the block copolymer of Formula (II) is a diblock copolymer. In some embodiments, the block copolymer of Formula (II) is a block copolymer comprising a hydrophilic polymer segment and a hydrophobic polymer segment.

[0036] The hydrophilic polymer segment comprises poly(ethylene oxide) (PEO). In some embodiments, the hydrophilic polymer segment is about 2 kDa to about 10 kDa in size. In some embodiments, the hydrophilic polymer segment is about 2 kDa to about 5 kDa in size. In some embodiments, the hydrophilic polymer segment is about 3 kDa to about 8 kDa in size. In some embodiments, the hydrophilic polymer segment is about 4 kDa to about 6 kDa in size. In some embodiments, the hydrophilic polymer segment is about 5 kDa in size.

[0037] In some embodiments, the block copolymer comprises a hydrophobic polymer segment. In some embodiments, the hydrophobic polymer segment comprises a tertiary amine. In some embodiments, the hydrophobic polymer segment comprises:

[0038] [ka] (wherein the total number of x's is about 50 to 200) In some embodiments, x is about 60 to 150. In some embodiments, x is an integer between about 60 and about 150. In some embodiments, the hydrophilic segment comprises dibutylamine.

[0039] In some embodiments, n repeating polyethylene oxide repeat units are present. In some embodiments, n is 90 to 140. In some embodiments, n is 95 to 130. In some embodiments, n is 100 to 120. In some embodiments, n is 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120. In some embodiments, n is 114. In some embodiments, n is 113.

[0040] In some embodiments, y is 0 to 3. In some embodiments, y is 0.5 to 2.5. In some embodiments, y is 1.5 to 2.5. In some embodiments, y is 0.5 to 1.5. In some embodiments, y is 0.5, 1, 1.5, 2, 2.5, or 3. In some embodiments, y is 1, 2, or 3. In some embodiments, y is 0.5. In some embodiments, y is 1.5. In some embodiments, y is 0.

[0041] In some embodiments, z is 0 to 3. In some embodiments, z is 1.5 to 2.5. In some embodiments, z is 1, 1.5, 2, 2.5, or 3. In some embodiments, z is 1, 2, or 3. In some embodiments, z is 1.5. In some embodiments, z is 0.

[0042] In some embodiments, the copolymer block units (x, y, and z) can occur in any sequence or configuration. In some embodiments, x, y, and z occur sequentially as described in formula (II).

[0043] In certain embodiments, the block copolymer comprises a fluorescent dye conjugated via an amine. In some embodiments, the fluorescent dye is a pH-insensitive dye. In some embodiments, the fluorescent dye is a cyanine dye or a derivative thereof. In some embodiments, the fluorescent dye is indocyanine green (ICG). Indocyanine green (ICG) is used in medical diagnostics.

[0044] In some embodiments, the block copolymer is not conjugated to a fluorescent dye or derivative thereof, hi some embodiments, the block copolymer is not conjugated to indocyanine green (ICG).

[0045] In some embodiments, the block copolymer of Formula (II) is a poly(ethylene oxide)-b-poly(dibutylaminoethyl methacrylate-r-aminoethyl methyl acrylate hydrochloride) copolymer indocyanine green and acetic acid conjugate. In some embodiments, the block copolymer of Formula (II) is a PEO 90~140 -bP(DBA 60~150 -r-ICG 0~3 -r-AMA 0~3 ), (Compound 1).

[0046] In some embodiments, X 1 is an end group. In some embodiments, the end capping group is the product of an atom transfer radical polymerization (ATRP) reaction. In some embodiments, X 1 is a halogen. In some embodiments, X 1 is Br. In some embodiments, X 1 is —OH. In some embodiments, X 1 is an acid. In some embodiments, X 1 is —C(O)OH. In some embodiments, X 1 is H.

[0047] The term "r" represents the connection between different block copolymer units / segments (e.g., represented by x, y, and z). In some embodiments, each r independently represents the carbon atom of the unit / segment. The join that joins , or alkyl group -(CH2) n - (wherein n is 1 to 10) )in In some embodiments, the copolymer block segments / units (e.g., represented by x, y, and z) can occur in any order, sequence, or configuration. In some embodiments, the copolymer block units occur sequentially as described in formula (II).

[0048] In some embodiments, the block copolymer of Formula (II) has Formula (II-a):

[0049] [ka] or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0050] In some embodiments, the block copolymer of Formula (II) is in the form of a micelle or nanoparticle. The size of the micelle is typically on the nanometer scale (i.e., between about 1 nm and 1 μm in diameter). In some embodiments, the micelles have a size of about 10 to about 200 nm. In some embodiments, the micelles have a size of about 20 to about 100 nm. In some embodiments, the micelles have a size of about 30 to about 50 nm. In some embodiments, the micelles have a diameter of less than about 1 μm. In some embodiments, the micelles have a diameter of less than about 100 nm. In some embodiments, the micelles have a diameter of less than about 50 nm.

[0051] In another aspect, provided herein is a pH-responsive composition comprising one or more block copolymers of Formula (II).

[0052] In some embodiments, the pH-responsive composition has a pH transition point and an emission spectrum. In some embodiments, the pH transition point is between 4 and 8 or between 6 and 7.5. In some embodiments, the pH transition point is between 4.8 and 5.5. In some embodiments, the pH transition point is about 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. In some embodiments, the pH transition point is 4.8. In some embodiments, the pH transition point is 4.9. In some embodiments, the pH transition point is 5.0. In some embodiments, the pH transition point is 5.1. In some embodiments, the pH transition point is 5.2. In some embodiments, the pH transition point is 5.3. In some embodiments, the pH transition point is 5.4. In some embodiments, the pH transition point is 5.5.

[0053] In some embodiments, the pH-responsive composition has an emission spectrum between 700 and 900 nm. In some embodiments, the pH-responsive composition has an emission spectrum between 750 and 800 nm. In some embodiments, the pH-responsive composition has an emission spectrum between 750 and 850 nm.

[0054] In some embodiments, the pH-responsive composition has a pH transition range (ΔpH 10~90% In some embodiments, the pH responsive composition has a pH transition range of less than 1 pH unit. In some embodiments, the pH responsive composition has a pH transition range of less than 0.25 pH units. In some embodiments, the pH responsive composition has a pH transition range of less than 0.15 pH units.

[0055] In some embodiments, the composition has a fluorescence activation ratio. <pH t The normalized fluorescence intensity obtained from a formulation in a buffer with pH > pH (pH transition of the formulation) t In some embodiments, the fluorescence activation ratio is greater than 25. In some embodiments, the fluorescence activation ratio is greater than 50.

[0056] II. Pharmaceutical Compositions The pharmaceutical compositions disclosed herein comprise one or more pH-responsive micelles and / or nanoparticles comprising a block copolymer and the fluorescent dye indocyanine green. The block copolymer comprises a hydrophilic polymer segment and a hydrophobic polymer segment, and the hydrophobic polymer segment contains ionizable amine groups that confer pH sensitivity. This pH sensitivity can be utilized to provide pharmaceutical compositions suitable as diagnostic tools for imaging (e.g., to aid in tumor removal and staging).

[0057] In one aspect, provided herein is a pharmaceutical composition comprising a micelle, the micelle comprising: 1) Formula (II):

[0058] [ka] (In the formula, X 1 is a halogen, —OH, or —C(O)OH; n is 90 to 140, x is between 50 and 200; y is 0 to 3, z is 0 to 3) or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and 2) Contains stabilizers.

[0059] In some embodiments, the pharmaceutical composition comprises a micelle, wherein the micelle comprises one or more block copolymers having a structure of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, is a micelle-based fluorescent imaging agent. In some embodiments, the block copolymer of Formula (II) is a poly(ethylene oxide)-b-poly(dibutylaminoethyl methacrylate-r-aminoethyl methyl acrylate hydrochloride) copolymer indocyanine green and acetic acid conjugate. In some embodiments, the block copolymer of Formula (II) is a PEO 90-140 -bP(DBA 60-150 -r-ICG 0-3 -r-AMA 0-3 ), (Compound 1). In some embodiments, the block copolymer is a copolymer capable of forming micelles or nanoparticles.

[0060] In some embodiments, the pharmaceutical composition comprises about 1 mg / mL to about 5 mg / mL of the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the pharmaceutical composition comprises about 1 mg / mL, about 1.5 mg / mL, about 2 mg / mL, about 2.5 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, or about 5 mg / mL of the block copolymer of Formula (II).

[0061] In some embodiments, the pharmaceutical composition comprises about 3.0 mg / mL of the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0062] In some embodiments, the pharmaceutical composition comprises about 0.1 mg / kg to about 8 mg / kg of the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the pharmaceutical composition comprises about 0.5 mg / kg to about 7 mg / kg of the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the pharmaceutical composition comprises about 0.1 mg / kg to about 3 mg / kg of the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the pharmaceutical composition comprises about 0.1 to about 1.2 mg / kg of the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0063] In some embodiments, the pharmaceutical composition comprises about 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, or 7 mg / kg of the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the pharmaceutical composition comprises about 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 0.8 mg / kg, 1 mg / kg, 1.2 mg / kg, 1.4 mg / kg, 1.6 mg / kg, 1.8 mg / kg, 2 mg / kg, 2.5 mg / kg, or 3 mg / kg of the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the pharmaceutical composition comprises about 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 0.8 mg / kg, 1 mg / kg, or 1.2 mg / kg of the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the pharmaceutical composition comprises about 0.1 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 0.3 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 0.5 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 0.8 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 1 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 1.2 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 1.4 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 1.6 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 1.8 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 2 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 2.5 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 3 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 3.5 mg / kg of the block copolymer of Formula (II).In some embodiments, the pharmaceutical composition comprises about 4 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 5 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 6 mg / kg of the block copolymer of Formula (II). In some embodiments, the pharmaceutical composition comprises about 7 mg / kg of the block copolymer of Formula (II).

[0064] In some embodiments of the pharmaceutical compositions disclosed herein, the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, is substantially pure. In some embodiments of the pharmaceutical compositions disclosed herein, the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, is substantially free of impurities. In some embodiments of the pharmaceutical compositions disclosed herein, substantially free of impurities is defined as an impurity content of less than about 10%, about 5%, about 3%, about 1%, about 0.5%, about 0.1%, or about 0.05%. In some embodiments of the pharmaceutical compositions disclosed herein, substantially free of impurities is defined as an impurity content of less than about 1%. In some embodiments of the pharmaceutical compositions disclosed herein, substantially free of impurities is defined as an impurity content of less than about 0.5%. In some embodiments of the pharmaceutical compositions disclosed herein, substantially free of impurities is defined as an impurity content of less than about 0.1%.

[0065] In some embodiments of the pharmaceutical compositions disclosed herein, the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, is at least about 90%, about 95%, about 98%, or about 99% pure.

[0066] In some embodiments of the pharmaceutical compositions disclosed herein, the block copolymer of Formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, is at least about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, or about 100% pure.

[0067] The term "stabilizer" is intended to mean an agent that, when added to a biologically active material, prevents or delays the loss of biological activity of the material over time compared to when the material is stored in the absence of the stabilizer. Some of these additives have been found to extend the shelf life of biologically active materials by several months or longer when stored in an essentially dehydrated form at ambient temperatures. Additionally, a variety of cryoprotective additives and agents, when used as excipients, aid and preserve biological activity when biological materials are dried or frozen. Protective substances are water-soluble sugars, such as monosaccharides, disaccharides, trisaccharides, water-soluble polysaccharides, sugar alcohols, polyols, or mixtures thereof. Examples of monosaccharides, disaccharides, and trisaccharides include, but are not limited to, glucose, mannose, glyceraldehyde, xylose, lyxose, talose, sorbose, ribulose, xylulose, galactose, fructose, sucrose, trehalose, lactose, maltose, and raffinose. Among water-soluble polysaccharides are certain water-soluble starches and celluloses. An example of a sugar alcohol is glycerol. Other substances that function as stabilizers include, for example, amino acids, such as arginine, and proteins, such as albumin.

[0068] In some embodiments, the pharmaceutically acceptable excipient is a cryoprotectant or stabilizer. In some embodiments, the pharmaceutically acceptable excipient is a stabilizer. In some embodiments, the stabilizer is a sugar, a sugar derivative, a surfactant, or a salt.

[0069] In some embodiments, the stabilizer is a monosaccharide, a disaccharide, a trisaccharide, a water-soluble polysaccharide, a sugar alcohol, or a polyol, or a combination thereof. In some embodiments, the stabilizer is fructose, galactose, glucose, lactose, sucrose, trehalose, maltose, mannitol, sorbitol, ribose, dextrin, cyclodextrin, maltodextrin, raffinose, or xylose, or a combination thereof. In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is trehalose dihydride.

[0070] In some embodiments, the pharmaceutical composition comprises about 0.5% w / v to about 25% w / v, about 1% to about 20% w / v, about 5% to about 15% w / v, about 6% to about 13% w / v, about 7% to about 12% w / v, or about 8% to about 11% w / v of a stabilizer. In some embodiments, the pharmaceutical composition comprises about 7% to about 12% w / v of a stabilizer. In some embodiments, the pharmaceutical composition comprises about 8% to about 11% w / v of a stabilizer.

[0071] In some embodiments, the pharmaceutical composition comprises about 5% w / v, about 6% w / v, about 7% w / v, about 8% w / v, about 9% w / v, about 10% w / v, about 11% w / v, about 12% w / v, about 13% w / v, about 14% w / v, or about 15% w / v of the stabilizer. In some embodiments, the pharmaceutical composition comprises about 9% w / v of the stabilizer. In some embodiments, the pharmaceutical composition comprises about 10% w / v of the stabilizer. In some embodiments, the pharmaceutical composition comprises about 11% w / v of the stabilizer. In some embodiments, the pharmaceutical composition comprises about 12% w / v of the stabilizer. In some embodiments, the pharmaceutical composition comprises about 13% w / v of the stabilizer. In some embodiments, the pharmaceutical composition comprises about 14% w / v of the stabilizer. In some embodiments, the pharmaceutical composition comprises about 15% w / v of the stabilizer.

[0072] In some embodiments, the pharmaceutical composition further comprises a liquid carrier. In some embodiments, the liquid carrier is an aqueous solution. In some embodiments, the liquid carrier is selected from sterile water, sterile water for injection (SWFI), saline, half normal saline, dextrose (e.g., aqueous dextrose; e.g., 5% dextrose in water, D5W), or lactated Ringer's solution (RL), or a combination thereof (e.g., 50% dextrose and 50% saline). In some embodiments, the liquid carrier is selected from sterile water.

[0073] In some embodiments, the pharmaceutical composition has the structure of Formula (II):

[0074] [ka] (In the formula, X 1 is Br, n is 90 to 140, x is between 60 and 150; y is 0 to 3, z is 0 to 3) or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and Contains approximately 10% w / v trehalose in water.

[0075] Pharmaceutical compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration. Exemplary routes of administration are described herein.

[0076] In some embodiments, the pharmaceutical compositions disclosed herein are in a form for oral, intravenous (IV), intramuscular, subcutaneous, intratumoral, or intradermal administration. In some embodiments, the pharmaceutical compositions are formulated for oral, intramuscular, subcutaneous, or intravenous administration. In some embodiments, the pharmaceutical compositions are formulated for intratumoral administration. In some embodiments, the pharmaceutical compositions are formulated for intravenous administration. In some embodiments, the pharmaceutical compositions are formulated as an aqueous solution or suspension for intravenous (IV) administration. In some embodiments, the pharmaceutical compositions are formulated for administration as a single dose. In some embodiments, the pharmaceutical compositions are formulated for administration as multiple doses. In some embodiments, the pharmaceutical compositions disclosed herein are formulated for administration as an IV bolus.

[0077] In some embodiments of the pharmaceutical composition in an IV dosage form, the pH is about 3.5 to about 8.5. In some embodiments, the pH of the IV dose is about 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5.

[0078] Aqueous suspensions contain the active substance mixed with suitable excipients for its manufacture. Such excipients can be suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic; dispersing or wetting agents, such as naturally occurring phosphatides (e.g., lecithin), or alkylene oxide condensates with fatty acids (e.g., polyoxyethylene stearates), or ethylene oxide condensates with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), or ethylene oxide condensates with fatty acids and partial esters derived from hexitols (e.g., polyoxyethylene sorbitol monooleate), or ethylene oxide condensates with fatty acids and partial esters derived from hexitol anhydrides (e.g., polyethylene monosorbitan oleate). Aqueous suspensions can also contain one or more preservatives.

[0079] Oily suspensions can be formulated by suspending the active ingredient in vegetable oils, such as peanut oil, olive oil, sesame oil or coconut oil, or mineral oils, such as liquid paraffin.Oily suspensions can contain thickening agents, such as beeswax, hard paraffin or cetyl alcohol.Sweeteners, such as those described above, and flavoring agents can be added to obtain a palatable oral preparation.

[0080] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, and optionally one or more suspending agents and / or preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.

[0081] The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin, or a mixture thereof. Suitable emulsifiers may be naturally occurring gums, such as gum arabic or gum tragacanth; naturally occurring phosphatides, such as soybean, lecithin, and esters or partial esters derived from fatty acids; hexitol anhydrides, such as sorbitan monooleate; and condensation products of partial esters with ethylene oxide, such as polyoxyethylenesorbitan monooleate.

[0082] Pharmaceutical compositions typically contain a therapeutically effective amount of a block copolymer of Formula (II) or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and one or more pharmaceutically and physiologically acceptable formulation agents. Suitable pharmaceutically or physiologically acceptable diluents, carriers, or excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methylparaben, ethyl or n-propyl, p-hydroxybenzoate), emulsifiers, suspending agents, dispersing agents, solvents, fillers, extenders, surfactants, buffers, vehicles, diluents, and / or adjuvants. For example, a suitable vehicle is saline solution or citrate-buffered saline, possibly supplemented with other materials commonly present in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin is another exemplary vehicle. Those skilled in the art will readily recognize that a variety of buffers that can be used in pharmaceutical compositions and dosage forms are contemplated herein. Typical buffer solutions include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. For example, buffer solutions can be water-soluble materials such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof. Acceptable buffering agents include, for example, Tris buffer; N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES); 2-(N-morpholino)ethanesulfonic acid (MES); 2-(N-morpholino)ethanesulfonic acid sodium salt (MES); 3-(N-morpholino)propanesulfonic acid (MOPS); and N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

[0083] After the pharmaceutical composition has been formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form, a lyophilized form that requires reconstitution before use, a liquid form that requires dilution before use, or other acceptable form. In some embodiments, the pharmaceutical composition is provided in a single-use container (e.g., a single-use vial, ampule, syringe, or auto-injector), while in other embodiments, a multi-use container (e.g., a multi-use vial) is provided.

[0084] The formulation can also include carriers that protect the composition from rapid degradation or elimination from the body, such as controlled-release formulations, including liposomes, hydrogels, prodrugs, and microencapsulated delivery systems. For example, time-delay materials such as glyceryl monostearate or glyceryl stearate, alone or in combination with a wax, can be utilized. Any drug delivery device can be used to deliver the block copolymer of formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, including implants (e.g., implantable pumps) and catheter systems, slow injection pumps, and devices, all of which are well known to those skilled in the art.

[0085] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. Such suspensions can be formulated by known techniques using suitable dispersing or wetting agents and suspending agents, as described herein. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable diluents, solvents, and dispersion media that can be employed include water, Ringer's solution, isotonic sodium chloride solution, Cremophor® EL (BASF, Parippany, NJ) or phosphate-buffered saline (PBS), ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), sterile water for injection (SWFI), D5W, and suitable mixtures thereof. Additionally, sterile, fixed oils are conventionally employed as solvents or suspending media; for this purpose, any bland, fixed oil, including synthetic mono- or diglycerides, may be employed. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Prolonged absorption of certain injectable formulations can be achieved by including an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0086] III. Method of Use In some embodiments, the pharmaceutical compositions described herein are used in pH-responsive compositions. In some embodiments, the pH-responsive compositions are used to image physiological and / or pathological processes involving changes in intracellular or extracellular pH (e.g., the acidic pH of cancerous tumors). In some embodiments, the pharmaceutical composition micelles described herein are useful for detecting primary and metastatic tumor tissue (including peritoneal metastases and lymph nodes), resulting in reduced tumor recurrence and reoperation rates. In some embodiments, the pH-sensitive contrast agents enable tumor detection from surrounding normal tissue due to their high tumor to background fluorescence response ratio (CNR and TBR).

[0087] Aerobic glycolysis, known as the Warburg effect, occurs in all solid tumors, where cancer cells preferentially take up glucose and convert it to lactate or other acids. Lactate or other acids are preferentially accumulated in the extracellular space by monocarboxylate transporters or other transporters. The resulting acidification of the extracellular space promotes extracellular matrix remodeling for further tumor invasion and metastasis.

[0088] Intraoperative real-time fluorescence imaging helps surgeons detect or delineate tumor versus normal tissue or metastatic disease from, for example, affected lymph nodes, with the goal of achieving negative margins and complete tumor resection, and to aid in staging. These improved surgical outcomes translate into significant clinical benefits, such as reduced tumor recurrence and reoperation rates, avoidance of unnecessary surgeries, preservation of function, and cosmesis.

[0089] Another major purpose of cancer surgery is to aid in pathological staging for treatment decisions. Due to occult lymph node metastasis, lymph node status is a key component of cancer staging. Because simple node sampling during surgery underestimates nodal metastasis, selective, comprehensive, and localized nodal dissection has become the standard of care (SOC) for head and neck cancer. Regarding colorectal cancer, for example, up to 25% of "node-negative" patients die from recurrence and metastasis, indicating that the presence of residual occult disease and lymph node metastasis has increased prognostic value, especially for stage II colorectal patients. Accurate detection of nodal metastasis in these patients can lead to upstaging and enhanced adjuvant treatment, resulting in better matching of therapy to disease.

[0090] Thus, techniques that can selectively and accurately improve intraoperative visualization of tumor margins, occult tumor, and tumor-positive lymph nodes and other metastatic disease could potentially improve the completeness of surgical resection, the appropriateness of adjuvant therapy selection, pathological staging, and oncological outcomes for patients with solid tumors.

[0091] Some embodiments provided herein describe block copolymers that form micelles at physiological pH (7.35-7.45). In some embodiments, the block copolymers described herein are conjugated to an ICG dye. In some embodiments, the micelles are 2×10 7 In some embodiments, the micelles have a molecular weight of greater than about 2.7 x 10 Daltons. 7 In some embodiments, the ICG dye is sequestered within the micelle core at physiological pH (7.35-7.45) (e.g., in the blood circulation), which results in fluorescence quenching. In some embodiments, when the micelles encounter an acidic environment (e.g., tumor tissue), the micelles have an average molecular weight of approximately 3.7 x 10 4 The micelles dissociate into individual compounds with different daltons, allowing for activation of the fluorescent signal from the ICG dye, resulting in specific fluorescence in acidic environments (e.g., tumor tissue). In some embodiments, the micelles dissociate at a pH below the pH transition point (e.g., the acidic conditions of the tumor microenvironment).

[0092] In some embodiments, the fluorescence response is very strong due to a sharp phase transition that occurs between hydrophobicity-driven micellar self-assembly (non-fluorescent OFF state) and the concerted dissociation of these micelles (fluorescent ON state) at a predefined low pH.

[0093] In some embodiments, the micelles described herein have a pH transition point and an emission spectrum. In some embodiments, the pH transition point is between 4 and 8 or between 6 and 7.5. In other embodiments, the pH transition point is between 4.8 and 5.5. In certain embodiments, the pH transition point is about 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. In some embodiments, the emission spectrum is between 700 and 900 nm. In some embodiments, the emission spectrum is between 750 and 850 nm.

[0094] In some cases, the pH-sensitive micelle compositions described herein have a narrow pH transition range. In some embodiments, the micelles described herein have a pH transition range (ΔpH10~90% In various embodiments, the micelles have a pH transition range of less than about 0.9 pH units, less than about 0.8 pH units, less than about 0.7 pH units, less than about 0.6 pH units, less than about 0.5 pH units, less than about 0.4 pH units, less than about 0.3 pH units, less than about 0.2 pH units, or less than about 0.1 pH units. In some embodiments, the micelles have a pH transition range of less than about 0.5 pH units. In some embodiments, the pH transition range is less than 0.25 pH units. In some embodiments, the pH transition range is less than 0.15 pH units.

[0095] In some embodiments, the pH-sensitive composition has a fluorescence activation ratio. In some embodiments, the fluorescence activation ratio is greater than 25. In some embodiments, the fluorescence activation ratio is greater than 50.

[0096] In some embodiments, when the intracellular environment is to be imaged, the cell is contacted with the micelle under conditions appropriate to cause uptake of the micelle. In some embodiments, the intracellular environment is a portion of a cell. In some embodiments, the portion of a cell is a lysosome or an endosome. In some embodiments, the extracellular environment is the environment of a tumor or vasculature cell. In some embodiments, the extracellular environment is intravascular or extravascular. In some embodiments, imaging the pH of the intracellular or extracellular environment comprises imaging of metastatic disease. In some embodiments, the metastatic disease is cancer. In some embodiments, the tumor is from a solid cancer. In some embodiments, the tumor is from a non-solid cancer. In some embodiments, imaging the pH of the tumor environment comprises imaging of lymph nodes or nodes. In some embodiments, imaging the pH of the tumor environment allows for determining tumor size or tumor margins during surgery.

[0097] In another aspect, a method for imaging pH of an intracellular or extracellular environment, the method comprising: (a) contacting an intracellular or extracellular environment with a block copolymer or pharmaceutical composition disclosed herein; and (b) detecting one or more optical signals from the intracellular or extracellular environment, the detected optical signals indicating that the micelles comprising one or more block copolymers of Formula (II) have reached their pH transition point and dissociated. Includes:

[0098] In some embodiments, the optical signal is a fluorescent signal.

[0099] In some embodiments, the extracellular environment is a tumor or vasculature cell, hi some embodiments, the extracellular environment is intravascular or extravascular.

[0100] In some embodiments, the pH of the intracellular or extracellular environment comprises imaging the pH of the tumor environment. In some embodiments, imaging the pH of the tumor environment comprises imaging a lymph node or nodes. Sentinel lymph nodes are the first lymph node or group of nodes that drain a cancer and are the first organ reached by metastatic cancer cells from a tumor. In some embodiments, imaging the pH of a lymph node or nodes informs surgical removal of the lymph node. In some embodiments, imaging the pH of a lymph node or nodes informs staging of cancer metastasis. In some embodiments, imaging the pH of a lymph node or nodes enables patient management.

[0101] In some embodiments, imaging the pH of the tumor environment allows for determination of tumor size or tumor margins. In some embodiments, imaging the pH of the tumor environment allows for tumor staging. In some embodiments, imaging the pH of the tumor environment allows for addressing patient outcomes. In some embodiments, imaging the pH of the tumor environment allows for more accurate removal of tumors during surgery. In some embodiments, imaging the pH of the tumor environment allows for detection of residual metastatic disease. In some embodiments, imaging the pH of the tumor environment provides information for the determination of satellite, multifocal, or occult tumors.

[0102] In some embodiments, imaging the pH of the tumor environment provides information for the detection of subclinical disease.

[0103] In some embodiments, the pharmaceutical composition is administered to a patient in need thereof prior to imaging of the tumor, hi some embodiments, the pharmaceutical composition is administered to a patient in need thereof prior to surgery, prior to imaging of the tumor for staging.

[0104] In some embodiments, the pharmaceutical composition is administered to a patient in need thereof before surgery. In some embodiments, the pharmaceutical composition is administered to a patient in need thereof after surgery. In some embodiments, the surgery is a tumor resection.

[0105] In another aspect, there is provided a method of removing a tumor in a patient in need thereof, the method comprising: (a) detecting one or more optical signals from a tumor or a sample thereof obtained from a patient administered an effective dose of a block copolymer or pharmaceutical composition disclosed herein, wherein the detected optical signals indicate the presence of a tumor; and (b) removing the tumor through surgery Includes:

[0106] In some embodiments, the light signal is indicative of the margins of the tumor.

[0107] In some embodiments, the optical signal is a fluorescent signal.

[0108] In some embodiments, the tumor is at least 90% removed.

[0109] In some embodiments, the tumor is at least 95% removed.

[0110] In some embodiments, the tumor is at least 99% removed.

[0111] In some embodiments, the tumor is resected with clean margins. In some embodiments, the clean margins are non-fluorescent tissue. In some embodiments, the non-fluorescent tissue is non-cancerous tissue. In some embodiments, a lack of fluorescence in the wound bed after resection of the tumor or lymph nodes indicates removal of the tumor.

[0112] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a pan-tumor. In some embodiments, the solid tumor is of cancer origin. In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, esophageal cancer, colorectal cancer, brain cancer, or skin cancer (including melanoma and sarcoma). In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), esophageal cancer, or colorectal cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma (NHSCC). In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is skin cancer treatable with Mohs surgery.

[0113] In another aspect, there is provided a method of treating cancer, the method comprising: (a) detecting one or more optical signals in a cancer patient in need of cancer treatment, the cancer patient having been administered an effective dose of a block copolymer or pharmaceutical composition disclosed herein, wherein the detected optical signals indicate the presence of a cancerous tumor; and (b) removing the cancerous tumor, thereby treating the cancer. Includes:

[0114] In some embodiments, the method further comprises imaging the body cavity of the cancer patient, or imaging the cancerous tumor or a slice or sample thereof (e.g., fresh or formalin-fixed), optionally by back-table fluorescence-guided imaging after removal from the patient. In some embodiments, the method of treating cancer further comprises imaging the cancerous tumor after removal to ensure a clean border. In some embodiments, a clean border is indicated by the absence of tumor in the wound bed. In some embodiments, a clean border is indicated when no fluorescence is detected in the sample or in the wound bed. In some embodiments, a clean border indicates that the entire cancerous tumor has been removed. In some embodiments, a clean border indicates that all of the cancer has been removed.

[0115] In another aspect, a method of minimizing cancer recurrence for at least five years, the method comprising: (a) detecting one or more optical signals in a cancer patient in need thereof who has been administered an effective dose of a block copolymer or pharmaceutical composition disclosed herein, wherein the detected optical signals indicate the presence of a cancerous tumor; and (b) treating the cancer to minimize recurrence if one or more optical signals are detected. Includes:

[0116] In another aspect, there is provided a method for detecting a cancerous tumor, the method comprising: (a) detecting one or more optical signals in a cancer patient in need of cancer treatment who has been administered an effective dose of a block copolymer or pharmaceutical composition disclosed herein, wherein the presence of a tumor indicates a recurrence of the cancer; and (b) Treating the recurrence of cancer Includes:

[0117] In some embodiments, the tumor is of cancer origin. In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, esophageal cancer, colorectal cancer, brain, or skin (including melanoma and sarcoma). In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), esophageal cancer, or colorectal cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is prostate cancer.

[0118] In some embodiments, the method further comprises imaging the tumor using an intraoperative or endoscopic camera. In some embodiments, the intraoperative camera is a near-infrared (NIR) camera. In some embodiments of the methods disclosed herein, the intraoperative or endoscopic camera is an indocyanine green compatible camera.

[0119] dosage In some embodiments, the pharmaceutical composition is administered to a patient in need thereof. In some embodiments, the patient in need thereof is a mammal. In some embodiments, the patient in need thereof is a human. In some embodiments, the mammal is not a human. In some embodiments, the mammal is a dog, cat, cow, pig, rabbit, or horse. In some embodiments, the mammal is a dog or cat. In some embodiments, the mammal is a cat. In some embodiments, the mammal is a horse. In some embodiments, the mammal is a cow. In some embodiments, the mammal is a pig. In some embodiments, the mammal is a rabbit. In some embodiments, the mammal is a dog.

[0120] The block copolymer of Formula (II) of the present disclosure, or a hydrate, solvate, tautomer, or pharmaceutically acceptable salt thereof, may be in the form of a composition suitable for administration to a subject. Generally, such a composition is a "pharmaceutical composition" comprising the block copolymer of Formula (II) or a hydrate, solvate, tautomer, or pharmaceutically acceptable salt thereof and one or more pharmaceutically or physiologically acceptable diluents, carriers, or excipients. In certain embodiments, the block copolymer of Formula (II) or a hydrate, solvate, tautomer, or pharmaceutically acceptable salt thereof is present in a therapeutically acceptable amount. Pharmaceutical compositions can be used in the methods of the present invention. Thus, for example, pharmaceutical compositions can be administered to a subject ex vivo or in vivo to carry out the therapeutic and prophylactic methods and uses described herein.

[0121] In some embodiments, the pharmaceutical composition is administered about 1 to 2 weeks before surgery. In some embodiments, the pharmaceutical composition is administered about 2 weeks before surgery. In some embodiments, the pharmaceutical composition is administered about 1 week before surgery. In some embodiments, the pharmaceutical composition is administered about 16 hours to about 80 hours before surgery. In some embodiments, the pharmaceutical composition is administered about 24 hours to about 32 hours before surgery. In some embodiments, the pharmaceutical composition is administered about 16 hours to about 32 hours before surgery. In some embodiments, the pharmaceutical composition is administered about 1 hour to about 5 hours before surgery. In some embodiments, the pharmaceutical composition is administered about 3 hours to about 9 hours before surgery.

[0122] In some embodiments, the pharmaceutical composition is administered at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 24 hours, at least 28 hours, at least 32 hours, at least 80 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 1 week, or at least 2 days before surgery.

[0123] In some embodiments, the pharmaceutical composition is administered about 1 to 2 weeks before imaging of the tumor. In some embodiments, the pharmaceutical composition is administered about 2 weeks before imaging of the tumor. In some embodiments, the pharmaceutical composition is administered about 1 week before imaging of the tumor. In some embodiments, the pharmaceutical composition is administered about 16 hours to about 80 hours before imaging of the tumor. In some embodiments, the pharmaceutical composition is administered about 24 hours to about 32 hours before imaging of the tumor. In some embodiments, the pharmaceutical composition is administered about 16 hours to about 32 hours before imaging of the tumor. In some embodiments, the pharmaceutical composition is administered about 3 hours to about 9 hours before imaging of the tumor. In some embodiments, the pharmaceutical composition is administered about 1 hour to about 5 hours before imaging of the tumor. In some embodiments, the pharmaceutical composition is administered about 1 hour to about 32 hours, about 2 hours to about 32 hours, 16 hours to about 32 hours, or about 20 hours to about 28 hours before imaging of the tumor.

[0124] In some embodiments, the pharmaceutical composition is administered at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 24 hours, at least 28 hours, at least 32 hours, at least 80 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 1 week, or at least 2 weeks prior to imaging of the tumor.

[0125] In some embodiments, the pharmaceutical composition of the block copolymer of Formula (II) or a hydrate, solvate, tautomer, or pharmaceutically acceptable salt thereof described herein is provided at the maximum tolerated dose (MTD) for the block copolymer of Formula (II). In other embodiments, the amount of the pharmaceutical composition of the block copolymer of Formula (II) or a hydrate, solvate, tautomer, or pharmaceutically acceptable salt thereof administered is about 10% to about 90% of the maximum tolerated dose (MTD), about 25% to about 75% of the MTD, or about 50% of the MTD. In some other embodiments, the amount of the block copolymer of Formula (II) or hydrate, solvate, tautomer, or pharmaceutically acceptable salt thereof pharmaceutical composition administered is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or higher of the MTD for the block copolymer of Formula (II), or any range derivable therein.

[0126] definition Unless otherwise stated, the following terms used in this application have the definitions given below. The use of the term "including" and other forms, such as "include," "includes," and "included," is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0127] "Pharmaceutically acceptable," as used herein, refers to a relatively non-toxic material, e.g., a carrier or diluent, that does not abrogate the biological activity or properties of the block copolymer, i.e., the material may be administered to an individual in a harmful manner without causing adverse biological effects or interacting with any of the components of the composition in which it is contained.

[0128] The term "pharmaceutically acceptable salt" refers to a form of a therapeutically active agent consisting of the cationic form of the therapeutically active agent combined with a suitable anion, or in alternative embodiments, the anionic form of the therapeutically active agent combined with a suitable cation. Handbook of Pharmaceutical Salts: Properties, Selection and Use. International Union of Pure and Applied Chemistry, Wiley-VCH, 2002; S.M. Berge, L.D. Bighley, D.C. Monkhouse, J. Pharm. Sci., 1977, Vol. 66, pp. 1-19; P.H. Stahl and C.G. Wermuth, eds., Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zuich: Wiley-VCH / VHCA, 2002. Pharmaceutical salts are generally more soluble and rapidly dissolving in gastric and intestinal fluids than non-ionic species, making them useful in solid dosage forms. Furthermore, their solubility is often a function of pH, allowing for selective dissolution in one part of the gastrointestinal tract or another, an ability that can be manipulated as an aspect of delayed- and sustained-release behavior. Also, salt-forming molecules can be in equilibrium with neutral forms, allowing for tailored passage through biological membranes.

[0129] In some embodiments, pharmaceutically acceptable salts are obtained by reacting the block copolymer of Formula (II) with an acid. In some embodiments, the block copolymer of Formula (A) (i.e., in free base form) is basic and is reacted with an organic or inorganic acid. Inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and metaphosphoric acid. Organic acids include, but are not limited to, 1-hydroxy-2-naphthoic acid; 2,2-dichloroacetic acid; 2-hydroxyethanesulfonic acid; 2-oxoglutaric acid; 4-acetamidobenzoic acid; 4-aminosalicylic acid; acetic acid; adipic acid; ascorbic acid (L); aspartic acid (L); benzenesulfonic acid; benzoic acid; camphoric acid (+); camphor-10-sulfonic acid (+); capric acid (decanoic acid); caproic acid (hexanoic acid); caprylic acid (octanoic acid); carbonic acid; cinnamic acid; citric acid; cyclamic acid; dodecyl sulfate; ethane-1,2-disulfonic acid. Sulfonic acid; ethanesulfonic acid; formic acid; fumaric acid; galactaric acid; gentisic acid; glucoheptonic acid (D); gluconic acid (D); glucuronic acid (D); glutamic acid; glutaric acid; glycerophosphoric acid; glycolic acid; hippuric acid; isobutyric acid; lactic acid (DL); lactobionic acid; lauric acid; maleic acid; malic acid (-L); malonic acid; mandelic acid (DL); methanesulfonic acid; naphthalene-1,5-disulfonic acid; naphthalene-2-sulfonic acid; nicotinic acid; oleic acid; oxalic acid; palmitic acid; pamoic acid; phosphoric acid; proprionic acid; pyroglutamic acid (-L); salicylic acid; sebacic acid; stearic acid; succinic acid; sulfuric acid; tartaric acid (+L); thiocyanic acid; toluenesulfonic acid (p); and undecylenic acid.

[0130] In some embodiments, the block copolymer of Formula (II) is prepared as a chloride, sulfate, bromide, mesylate, maleate, citrate, or phosphate salt.

[0131] In some embodiments, pharmaceutically acceptable salts are obtained by reacting a block copolymer of Formula (II) with a base. In some embodiments, the block copolymer of Formula (II) is acidic and reacts with a base. In such situations, the acidic protons of the block copolymer of Formula (II) are replaced with metal ions, such as lithium, sodium, potassium, magnesium, calcium, or aluminum ions. In some cases, the block copolymers described herein coordinate with organic bases, such as, but not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, meglumine, N-methylglucamine, dicyclohexylamine, and tris(hydroxymethyl)methylamine. In other cases, the block copolymers described herein form salts with amino acids, such as, but not limited to, arginine and lysine. Acceptable inorganic bases used to form salts with block copolymers containing acidic protons include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium hydroxide, and lithium hydroxide. In some embodiments, the block copolymers provided herein are prepared as sodium, calcium, potassium, magnesium, melamine, N-methylglucamine, or ammonium salts.

[0132] Reference to pharmaceutically acceptable salts should be understood to include solvent addition forms. In some embodiments, solvates contain stoichiometric or non-stoichiometric amounts of solvent and are formed during the crystallization process with a pharmaceutically acceptable solvent, such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of the compounds described herein are conveniently prepared or formed during the processes described herein. In addition, the compounds provided herein optionally exist in unsolvated as well as solvated forms.

[0133] The methods and formulations described herein include the use of N-oxides (where appropriate) of block copolymers having the structure of Formula (II), or pharmaceutically acceptable salts, as well as active metabolites of these compounds that have the same type of activity.

[0134] In another embodiment, the compounds described herein are labeled isotopically (e.g., with a radioisotope) or by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0135] The compounds described herein include isotopically labeled compounds that are identical to those listed in the various formulas and structures presented herein, except for the fact that one or more atoms have been replaced by an atom having an atomic mass or mass number different from that normally found in nature. Examples of isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, iodine, and phosphorus, such as, for example: 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, 36 Cl, 123 I, 124 I, 125 I, 131 I, 32 P and 33 In one embodiment, the compounds described herein are isotopically labeled, e.g., with a radioactive isotope, e.g., 3 H and 14 Incorporation of C is useful in drug and / or substrate tissue distribution assays. In one aspect, substitution with isotopes such as deuterium affords certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements.

[0136] As used herein, the terms "pH-responsive system," "pH-responsive composition," "micelle," "pH-responsive micelle," "pH-sensitive micelle," "pH-activatable micelle," and "nano-particles of pH-activatable micelles (pHAM)" are used interchangeably herein to refer to micelles containing one or more compounds that dissociate depending on pH (e.g., above or below a certain pH). As a non-limiting example, at a certain pH, the block copolymer of Formula (II) is substantially in the form of micelles. As the pH changes (e.g., decreases), the micelles begin to dissociate, and as the pH changes further (e.g., decreases further), the block copolymer of Formula (II) exists in a substantially dissociated (non-micellar) form.

[0137] As used herein, "pH transition range" refers to the pH range over which micelles dissociate.

[0138] As used herein, "pH transition value" (pH) refers to the pH at which one-half of the micelles separate.

[0139] "Nanoprobe" as used herein refers to a pH-sensitive micelle that includes an imaging labeling moiety. In some embodiments, the labeling moiety is a fluorescent dye. In some embodiments, the fluorescent dye is indocyanine green dye.

[0140] The terms "administer," "administering," "administration," and the like, as used herein, refer to methods that can be used to enable delivery of a compound or composition to a desired site of biological action. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injection (intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intratumor, or infusion), topical, and rectal administration. Those of skill in the art are familiar with administration techniques that can be utilized with the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally. In some embodiments, the compositions described herein are administered intravenously.

[0141] Terms such as "co-administration," as used herein, are intended to encompass administration of selected therapeutic agents to a single patient and are intended to include therapeutic regimens in which the agents are administered by the same or different routes of administration or at the same or different times.

[0142] The terms "effective amount" or "therapeutically effective amount," as used herein, refer to a sufficient amount of an agent or compound administered, which will relieve to some extent one or more of the symptoms of the disease or condition being treated. Results include reduction and / or alleviation of the signs, symptoms, or causes of the disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a compound disclosed herein that is required to obtain a clinically significant reduction in disease symptoms. An appropriate "effective amount" in any individual case is optionally determined using techniques, for example, a dose escalation study.

[0143] The terms "enhance" or "enhancing," as used herein, means to increase or prolong either in potency or duration a desired effect. Thus, in regard to enhancing the effect of therapeutic agents, the term "enhance" refers to the ability to increase or prolong, either in potency or duration, the effect of other therapeutic agents on a system. An "enhancing-effective amount," as used herein, refers to an amount sufficient to enhance the effect of another therapeutic agent in a desired system.

[0144] The term "subject" or "patient" includes mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees, and other ape and monkey species; domestic animals such as cows, horses, sheep, goats, and pigs; farm animals such as rabbits, dogs, and cats; and test animals, including rodents, such as rats, mice, and guinea pigs. In one aspect, the mammal is a human.

[0145] The terms "treat," "treating," or "treatment," as used herein, include alleviating, attenuating, or ameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting a disease or condition, e.g., arresting the development of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating symptoms caused by a disease or condition, or prophylactically and / or therapeutically arresting symptoms of a disease or condition.

[0146] The use of the term "or" in the claims is used to mean "and / or," although the present disclosure supports the definition that refers to alternatives only and "and / or," unless expressly indicated to refer to alternatives only or that the alternatives are mutually exclusive. Throughout this application, the term "about" is used to indicate a value that includes the standard deviation of error for the device or method being utilized to determine the value, e.g., about ±10% of the stated number, or 10% below the lower limit and 10% above the upper limit, for values ​​recited for a stated range. In accordance with long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising," for example, in the claims or specification, refer to one or more unless specifically indicated otherwise. [Example]

[0147] Example 1. Synthesis of block copolymers The block copolymers of formula (II) described herein are synthesized using standard synthetic techniques or methods known in the art.

[0148] Unless otherwise indicated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are utilized. Block copolymers are prepared using standard organic chemistry techniques, such as those described in March's Advanced Organic Chemistry, 6th Edition, John Wiley and Sons, Inc.

[0149] Some abbreviations used herein are as follows: DCIS ductal carcinoma in situ DCM: dichloromethane DMAP: 4-dimethylaminopyridine DMF: dimethylformamide DMF-DMA: N,N-dimethylformamide dimethyl acetal EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EtOAc: ethyl acetate EtOH: ethanol ICG-OSu: Indocyanine green succinamide ester MeOH: Methanol PMDETA: N,N,N',N",N"-pentamethyldiethylenetriamine TEA: Triethylamine Area under the AUC curve AUC all AUC from time=0 to the last time point (including concentration=0) AUC last Time = AUC from 0 to the last time point with a reportable concentration AUC 0~24hr AUC from 0 to 24 hours BC Breast Cancer BLS Breadloaf Slide BQL Below the limit of quantification C 10m Plasma concentration at 10 minutes C max maximum plasma concentration CNR Contrast to Noise Ratio CRC colorectal cancer EC Esophageal cancer FFPE or FF Formalin-fixed paraffin-embedded or formalin-fixed GMP: Standards for pharmaceutical manufacturing management and quality control GLP: Good Laboratory Practice GPC Gel Permeation Chromatography HNSCC head and neck squamous cell carcinoma Hr time ISR Reproducibility evaluation of measurements on real samples IV (intravenous) kg kilogram LLOQ Lower limit of assay quantification MFI Mean Fluorescence Intensity mg milligram mL milliliter μg microgram NC Not calculated NR Not reported OC ovarian cancer PK Pharmacokinetics PPV positive predictive value PrC prostate cancer ROI Region of Interest r 2 adj Coefficient of determination adjusted for sample size SEC size exclusion chromatography SOC standard of care SOP Standard Operating Procedure TBR tumor-to-background ratio T 1 / 2 Half-life T max Time to maximum concentration

[0150] A five-step process was used to synthesize the block copolymer of formula (II). Steps 1–4 were carried out in a controlled manufacturing environment. Intermediate 8 (polydibutylamine, PDBA) was synthesized by atom transfer radical polymerization (ATRP, step 4) of 3 (PEG-Br, macroinitiator), 7 ((dibutylamino)ethyl methacrylate, DBA-MA), and 4 (aminoethyl methyl acrylate hydrochloride, AMA-MA). The final step involved the preparation of compound 1 by covalently attaching 8 (the diblock copolymer backbone of PDBA) to 9 (ICG fluorophore, ICG-OSu). In step 5, all raw materials, solvents, and reagents used were either National Formulary (NF) or United States Pharmacopeia (USP) approved, except for intermediate 9 (ICG-OSu), which was supplied as GMP material. As a precaution, compound 1 was stored at −80°C ± 15°C and protected from light.

[0151] Schemes 1 and 2 provide a process flow chart followed by a detailed description of the manufacturing process.

[0152] [ka]

[0153] Step 1 Synthesis: Poly(ethylene glycol) methyl ether (PEG-OH) 1a, trimethylamine, and 4-(dimethylamino)pyridine (DMAP) in dichloromethane (CHCl) were cooled in an ice bath. α-Bromoisobutyryl bromide 1b in dichloromethane was then added dropwise to the flask while the flask was kept in the ice bath. The reaction mixture was allowed to warm to room temperature (RT) and stirred for 16 h.

[0154] Purification: The reaction mixture was then slowly added under stirring to a beaker containing a 10-fold excess volume of diethyl ether to precipitate crude product 3. The crude product was then filtered and dried in a vacuum oven. The dried crude product 3 was recrystallized five times from ethanol and dried in a vacuum oven to obtain purified 3 (PEG-Br macroinitiator). Typical yields are 40%-70%, with purities >93% (high-performance liquid chromatography [HPLC] area%).

[0155] Step 2: Recrystallization: The crude product 2-aminoethyl methacrylate hydrochloride (AMA-MA monomer), 2-propanol 4a, and ethyl acetate were combined and heated to 70 °C until the solid dissolved. The solution was filtered through a preheated Buchner funnel containing Celite. The filtered solution was allowed to cool to room temperature and then further cooled to 2–8 °C to crystallize over a period of 8–16 h. The resulting crystalline solid was warmed to room temperature, then filtered and washed three times with cold ethyl acetate. The isolated crystalline product was dried under vacuum to give purified 4, which was stored at −80 °C for use in Step 4. Typical yields were 40–70%, and purity was indicated by solubility in use tests and a sharp melting point (≤3 °C) ranging from 102–24 °C.

[0156] Step 3: Synthesis: 2-(Dibutylamino)ethanol (DBA-EtOH, 5), trimethylamine, copper(I) chloride (CuCl), and dichloromethane were combined in a flask and cooled in an ice bath. Methacryloyl chloride 6 was then added dropwise to the flask while the flask was maintained in the ice bath. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. The reaction mixture was then cooled in an ice bath and filtered. The filtrate was transferred to a separatory funnel, and the organic phase was washed twice with a saturated aqueous solution of sodium bicarbonate (NaHCO), followed by one wash with DI water. The organic phase was then dried over anhydrous sodium sulfate (NaSO), filtered, and the solvent was removed in vacuo using a rotary evaporator to yield the monomer product 7 as a liquid.

[0157] Purification: Additional CuCl was added as a stabilizer, and the product was purified by vacuum distillation. The clear to yellowish distillate 7 (DBA-MA) was transferred to an amber vial and stored at -80 °C for use in Step 4. Typical yields are 30%-60% with purities >93% (HPLC area %).

[0158] [ka]

[0159] Step 4: Synthesis: Intermediate 3 was added to a flask and dissolved in a mixture of dimethylformamide (DMF) and 2-propanol by gently heating the flask. The contents of the flask were allowed to cool to room temperature, and 4 and 7 (AMA-MA and DBA-MA monomers, respectively) were added to the solution, followed by N,N,N',N",N"-pentamethyldiethylenetriamine (PMDETA). The reaction mixture was stirred and then subjected to four freeze-pump-thaw cycles under nitrogen to remove air (oxygen). While further frozen, the reaction mixture was treated with copper(I) bromide (CuBr) and subjected to three vacuum and nitrogen flush cycles to ensure removal of trapped air. The reaction mixture was then warmed to 40 °C in an oil bath. The reaction mixture was allowed to react for an additional 16 h. Upon completion of the reaction, the mixture was diluted with tetrahydrofuran and filtered through a bed of aluminum oxide (Al2O3). The solvent was removed from the filtrate using rotary evaporation and dried under vacuum.

[0160] Purification: The dried crude product was dissolved in methanol and purified by tangential flow filtration through a 10k MWCO Pellicon® 2 Mini Filter cassette with methanol. The solvent was then removed using rotary evaporation. The purified intermediate 8 (PEO 113 -b-(DBA 80~150 -r-AMA 1~3), PDBA) was dried under vacuum and stored at -80 °C for use in Step 5. Typical yields are 60%-90% with purities >93% (HPLC area %). In some cases, the product is a mixture of conjugated and unconjugated polymer.

[0161] Step 5: Synthesis: Intermediate 8 (PDBA) was dissolved in methanol (MeOH) with the aid of a sonication bath. The methanol solution was then added to 9 (ICG-OSu). The reaction was stirred at room temperature for 16 hours while protected from light. At the end of the reaction, a 6-fold excess of acetic anhydride was added to the reaction mixture and allowed to mix for 1-1.5 hours to produce crude compound 1.

[0162] Purification: The crude product was purified by tangential flow filtration through a 10k Pellicon® 2 Mini Ultrafiltration module with methanol. The solvent of the filtered solution was removed in vacuo to yield compound 1, which was protected from light and stored at -80°C. Typical yields are >70% with a purity of NLT 95% (SEC).

[0163] Analysis: Relative molar mass distribution analysis was performed via a custom gel permeation chromatography (GPC) method using refractive index (RI) detection and two Agilent PLgel Mixed-D 300 x 7.5 mm columns. Molar mass distribution was calculated by comparing sample chromatograms to a calibration curve constructed from polystyrene standards ranging from 580 to 1,074,000 g / mol.

[0164] Example 2. Storage of Compound 1 The current presentation of Compound 1 for infusion is a 3 mg / mL green aqueous solution stored at −80° C. The vial is thawed to room temperature and then administered intravenously at 15 mg / min for Phase 1 and 30 mg / min for Phase 2.

[0165] Example 3. Stability of Compound 1 Stability data indicates that Compound 1, 3.0 mg / mL injection, is stable at -80°C under long-term storage conditions for up to 24 months (current shelf life). No significant changes were observed in assay or related substances and impurity levels, or in any of the other attributes tested at storage conditions. Updated stability results are provided in Tables 1 and 2.

[0166] [Table 1]

[0167] [Table 2]

[0168] Example 4. PK effects in humans Phase 1 study objectives Phase 1 was a single-investigator, non-randomized, open-label, single-arm, cross-sectional study to evaluate the safety, PK profile, and imaging feasibility of Compound 1 in patients with solid tumors requiring surgical resection. The primary objective of this study was to investigate the safety, PK, and feasibility of Compound 1 as an intraoperative optical contrast agent for detecting tumors and metastatic lymph nodes in solid tumors. The study was designed to determine the optimal dose range of Compound 1 for sufficient TBR and CNR of intraoperatively obtained fluorescence at 24 (±8) hours post-dose using an ICG-compatible camera and imaging device and with ex vivo specimens.

[0169] Phase 1 enrolled 30 patients with solid tumors (HNSCC, breast cancer, esophageal cancer, or colorectal cancer) with biopsy-confirmed diagnosis of their respective tumor types and scheduled for surgical removal of their tumors. The study design included a standard 3 + 3 design for a dose-escalation portion (Phase 1a; N=18 maximum) followed by a dose-escalation portion (Phase 1b; N=15). All patients received a single IV dose of Compound 1, followed by scheduled surgery approximately 24 hours after Compound 1 infusion.

[0170] Phase 1a was a dose-finding study conducted in five cohorts of three patients each. The dose levels evaluated were 0.3, 0.5, 0.8, 0.1, and 1.2 mg / kg, in that order. Dose escalation between cohorts occurred after the last patient in the previous cohort completed the 10-day safety assessment. Safety, PK, and imaging feasibility were evaluated in both the Phase 1a and Phase 1b portions of the study. Patient safety is assessed during the study and up to 10 days post-dose.

[0171] During surgery, intraoperative images of Compound 1 fluorescence were obtained from the primary tumor and metastatic lymph nodes, as well as surrounding tissues, including normal, non-cancerous tissue, using a near-infrared (NIR) camera. This could be in vivo and / or ex vivo imaging of the resected specimen. If deemed safe by the surgeon, up to 10 study-related biopsies were taken from areas with Compound 1 fluorescence that were not otherwise clinically suspicious of tumor. Multiple NIR cameras were used to assess the feasibility of imaging tumors with Compound 1.

[0172] Tumor samples were processed for histology according to standard pathology practices used in clinical cancer care. Selected histological features required for clinical decision-making provided a diagnosis for margins. Fluorescence images were collected from tumor and lymph node samples and study-associated biopsies. Margin widths and the number of positive margins were noted and correlated with the location of fluorescence within the margins. From this, correlations between Compound 1 fluorescence and histopathology were calculated.

[0173] Trends and Demographic Data All patients received a single dose of Compound 1 and completed the study. All patients were included in imaging, PK, and safety analyses.

[0174] Thirty patients with four different tumor types (HNSCC, n=13; BC, n=11 patients; CRC, n=3; EC, n=3) undergoing scheduled surgery received a single dose of ONM-100 24 (±8) hours before their planned surgery (Table 3).

[0175] In Phase 1a, a total of 3 male and 12 female patients were enrolled in the study, with ages between 34 and 80 years and body mass indexes between 17.4 and 37.1 kg / m². All patients were Caucasian (Caucasian race); none were of Hispanic or Latino ethnicity. A total of 8 patients had a diagnosis of HNSCC and 7 patients had a diagnosis of BC.

[0176] In Phase 1b, a total of 5 male and 10 female patients were enrolled in the study, with ages between 45 and 85 years and body mass indexes between 18.9 and 39.4 kg / m². All patients were Caucasian (Caucasian race); none were of Hispanic or Latino ethnicity. A total of 5 patients were diagnosed with HNSCC, 4 patients with BC, 3 patients with CRC, and 3 patients with EC. The mean age in Phase 1b (68 years) was higher than in Phase 1a (58 years).

[0177] [Table 3]

[0178] Pharmacokinetic results Study Design: A single IV dose of Compound 1 was administered as a 1- to 5-minute IV infusion to patients in five cohorts of three patients per cohort (0.1, 0.3, 0.5, 0.8, and 1.2 mg / kg) in Phase 1a and to 15 patients at the 1.2 mg / kg dose in Phase 1b. Patient demographics, including tumor type, are presented in Tables 4 and 5 for Phase 1a and Table 5 for Phase 1b. Intertek Pharmaceutical Services (San Diego, CA) determined plasma concentrations of Compound 1 using a validated direct fluorescence reader assay. Pacific BioDevelopment (Davis, CA) performed the PK analysis.

[0179] Sample Collection: Blood samples were collected pre-infusion and 10 minutes and at 0.5, 1, 3, 8, 24, 48, 72, and 240 hours post-infusion.

[0180] [Table 4]

[0181] [Table 5]

[0182] Analysis: Plasma concentration versus time profiles were generated for each patient. Pharmacokinetic parameters were predicted using Phoenix WinNonlin (version 8.0). Per SOP, concentrations reported as BQL were set to 0, except for the 0.5 hour sample for subject #ON1102, which was set to LLOQ / 2 (a value of 5 μg / mL was used for parameter calculation), as were the 24 and 48 hour samples.

[0183] The prediction parameter is C max , T max , T 1 / 2 , AUC last , AUC all and AUC 0~24hr If there are less than three data points at the end of the curve, the program 1 / 2 (NC) was not calculated. The coefficient for determining the terminal gradient was 0.8, T 1 / 2 The AUCs extrapolated to infinity were not reported for any data set. In all cases, the extrapolated AUC (%) was greater than 20%, and therefore, AUC inf The concentration at 10 minutes, or the first measurement point (C10m), was also reported for each patient.

[0184] The area under the plasma concentration versus time curve from dosing to the last time point was calculated using the measured concentration (AUC last ) were predicted by the linear trapezoidal method. The last three or more time points were used to predict the elimination rate constant (λz), which was used to calculate the area under INF) until the terminal half-life (T 1 / 2 ) and AUC were predicted from the following equations: T 1 / 2 =ln(2) / λz AUC INF =AUC 0~t +C t / λz (In the formula, C t is the last measurable concentration).

[0185] Phase 1a Patient demographic data for Phase 1a are presented in Table 4. Individual plasma concentrations are presented in Table 6. Individual pharmacokinetic parameter estimates and group summary statistics are presented in Table 6. Plots of mean plasma concentrations (log and linear) versus time are presented in Figures 1A-1B.

[0186] Compound 1 was not measurable in any subject samples following the 0.1 mg / kg dose.

[0187] Exposure was dose-related. C max , AUC last , AUC all and AUC 0~24hr The higher the dose, the higher the concentration and AUC 10 minutes after administration. 0~24hr are plotted against dose in Figures 2 and 3, respectively. The plots show the results of linear regressions performed on the parameter versus dose data. Data from the 0.1 mg / kg dose group, where all plasma values ​​were reported as BQLs, are excluded from these plots. The study was not powered to perform statistical analysis for dose proportionality. However, the linear regressions indicate a strong correlation between exposure and dose.

[0188] average C 10 The values ​​were 12.0, 17.3, 19.8, and 31.7 μg / mL for the 0.3, 0.5, 0.8, and 1.2 mg / kg doses, respectively. Mean AUC 0~24hrThe mean terminal half-life values ​​were quantifiable only for the 0.8 and 1.2 mg / kg dose groups, and were 79.0 and 36.5 hours, respectively.

[0189] [Table 6] TIFF0007752110000017.tif16164

[0190] Phase 1b Patient demographic data for Phase 1b are presented in Table 7. Individual plasma concentrations for patients are shown in Table 8. Individual pharmacokinetic parameter estimates and group summary statistics are presented in Table 9. Plots of individual plasma concentrations (log and linear) versus time are presented in Figures 4A-4B.

[0191] average C 10m was 33.2 μg / mL, and the mean AUC 0~24hr The mean terminal half-life was 46.4 hours.

[0192] [Table 7]

[0193] [Table 8]

[0194] [Table 9]

[0195] Combined Phase 1a and Phase 1b data Mean plasma concentrations were plotted versus time by dose group for all patients in Phase 1a and Phase 1b combined and are presented in Figure 5A (log plot) and Figure 5B (linear plot). 10m ) and AUC 0~24hr Plots of dose versus dose are plotted in Figures 6 and 7. The data support the observation made based on the Phase 1a data that exposure was proportional to dose.

[0196] Individual patient pharmacokinetic parameters and summary statistics, organized by tumor type, for all patients in Phase 1a and Phase 1b treated at 1.2 mg / kg are presented in Table 10. There were no apparent differences in predicted pharmacokinetic parameters based on tumor type. 10m The values ​​ranged from 31.2 to 35.5 μg / mL, and the AUC 0~24hr Values ​​ranged from 585 to 677 μg-h / mL.

[0197] The mean plasma concentration versus time plots for each tumor type are shown in Figure 8A (log plot) and Figure 8B (linear plot). These plots illustrate that there were no clear differences in Compound 1 pharmacokinetics among the tumor types tested. Individual plasma concentration versus time plots for each tumor type are presented in Figures 8C-8F (log plot) and Figures 8G-8J (linear plot).

[0198] [Table 10]

[0199] Although the study was not powered to perform statistical analysis for dose proportionality, C10 appeared to be dose proportional at 0.3 to 1.2 mg / kg (Figure 6), with AUC 0~hr appears to be dose-proportional to 1.2 mg / kg (Figure 7).

[0200] Example 5. Fluorescence image acquisition and image processing Intraoperative images and videos of "open surgery" were obtained using either the NOVADAQ SPY Elite or the SurgVision Explorer Air. The camera distance to the tumor was approximately 20 cm for the Explorer Air and 30 cm for the NOVADAQ SPY, as instructed in the manual. The NOVADAQ SPY camera can only generate fluorescent video, which can be converted into images during post-processing. The settings for raw data acquisition for this camera were fixed. For the Explorer Air, an attempt was made to use the same settings (exposure time and gain) for each patient to allow direct comparison between images obtained from both systems; however, adjustments were required depending on the amount of visible fluorescence during surgery and, in some cases, due to saturation of the camera system. In some patients, when open surgery was not performed, an Olympus NIR laparoscope and Da Vinci Firefly camera system were used. The systems were used according to the manufacturer's instructions.

[0201] Initially, pre-excision fluorescent images and / or videos of the tumor and surrounding area were generated. After surgical excision, images of the wound bed were obtained. If fluorescent areas were visible within the wound bed, biopsies were taken, if possible, and the excised specimens were imaged on all sides on the back table in the operating room. If applicable, lymph nodes were imaged in situ and on the back table, and then the wound bed was imaged again at lymph node dissection.

[0202] Designated imaging study staff performed the fluorescence imaging. The pre-excision images were blinded to the surgeon to avoid any bias against standard surgery, but the surgeon was able to view a second monitor for the white light image while performing the surgery. The surgeon was assisted with imaging at the wound bed and back table. During the imaging procedure, ambient light in the operating room was switched off to prevent possible interaction with the fluorescence imaging procedure itself.

[0203] Images were processed using Fiji (ImageJ, version 2.0.0). Images were scaled for each patient based on maximum and minimum fluorescence intensity per pixel.

[0204] Intraoperative back-table image acquisition and postoperative image acquisition During all phases of tissue processing, samples were stored in the dark whenever possible to prevent possible photobleaching of the contrast agent.

[0205] Immediately after resection, the entire specimen was imaged (intraoperative back-table imaging) using both the designated intraoperative camera system and the LI-COR PEARL® Trilogy system within a maximum of 60 minutes after surgical resection of the specimen, covering all six resection planes (e.g., anterior, posterior, lateral, medial, caudal, and cranial). Combined imaging time with both devices was a maximum of 5 minutes. The specimen was marked with blue and black ink to mark the resection planes. The two-ink restriction did not affect the SOC for tissue processing by the pathologist, but if a third ink color was required, green ink was used to define the intended additional pathological resection margins.

[0206] The timing of postoperative tissue slice imaging was adapted to accommodate differences in SOC for sample processing of different tumor types. Briefly, BC samples were freshly sectioned on the day of surgery and then formalin-fixed, while other tumor types were sectioned after formalin fixation of radical resection specimens 1–3 days after surgery. Typically, surgical specimens were serially sectioned into ±0.5 cm thick tissue slices. For orientation purposes, white-light photographs were taken during and immediately after sectioning. After sectioning, fluorescence imaging of both sides of each tissue slice was performed in a light-blocking environment (LI-COR PEARL® Trilogy system). Thus, BC slices were imaged approximately 120 min after resection, while other tumor types were sectioned and imaged at a later date after resection and formalin fixation.

[0207] Each BLS was formalin-fixed overnight in 4% paraformaldehyde / phosphate-buffered saline. A pathologist then macroscopically sampled a portion of the BLS (FFPE-embedded) for further analysis according to the SOC and for preparation of 4 μm slices for hematoxylin and eosin (H / E) staining to delineate tumor tissue for histopathological correlation. Additional FFPE blocks could be embedded based on fluorescent imaging of the BLS, in addition to the pathologist's traditional macroscopic visual inspection of the SOC. A standardized workflow was implemented to cross-correlate the final histopathological results with the recorded fluorescent images of the tissue slices of interest. After 7–14 days, the FFPE blocks were scanned using an Odyssey Flatbed Scanner (LI-COR Bioscience).

[0208] Example 6. Histological correlation After the SOC pathology procedures were performed (approximately 7–10 days for Phase 1a and 7–14 days for Phase 1b), H / E slices were reviewed and discussed with a board-certified pathologist specialized in each tumor type.

[0209] Example 7. Postoperative fluorescence measurement Adobe Illustrator and Fiji (ImageJ) were used to create correlations between H / E slices and fluorescence images (i.e., breadloaf slices or BLS). Based on histopathological results, regions of interest (ROIs) containing tumor and background were precisely and manually drawn, and for each patient, CNR was calculated for each LI-COR PEARL image of a separate BLS. The median CNR was calculated based on all available BLS containing tumor. Fluorescence measurements were performed using Fiji (ImageJ) for the following: Mean fluorescence intensity (MFI; fluorescence intensity per pixel) Contrast (MFI of tumor tissue) MFI of tissues that do not contain noise tumors (e.g., healthy muscle, fibrosis, fat) Standard deviation of noise CNR (Contrast to Noise Ratio): CNR = (Fluorescence (tumor) - Fluorescence (normal tissue)) / Standard Deviation Fluorescence (normal tissue) · TBR (tumor-to-background fluorescence ratio): TBR = Fluorescence (tumor) / Fluorescence (normal tissue)

[0210] Intraoperative fluorescence measurement Macroscopic correlations were made between the visible white light and corresponding fluorescence images of the tumor area. After drawing ROIs containing the macroscopic tumor and ROIs containing the background, the MFIs of both the tumor and background areas were calculated. Following the calculations described above, the fluorescence ratios (CNR, TBR) were calculated for each patient (three measurements per patient).

[0211] Example 8. Statistical Methods The feasibility evaluation of Compound 1 for intraoperative imaging of solid tumors and lymph node metastases included quantification of the fluorescence signal CNR, sensitivity, and localization pattern of Compound 1 fluorescence. Furthermore, the safe dose range corresponding to a sufficient CNR was calculated by combined evaluation of intraoperative in vivo and ex vivo fluorescence signals (NOVADAQ imaging system) together with ex vivo examinations (e.g., histology, NIR flatbed scanning).

[0212] Example 9. Patient Demographics and Sample Characteristics To evaluate the feasibility of tumor-independent imaging using Compound 1, 15 additional patients with four different tumor types (HNSCC, BC, EC, or CRC) were administered Compound 1 (1.2 mg / kg) at the optimal dose selected from Phase 1a in Phase 1b. Patients in Phase 1b had HNSCC (n=5), BC (n=4), EC (n=3), and CRC (n=3). Sample characteristics are presented in Table 11.

[0213] [Table 11] TIFF0007752110000023.tif185170

[0214] Example 10. Fluorescence Imaging Results Phase 1a Fluorescence imaging results for the completed Phase 1a dose-escalation portion of the Phase 1 study are available for all 15 patients; three patients each in Cohort 1 (0.3 mg / kg), Cohort 2 (0.5 mg / kg), Cohort 3 (0.8 mg / kg), Cohort 4 (0.1 mg / kg), and Cohort 5 (1.2 mg / kg), and for more than 15 patients in Phase 1b, 1.2 mg / kg.

[0215] Fluorescence images Intraoperative (Figure 9A) and postoperative (Figure 9B) images from three patients dosed in Cohort 2 (0.5 mg / kg) and Cohort 5 (1.2 mg / kg) are presented. In Cohort 2, patients ON1104 and ON1106 had HNSCC, and patient ON1105 had breast cancer. In Cohort 5, patients ON1113 and ON1114 had HNSCC, and patient ON1115 had breast cancer.

[0216] Intraoperative imaging is defined as a combination of in vivo imaging performed within the time frame of surgery and back-table imaging of the entire specimen. The feasibility of intraoperative tumor imaging with Compound 1 was clearly demonstrated in all eight patients with HNSCC who received Compound 1 at doses between 0.1 and 1.2 mg / kg. Two of the tumors in seven BC patients were visualized with Compound 1. The remaining five BC tumors were deep-seated, surrounded by normal tissue, and not visible intraoperatively by fluorescent imaging with Compound 1. This is not surprising given the limited tissue penetration of NIR imaging. Importantly, none of these five BC tumors had positive margins. These results clearly demonstrate the feasibility of intraoperative imaging with Compound 1 in HNSCC and BC.

[0217] Postoperative tissue sample imaging clearly demonstrated the feasibility of Compound 1 imaging in tumor samples from all 15 patients. These images of Compound 1 show a sharp boundary between brightly fluorescent and darkly colored areas (Figures 10A, 15, and 16). The necrotic tumor core shows no fluorescence. For all patients, the fluorescent areas corresponded to H / E images with marked regions of interest. High tumor-to-background fluorescence ratios (CNR, TBR) were observed from areas identified as tumor or normal based on histopathological correlation. Similar images were obtained for all 15 patients in Phase 1a at each dose level tested.

[0218] Phase 1a Mean Fluorescence Intensity - Primary Tumor Intraoperative imaging In Phase 1a, all (8 of 8) HNSCC patient tumors and 2 of 7 BC patient tumors (patients with macroscopic tumors visible in vivo) showed Compound 1 fluorescence in vivo (dose range 0.1-1.2 mg / kg). In all of these patients, MFI from tumor tissue was above that from surrounding normal tissue.

[0219] Intraoperative fluorescence intensity cannot be standardized or compared between patients or dose levels due to the unique presentation of each surgical environment. Multiple variables, such as camera angle, camera-to-tissue distance, tumor location, and coverage by other tissues or fat, affect the absolute value of the fluorescence signal. Therefore, the ratio of in vivo fluorescence from tumor and non-tumor tissues was calculated for each patient. As shown in Figure 11A for CNR and Figure 11B for TBR, intraoperative TBR and CNR values ​​were high for all patients. This indicates a clear boundary in fluorescence intensity between tumor and normal tissue for each individual operation, a key factor that can potentially aid surgeons in real-time visualization of tumors during surgical resection. These ratios were variable and did not show any systematic increase or decrease with dose.

[0220] Postoperative imaging To correlate Compound 1 fluorescence with histopathological findings in tumor and normal tissues, fluorescence images of Compound 1 were captured from post-operative specimens (BLS specimens) prepared at each step of the standard pathology examination. Fluorescence intensities were compared across multiple specimens using a LI COR PEARL experimental camera capable of localizing imaging and fluorescence quantification.

[0221] Figure 12A shows the MFI of tumor and normal tissue regions confirmed by histology for multiple BLS selected by standard pathology examination for all 15 patients dosed at five dose levels (fresh samples from BC patients and formalin-fixed (FF) samples from NHSCC patients). Tumor MFI increased with dose. Normal tissue MFI also increased with dose. When plotted against each patient's plasma concentration at 10 minutes (Figure 12B), the MFI from histology confirmed that tumor and normal tissue samples showed clear boundaries (no overlap) for each patient (n = 15). This is a key factor important for real-time image-guided surgery, helping surgeons delineate tumors from background tissue. Similar to dose, MFI increased with increasing initial plasma concentration. These figures also demonstrate the absence of systematic trends in the fluorescent signal between formalin-fixed (FF) vs. fresh tissue or HNSCC vs. BC tissue.

[0222] Similar to intraoperative imaging, TBR (Figure 13A) and CNR (Figure 13B) calculated using postoperative fluorescence from tumor and normal regions confirmed by histology showed high variability and remained relatively constant with dose.

[0223] Phase 1a Summary In 15 patients undergoing surgery for single-agent BC or HNSCC cancer, intraoperative and postoperative fluorescence imaging was performed using open-field and closed-field NIR cameras after a single intravenous dose of Compound 1 administered 24 + 8 hours before surgery. Five different dose levels were evaluated, ranging from 0.1 to 1.2 mg / kg. These data demonstrate the feasibility of imaging tumors with Compound 1 in all HNSCC and BC patients. Compound 1 imaging was possible using multiple NIR cameras detecting ICG. For each patient, the MFI was well defined between tumor and normal tissues. The MFI for both tumor and normal tissues increased slightly over the dose range evaluated. The fluorescence ratios (CNR and TBR) were variable but high, further illustrating the sharp boundary between tumor and normal tissue fluorescence. The CNR and TBR did not show any systematic increase or decrease with dose and were very similar for BC and HNSCC tumors.

[0224] For the Phase 1b portion of the study, the highest dose (1.2 mg / kg) from the Phase 1a portion of the Phase 1 study was selected for further evaluation of the safety, PK, and imaging feasibility of Compound 1. In the Phase 1b portion of the study, 15 additional patients with four tumor types (BC, HNSCC, CRC, and EC) received Compound 1 (1.2 mg / kg) with a surgery / imaging window of 24 ± 8 hours post-dose.

[0225] The selection of the 1.2 mg / kg dose level of Compound 1 for the Phase 1b portion of the study was based on the following results from the Phase 1a portion: In the Phase 1a study, the safety profile was comparable at all dose levels tested, and higher doses did not raise any specific safety concerns or trends. Plasma exposure of Compound 1 increased proportionally with dose. Mean fluorescence intensity increased with plasma exposure of Compound 1. CNR and TBR values ​​were variable but remained high (ranging from 2 to 5) and did not decrease with dose for both in vivo tumor and postoperative sample fluorescence. These data support the use of higher fluorescence intensity along with the highest possible dose / exposure to evaluate imaging feasibility using additional tumor types and endoscopic cameras, as well as other potentially challenging scenarios, such as tumors covered by normal tissue, tumor locations with anatomical challenges, ductal carcinoma in situ, multifocal tumors, and small lymph node metastases.

[0226] In the Phase 1b portion of the study, the highest dose (1.2 mg / kg) from the Phase 1a portion of the Phase 1 study was selected to evaluate the safety, PK, and imaging feasibility of Compound 1. In the Phase 1b portion of the study, 15 additional patients with four tumor types (BC, HNSCC, CRC, and EC) received Compound 1 (1.2 mg / kg) at the time of surgery / imaging 24±8 hours post-dose.

[0227] Fluorescence images Compound 1 fluoresced intraoperatively in tumors in 10 / 15 patients, including 5 of 5 HNSCC, 3 of 4 BC, and 2 of 3 CRC. One deep rectal tumor and one deep BC tumor could not be visualized intraoperatively, due to limited penetration depth, which is not surprising for NIR imaging. Three intraluminal EC tumors were not detected by extraluminal imaging (one EC patient had a pathological complete response). As in Phase 1a, compound 1 fluorescence detected all positive margins in BC and HNSCC patients in Phase 1b. None of the intraluminal or deep tumors had positive margins on final pathology examination. As in Phase 1a, compound 1 fluoresced postoperatively in tissue slices from all patients and tumor types (including 2 of 3 EC patients with viable tumors). Postoperative images clearly show a sharp boundary between the bright fluorescent and blue / dark areas.

[0228] Phase 1b confirmed imaging feasibility in BC and HNSCC (similar to Phase 1a) and demonstrated imaging feasibility in other solid tumors with similar sharp boundaries between tumor and normal tissue.

[0229] Phase 1a and Phase 1b Mean Fluorescence Intensity - Primary Tumor Intraoperative imaging In the following analyses, data from all patients dosed at 1.2 mg / kg in Phase 1a and Phase 1b were combined. A total of 18 patients with HNSCC (n=7), BC (n=5), EC (n=3), and CRC (n=3) received Compound 1 at 1.2 mg / kg in Phase 1a and Phase 1b.

[0230] Intraoperative MFI, CNR, and TBR were calculated for tumors in patients where intraoperative imaging was possible (tumors from 11 of 18 patients, see Table 15). Intraoperative CNR and TBR values ​​were high for all patients, indicating a clear demarcation of fluorescence intensity between tumor and normal tissue for each operation. This is an important key factor that could potentially assist surgeons in delineating tumor from background in real time during surgical resection. These CNR and TBR results also demonstrate that the Phase 1b results are confirmation of the Phase 1a results.

[0231] Summary of Phase 1b Results Data from Phase 1b clearly demonstrate that compound 1 was well tolerated at a dose of 1.2 mg / kg and enabled fluorescent tumor visualization both intra- and postoperatively in BC, HNSCC, CRC, peritoneal metastases, and possibly EC (as demonstrated by postoperative imaging), supporting a tumor-independent mechanism of action of compound 1 for imaging of solid whole tumors. Peritoneal metastases were visualized in two patients using Compound 1 (NOVADAQ, Olympus, and PEARL) and extraluminal CRC (NOVADAQ). Ductal carcinoma in situ (DCIS) in patients with BC can be detected by Compound 1 both in vivo and post-operatively, indicating a positive direction for intraoperative guidance and decision-making. Lobular carcinoma (and lobular carcinoma in situ) was detected with compound 1 in patients with BC. Margin assessment on freshly excised specimens using Compound 1 appears possible (both for whole specimens and BLS).

[0232] The value of intraoperative imaging of EC using Compound 1 could not be assessed due to the fact that no images could be collected intraoperatively due to the lack of sensitivity of the minimally invasive camera system. However, EC tissue slices were visualized with Compound 1 imaging using a LI-COR Pearl camera. Optimizing Compound 1 dosing / schedule for imaging, as well as improvements in camera technology, may overcome this limitation.

[0233] The Phase 1b portion of the Phase 1 study further confirmed the imaging feasibility of Compound 1 using multiple NIR cameras designed to detect ICG.

[0234] Intraoperative fluorescence imaging with Compound 1 is clinically feasible at a dose of 1.2 mg / kg for both the SurgVision Open Air and NOVAQ SPY Elite fluorescence cameras.

[0235] Intraoperative visualization of tumors with Compound 1 using an Olympus Fluorescent Laparoscope and a DaVinci Robot with a firefly camera was difficult due to the lower sensitivity of both cameras compared to SurgVision and NOVADAQ SPY. Higher doses may be required for optimal imaging performance.

[0236] Example 11. Lymph node imaging After lymph node dissection, lymph nodes were identified by the attending pathologist and collected, if present. After collection, single lymph nodes were imaged using PEARL imaging before further processing. Images were processed using ImageJ (FiJi). Fluorescent images were reviewed by two independent investigators who were blinded to the histology for the presence of fluorescence. A pathologist who was blinded to the fluorescent images assessed whether lymph nodes were positive for tumor invasion or isolated tumor cells based on H / E staining.

[0237] Results per patient are presented in Table 12. Of 403 available lymph nodes from patients undergoing lymphadenectomy across four tumor types, 64 contained pathology-confirmed tumors (35 from a single patient), and Compound 1 fluoresced in 30 of these lymph nodes. Compound 1 did not fluoresce correctly in 293 of 339 pathology-negative lymph nodes.

[0238] [Table 12]

[0239] Overall performance characteristics are presented in Table 13. Overall sensitivity of compound 1 = (true positives) / (true positives + false negatives) = 30 / (30 + 34) = 0.47. Overall specificity for compound 1 = (true negatives) / (false positives + true negatives) = 293 / (46 + 293) = 0.86.

[0240] [Table 13]

[0241] Accurate intraoperative detection of metastatic lymph nodes is a critical, unmet need and technically challenging. It is hypothesized that at imaging times ≥ 24 hours, nonspecific fluorescence is likely present in lymph nodes due to spillover of primary tumor fluorescence into the lymph nodes. The low sensitivity may be due to the relatively low amount of Compound 1 in metastatic lymph nodes due to the smaller size (i.e., less absolute fluorescence) of lymph nodes compared to the primary tumor. Therefore, higher doses at earlier imaging times may provide improved diagnostic performance for fluorescent imaging of Compound 1 in primary tumors and metastatic lymph nodes.

[0242] Example 12. Fluorescence Imaging of Compound 1—Clinical Utility Fluorescence imaging with Compound 1 was possible for all patients with viable tumors (29 of 30 patients) and for all four tumor types evaluated (HNSCC, BC, CRC, or EC) (Figures 15 and 16). Intraoperatively (within 1 hour of surgery, combined in vivo and back-table imaging), all 13 HNSCC tumors, 5 of 11 superficial BC tumors, and 2 of 3 CRC tumors could be visualized with Compound 1 fluorescence. Six of 11 deep-seated BC tumors, 2 of 3 intraluminal EC tumors (one of the three EC tumors was confirmed to have a pathological complete response), and 1 of 3 CRC tumors (distant rectal tumor) could not be visualized. The absence of intraoperative fluorescence in some of these settings is likely due to limited NIR penetration depth when tumors are covered by normal tissue, the low sensitivity of current robotic and endoscopic cameras, optimal dose / schedule, and physical challenges to reach certain anatomical locations. Notably, none of these intracavitary or deep tumors had positive margins on final histopathological examination.

[0243] Post-operatively, all tumors, regardless of tumor type or volume, were fluorescent by standard fluorescent post-operative workflow analysis, while none of the healthy tissue samples were fluorescent.

[0244] 11A-11B and the quantitative fluorescence data clearly demonstrate that tumor fluorescence is well demarcated from background fluorescence. This ability of Compound 1 to help visualize tumors with sharp delineation from normal tissue for the four tumor types evaluated across multiple patients confirms the tumor-independent imaging feasibility of Compound 1 for image-guided surgery in solid tumors.

[0245] Fluorescence detection of tumor-positive margins Of the 24 total patients (HNSCC: 13; BC: 11), 9 patients (HNSCC: 6; BC: 3) had histologically confirmed tumor-positive surgical margins that were not detected during SOC surgery. Fluorescence-guided margin assessment was performed for each patient. Compound 1 imaging visualized these surgical margins in all patients, resulting in 100% sensitivity. All fluorescence-negative surgical margins correlated with final histopathological assessment (no false negatives). There were 5 false positives out of 15 (67% specificity), of which the tissue in which fluorescence was detected was not confirmed to be tumor by histopathological assessment. Five out of 14 (36%) patients had fluorescent tissue that was negative for tumor (PPV: 64%).

[0246] By tumor type, the sensitivity and specificity of Compound 1 for detecting patients with positive margins were 100% and 75%, respectively, for BC and 100% and 57% for HNSCC. Compound 1 fluorescence was negative in two of three EC patients and one of three CRC patients for whom histological margin status was available and negative. These initial data suggest tumor-independent diagnostic performance and demonstrate the feasibility of using Compound 1 imaging for accurate detection of tumor-positive margins during surgery.

[0247] Table 14 summarizes the pathology versus fluorescence correlation for margin status for individual patients for all four tumor types.

[0248] [Table 14] TIFF0007752110000027.tif96166

[0249] False positive fluorescence edge In three HNSCC patients (ON1108, ON1114, ON1121) and two BC patients (ON1123 and ON1151), false-positive fluorescent margins were detected that did not contain tumor on final histopathological examination. In the HNSCC patients, false-positive fluorescence corresponded to nerve tissue in one patient, salivary gland in another, and a fluorescent spot on the margin of the specimen in a third patient. In the two BC patients, false-positive fluorescent margins corresponded to the main fascia of the pectoral muscle and DCIS tissue, which were histologically classified as negative margins.

[0250] Compound 1 fluorescence was clearly detected in the skin of mastectomy patients during surgery as well as in ex vivo samples from mastectomy patients, in which Compound 1 fluorescence was observed in the nipple.

[0251] Example 13. Compound 1 detection of latent diseases Compound 1 fluorescence detected five additional occult lesions (one patient with HNSCC and four patients with BC) that would have been missed by preoperative, intraoperative, or postoperative pathology. In one patient (ON1113) with HNSCC who had positive surgical margins by both fluorescence and histopathology, satellite metastases were detected in the wound bed by Compound 1 fluorescence image-guided surgery that would have been missed by standard-of-care surgery.

[0252] In one BC patient (ON1151), both the wound bed and the specimen margin fluorescence at the back table were classified as false-positive results (i.e., histopathologically negative margins as defined by the Society of Surgical Oncology and the American Society for Radiation Oncology guidelines), and the fluorescence corresponded to DCIS, an entity with cancer cells within the canalicular wall, which may require additional surgery according to international guidelines, highlighting the clinical utility of detecting this lesion.

[0253] In three other BC patients, fluorescence imaging during histopathology processing detected additional cancers that would have been missed. Of these, patients ON1101 and ON1128 had additional satellite metastases of BC in tissue slices detected by Compound 1. In patient ON1115, Compound 1 detected a second primary tumor lesion (triple-negative BC) that was missed during preoperative workup and surgery.

[0254] Of the three patients with CRC, surgeons detected unexpected peritoneal metastases in one patient (ON1130) during surgery and through SOC procedures. The second CRC patient already presented with preoperative clinical suspicion of peritoneal metastases (ON1120). In both patients, the peritoneal metastases were fluorescent tumor-positive lesions (Figure 17) and were confirmed as malignant on final histopathological examination.

[0255] The ability to detect tumor-positive margins and occult disease across a range of tumor types with similar high sensitivity and specificity highlights the significant potential of Compound 1 image-guided surgery to aid clinical decision-making for surgical and postoperative patient management.

[0256] Diagnostic performance of compound 1 In this phase 1 study, intraoperative and postoperative imaging data were used for an initial analysis of the diagnostic performance of Compound 1. Performance parameters, such as MFI, CNR, and TBR, were calculated in vivo and in tissue slices to characterize Compound 1's ability to delineate tumor tissue from background. The sensitivity and specificity of detecting tumor tissue from adjacent normal tissue were assessed using tissue sample fluorescence and presented as receiver operating characteristic curves. The sensitivity, specificity, and PPV of Compound 1 fluorescence in detecting pathology-confirmed tumor-positive margins were obtained at the patient level.

[0257] Table 15 summarizes the in vivo and ex vivo CNR and TBR values ​​for all tumor types and patients for which in vivo imaging was possible or tissue slices that allowed fluorescence quantification were available. These ratios were variable but high, indicating that the MFI of tumor tissue was consistently higher than that of background tissue, an important factor for fluorescence-guided surgery. CNR and TBR values ​​did not show any systematic variation with dose or tumor type.

[0258] Intraoperative in vivo CNR and TBR In vivo CNR and TBR values ​​at 1.2 mg / kg were high across all tumor types for all patients for whom in vivo imaging was possible (11 of 18 patients: 7 of 7 HNSCC; 3 of 5 BC; 0 of 3 EC; and 1 of 3 CRC). Using only mucosal tumors directly exposed to surfaces where reliable assessment was possible (HNSCC), at 1.2 mg / kg, the median CNR was 5.6 (interquartile range 17.6) and the median TBR was 2.6 (interquartile range 1.4). These high CNR and TBR ratios imply sharp delineation of tumor tissue from background tissue for each patient's procedure, a key requirement for accurate image-guided surgery.

[0259] Intraoperative diagnostic performance in detecting surgical margins Compound 1 demonstrated 100% sensitivity in detecting patients with tumor-positive surgical margins with no false negatives. The specificity and PPV of Compound 1 for detecting patients with surgical margins were 67% and 64%, respectively. By tumor type, the sensitivity and specificity of Compound 1 for detecting patients with surgical margins were 100% and 75%, respectively, for BC and 100% and 57%, respectively, for HNSCC. Compound 1 fluorescence was negative in two of three EC patients and one of three CRC patients in whom histological margin status was available and negative. These initial data demonstrate tumor-independent diagnostic performance and the feasibility of using Compound 1 imaging for accurate detection of tumor-positive margins during surgery.

[0260] [Table 15] TIFF0007752110000029.tif52162

[0261] Postoperative MFI, CNR, TBR, and ROC curve Intraoperative fluorescence intensity cannot be standardized and compared across patients or dose levels due to the unique presentation of each surgical environment. Multiple variables, such as camera angle, camera-to-tissue distance, tumor location, and coverage by other tissues or fat, affect the absolute value of the fluorescence signal. To enable direct comparison of MFI across patients and doses, patient tissue slices were imaged with a LI-COR Pearl standardized closed-field camera using a standard postoperative workflow for fluorescence. In all patients with histopathologically proven viable tumor tissue, tumor tissue showed higher fluorescence signal intensity with sharp morphological delineation on the tissue slices compared with normal tissue, regardless of dose and tumor type. While MFI increased slightly with dose across the tested dose range, CNR and TBR remained variably high and did not show any systematic variation with dose or tumor type. ROC curve analysis performed at the measurement level on these tissue slices showed an area under the curve of 0.9726, P<0.0001, indicating excellent performance. These data support the sensitive, specific, and tumor-independent performance characteristics of Compound 1.

[0262] To further validate the intraoperative findings, ex vivo workflow analysis showed that tumor tissue from all subjects with histopathologically proven viable tumor tissue exhibited higher fluorescence signal intensity along with sharper morphological delineations in tissue slices compared with normal tissue, regardless of tumor type and dose cohort (Figure 16, panel y). The mean fluorescence intensity (MFI) of tumors increased with dose (Figure 20, panel a). In all cohorts, tumor MFI was significantly higher than that of nontumor tissue. The median tumor-to-background ratio (TBR) for all tissue slices (n = 97, from 26 subjects) was 4.5 with an interquartile range (IQR) of 3.1. The optimal dose for tumor detection and sensitivity in the phase 1b study was 1.2 mg / kg (TBR 4.5, IQR 3.0), and the MFI of tumor tissue in each dose group was significantly higher than that of the respective normal tissue in the available tissue slices. Receiver operating characteristic (ROC) curve analysis of these tissue slices showed an AUC of 0.9875 (Figure 20, panel g).

[0263] Example 14. Synergistic response of nanoscale macromolecules to tumor acidosis in response to image-guided cancer surgery In this first-in-human fluorescence image-guided surgery study, compelling in vivo and ex vivo data demonstrate that low pH generated by tumor acidosis can be utilized as a tumor-independent biomarker for cancer in patients with a variety of solid tumors, including HNSCC, BC, EC, and CRC. Compound 1, a pH-sensitive fluorescent contrast agent, was specifically and durably activated by tumor acidosis, sharply delineating tumor from normal tissue and, in some cases, providing information about occult cancer not obtainable from SOC: intraoperative detection of all positive margins (9 of 9), DCIS, and satellite cancers in patients with HNSCC, as well as ex vivo detection of three additional satellite lesions and a second primary in pathology specimens.

[0264] Successful clinical use of tumor pH for imaging is possible through the design of Compound 1 to overcome metabolic and phenotypic variability among different patients and tumors. It was possible to detect all histologically proven tumor-positive surgical margins (9 out of 9) using Compound 1 fluorescence imaging. Most importantly, there was no overlap between tumor and background fluorescence for any given patient. Suppression of background activation and complete and irreversible quenching at a threshold acidic pH due to the cooperative action of the pH-responsive unimers are described. This cooperation, not predicted by testing the individual unimers but emerging from multiple distinct polymers interacting as micelles, is responsible for the observed clinical effects.

[0265] conclusion Because surgeons usually already have extensive information about tumor location, precise, clear delineation of cancer location is required for clinical success. The ability of optical imaging output to improve surgical outcomes is predicated on delivering information that surgeons do not have from preoperative imaging and intraoperative testing. The additional information from Compound 1, not provided by SOC, could significantly impact clinical care.

[0266] This first-in-human Phase 1 study: Compound 1 fluorescence imaging was possible in all four tumor types evaluated (HNSCC, BC, CRC, or EC), demonstrating the feasibility of tumor-independent imaging with Compound 1, as expected by its mechanism of action. Compound 1 fluorescence demonstrated a sharp boundary between tumor and normal tissues confirmed by histological imaging, along with high CNR and TBR values, which are crucial factors for real-time image-guided surgery. Compound 1 imaging detected all nine patients with tumor-positive margins using in vivo wound bed imaging in conjunction with back-table imaging of the excised specimen within one hour of resection. In vivo wound bed imaging detected two other occult tumors that were missed at routine surgery and confirmed by standard pathology, demonstrating the potential for significant value of Compound 1 image-guided surgery in clinical decision-making and patient management. Compound 1 fluorescence was detectable by multiple NIR cameras used in the study (NOVADAQ SPY Elite, SurgVision Explorer Air, and LI-COR Pearl Imaging systems).

[0267] Thus, compound 1, an intravenously administered, pH-activatable, near-infrared (NIR) fluorescent contrast agent, allows both in vivo and back-of-the-table fluorescent visualization of solid tumors (HNSCC, BC, CRC, and EC) with clear delineation from normal tissue. The results demonstrate the ability of compound 1 to detect all tumor-positive surgical margins and occult disease in multiple patients that would otherwise be missed, demonstrating tumor-independent fluorescent visualization of tumors in all tumor types investigated. These data highlight the significant potential of compound 1 in clinical decision-making for treatment planning and patient management during the post-surgical period.

[0268] Example 15. Assessment of breast, HNSCC, prostate, and ovarian tumors 3-6 hours post-dose from multiple NIR camera systems and from multiple study sites in an early Phase 2 study During a Phase 2 clinical study, the ability to image tumors 3–6 hours after IV injection of Compound 1 was demonstrated in patients with breast, HNSCC, prostate, and ovarian cancer (Figures 22–26). The study also utilized data collected from different NIR cameras and from multiple sites. All patients received a single IV dose of Compound 1, followed by routine surgery approximately 3–6 hours after Compound 1 infusion. Pre- and post-resection intraoperative and back-table tumor visualization is shown in Figure 22 from a breast cancer patient (101-001; UPenn; VisionSense NIR camera) who received Compound 1 (2 mg / kg) 6 ± 3 hours prior to surgery and a HNSCC cancer patient (102-007; UTSW; NOVAQ SPY Elite NIR camera) who received Compound 1 (3 mg / kg) 6 ± 3 hours prior to surgery. In both cases, white-light images of the tumor / specimen before or after resection are juxtaposed with an overlay of the observed fluorescence and the white-light image, indicating the presence of tumor. Intraoperative / in vivo imaging of prostate cancer and post-tumor resection wound beds from two patients (102-008 and 102-009; UTSW; Da Vinci Firefly NIR camera with updated software / hardware) dosed with Compound 1 (3 mg / kg) 6 ± 3 hours before tumor resection are shown in Figure 23. In both cases, white-light imaging of the tumor / specimen and surgical wound bed before resection are juxtaposed with images of the observed fluorescence. The data show the absence of fluorescence from the tumor before resection and in the surgical wound bed after resection. A tumor from a patient (101-005) with ovarian cancer dosed with Compound 1 (3 mg / kg, 6 ± 3 hours) was imaged in vivo before resection, as shown in Figure 24. The white light image is juxtaposed with the overlay of the observed fluorescence and white light image, showing the presence of tumor. The data from Figures 22-26 demonstrate the ability of Compound 1 to image tumors 3-6 hours after dosing and the use of multiple types of NIR cameras and different clinical sites.

[0269] Example 16. Evaluation of tumor-selective imaging agents in dogs with solid neoplasms Materials and Methods: After evaluation and recruitment to the study, canine patients underwent (A) preoperative analysis to identify possible lesion types and (B) Compound 1 tracer 0.5–2.0 mg / kg was administered 18–78 hours before surgery. Intraoperative imaging was performed (C) before and after tumor removal (or limb amputation) using a Hamamatsu PDE or custom-built NIR camera. Resected tissues were imaged (D) with a LI-COR Pearl Imaging station, and tumor-to-normal tissue ratios were calculated, as appropriate. Resected tissues were then preserved (E) for histopathological review. Safety, in terms of adverse effects, was assessed separately via physical examination, clinical examination, and adverse event recording from infusion to discharge.

[0270] Results: A summary of data from the spayed or neutered canine patients recruited for the study is shown below (Table 16). Results are presented from a total of seven dogs, ranging in age from 4 to 2 years and weight from 20.9 to 9.5 kg, with different tumor types and a range of tumors, including cases with more than one tumor type. Doses tested so far ranged from 0.5 to 2.0 mg / kg. In almost all cases, some preoperative testing, such as radiography, bone biopsy, or fine needle aspiration and cytology, was performed and is noted in the table footnotes. Compound 1 was administered to the animals as described above ("Dose"), and 24 or 72 hours later ("Time"), surgery to remove the tumor began. Excised tissues were sent to a veterinary pathologist for lesion identification, which is noted in the table along with the anatomical location. Both acute and chronic adverse effects were monitored and noted from the time of injection until the animals were discharged and followed up (for suture removal).

[0271] [Table 16]

[0272] The results from the studies described in Table 16 demonstrated that: (i) no adverse effects were observed in any of the dogs at any stage from injection of Compound 1 to rehabilitation to post-surgery; (ii) based on combined data from pre-operative biopsies and histopathology, affected tissue was observed throughout the tumor, but fluorescent signals were observed in the expected locations for these affected tissues; and (iii) in one case, occult disease was identified during surgery to remove the primary tumor.

[0273] Results for canine patients are shown in Figures 27-32, along with white light and NIR fluorescence images using the LI-COR Pearl imaging station. Figure 27 shows a mast cell tumor resection. The white light image on the left shows the resected tissue; tumor tissue was also exposed by performing a vertical resection. The suspected cancerous tissue is clearly evident in the NIR fluorescence image on the right side of the image, distinguished from the resected distal tissue (arrows) on the right side of each image. Figure 28 shows an osteosarcoma resected from a canine patient by limb amputation and imaged with white light. Ex vivo imaging was performed using the Hamamatsu PDE and LI-COR Pearl. Figure 32 shows the detection of occult disease within a distal soft tissue sarcoma in a canine patient's lymph node. During surgical removal from a canine patient with a primary soft tissue sarcoma located in the left metatarsal region, a lymph node was observed to be fluorescent, removed, imaged intraoperatively with white light, and then imaged using a Hamamatsu PDE NIR camera in vivo and a LI-COR Pearl NIR Imaging station ex vivo.

[0274] Conclusions: A total of seven dogs with osteosarcoma, soft tissue sarcoma, mast cell tumor, ovarian cyst, and other diseased tissues were evaluated in the canine patient study. Results to date demonstrate: (i) no adverse effects in any dog ​​from the time of Compound 1 injection to discharge; (ii) correlation of Compound 1-induced cancer tissue location with data from physical examination, preoperative biopsy, and post-resection histopathology for all malignancies tested; and (iii) identification of occult disease (metastatic popliteal lymph node) in one canine patient study. Furthermore, fluorescence imaging was possible using three cameras, all of which detected ICG, suggesting that imaging can be performed with any camera capable of detecting ICG fluorescence. These results support the safety and efficacy of Compound 1 across a wide range of tumors with substantially different oncogenic genotypes and, depending on the dose regimen, are clinically relevant for human clinical trials.

[0275] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized to practice the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. Formula (II): 【Chemical 1】 (In the formula, X １ is a halogen, —OH, or —(O)OH; n is 90 to 140; each r is independently a bond connecting the carbon atoms of the units / segments or an alkyl group -(CH 2 ) m - where m is 1 to 10; x is 50 to 200; y is 1 to 3; z is 1 to 3. or a pharmaceutically acceptable salt, solvate, hydrate, or isotopic variant thereof.

2. X １ The block copolymer of claim 1 , wherein is a halogen.

3. X １ The block copolymer of claim 1 or 2, wherein is —Br.

4. The block copolymer according to any one of claims 1 to 3, wherein n is 100 to 120.

5. The block copolymer according to any one of claims 1 to 4, wherein n is 113.

6. The block copolymer according to any one of claims 1 to 5, wherein x is 60 to 150.

7. The block copolymer of any one of claims 1 to 6, wherein y is 1.

8. The block copolymer of any one of claims 1 to 7, wherein z is 1.

9. The block copolymer of any one of claims 1 to 7, wherein z is 2.

10. A composition or micelle comprising one or more block copolymers according to any one of claims 1 to 9.

11. 11. A pH-responsive composition comprising the micelle of claim 10, wherein the micelle has a pH transition point and an emission spectrum.

12. 12. The pH-responsive composition of claim 11, wherein the pH transition point is between 4.8 and 5.

5.

13. 12. The pH-responsive composition of claim 11, wherein the pH transition point is about 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.

5.

14. The pH-responsive composition according to any one of claims 11 to 13, having an emission spectrum between 700 and 900 nm.

15. pH transition range (ΔpH １０～９０％ The pH-responsive composition according to any one of claims 11 to 14, comprising:

16. 16. The pH-responsive composition of claim 15, wherein the pH transition range is less than 0.25 pH units.

17. 16. The pH-responsive composition of claim 15, wherein the pH transition range is less than 0.15 pH units.

18. 18. The pH-responsive composition of claim 11, having a fluorescence activation ratio of greater than 25.

19. The pH-responsive composition of any one of claims 11 to 17, having a fluorescence activation ratio of greater than 50.

20. A contrast agent comprising one or more block copolymers according to any one of claims 1 to 9.

21. 21. The contrast agent of claim 20, comprising a poly(ethylene oxide)-b-poly(dibutylaminoethyl methacrylate-r-aminoethyl methyl acrylate hydrochloride) copolymer indocyanine green and acetic acid conjugate.

22. A pharmaceutical composition comprising a micelle, the micelle comprising: 1) Formula (II): 【Chemistry 2】 (In the formula, X １ is a halogen, —OH, or —C(O)OH; n is 90 to 140; each r is independently a bond connecting the carbon atoms of the units / segments or an alkyl group -(CH 2 ) m - where m is 1 to 10; x is 50 to 200; y is 1 to 3; z is 1 to 3. or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and 2) Stabilizer A pharmaceutical composition comprising:

23. 23. The pharmaceutical composition of claim 22, wherein the stabilizing agent is a cryoprotectant.

24. 23. The pharmaceutical composition of claim 22, wherein the stabilizer is a sugar, a sugar derivative, a surfactant, or a salt.

25. 25. The pharmaceutical composition of claim 24, wherein the stabilizer is a sugar derivative.

26. 26. The pharmaceutical composition of claim 24 or 25, wherein the stabilizer is a monosaccharide, a disaccharide, a trisaccharide, a water-soluble polysaccharide, or a sugar alcohol, a polyol, or a combination thereof.

27. 27. The pharmaceutical composition of any one of claims 22 to 26, wherein the stabilizer is fructose, galactose, glucose, lactose, sucrose, trehalose, maltose, mannitol, sorbitol, ribose, dextrin, cyclodextrin, maltodextrin, raffinose, or xylose, or a combination thereof.

28. The pharmaceutical composition according to any one of claims 22 to 27, wherein the stabilizer is trehalose.

29. 29. The pharmaceutical composition of any one of claims 22 to 28, comprising from about 0.5% to about 25% w / v of a stabilizer.

30. 29. The pharmaceutical composition of any one of claims 22 to 28, comprising about 5% w / v to about 15% w / v of a stabilizing agent.

31. The pharmaceutical composition of any one of claims 22 to 30, further comprising a liquid carrier.

32. 31. The pharmaceutical composition of claim 30, wherein the liquid carrier is sterile water, saline, half normal saline, 5% dextrose in water (D5W), lactated Ringer's solution, or a combination thereof.

33. 33. The pharmaceutical composition of claim 31 or 32, wherein the liquid carrier is sterile water.

34. 34. The pharmaceutical composition of any one of claims 22 to 33, comprising about 1.0 mg / mL to about 5.0 mg / mL of the block copolymer of formula (II).

35. 34. The pharmaceutical composition of any one of claims 22 to 33, comprising about 1 mg / mL to about 5 mg / mL of the block copolymer of formula (II).

36. The pharmaceutical composition of any one of claims 22 to 33, comprising about 0.5 to about 7 mg / kg of the block copolymer of formula (II).

37. The pharmaceutical composition of any of claims 22 to 33, comprising about 1 mg / kg to about 7 mg / kg of the block copolymer of formula (II).

38. The pharmaceutical composition of any one of claims 22 to 33, comprising about 0.1 mg / kg to about 3 mg / kg of the block copolymer of formula (II).

39. 1) Formula (II): 【Chemistry 3】 (In the formula, X １ is -Br, n is 90 to 140; each r is independently a bond connecting the carbon atoms of the units / segments or an alkyl group -(CH 2 ) m - where m is 1 to 10; x is 60 to 150; y is 1 to 3; z is 1 to 3. or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and 2) Approximately 10% w / v trehalose in water A pharmaceutical composition comprising:

40. 40. The composition of any one of claims 22 to 39, formulated for oral, intramuscular, subcutaneous, intratumoral, or intravenous administration.

41. 40. The composition of any one of claims 22 to 39, formulated for intravenous administration.

42. 42. A pharmaceutical composition according to any one of claims 22 to 41 for use in a method for imaging the pH of an intracellular or extracellular environment, said method comprising: a) contacting an intracellular or extracellular environment with said pharmaceutical composition; and b) detecting one or more optical signals from the intracellular or extracellular environment, the detected optical signals indicating that the micelles comprising one or more block copolymers of formula (II) have reached their pH transition point and dissociated. The pharmaceutical composition comprising:

43. 43. The composition of claim 42, wherein the extracellular environment is intravascular or extravascular.

44. 43. The composition of claim 42, wherein imaging the pH of the intracellular or extracellular environment comprises imaging of metastatic disease.

45. 43. The composition of claim 42, wherein imaging the pH of the intracellular or extracellular environment comprises imaging the pH of the tumor environment.

46. 46. ​​The composition of claim 45, wherein imaging the pH of the tumor environment comprises imaging a lymph node or multiple lymph nodes.

47. 46. ​​The composition of claim 45, wherein imaging of a lymph node or nodes provides information for surgical removal of a tumor or staging of tumor metastasis.

48. 46. ​​The composition of claim 45, wherein imaging of the pH of the tumor environment allows for determination of tumor size.

49. 46. ​​The composition of claim 45, wherein imaging of the pH of the tumor environment allows for determination of tumor margins.

50. 46. ​​The composition of claim 45, wherein imaging of the pH of the tumor environment allows for more precise removal of the tumor during surgery.

51. 46. ​​The composition of claim 45, wherein imaging of the pH of the tumor environment allows for the determination of satellite tumors, multifocal tumors, or occult tumors.

52. 46. ​​The composition of claim 45, wherein imaging of the pH of the tumor environment allows for the detection of residual metastatic disease.

53. 46. ​​The composition of claim 45, wherein imaging of the lymph node or nodes allows for more precise removal of the lymph node or nodes during surgery.

54. The composition of any one of claims 42 to 53, wherein the method comprises administering the composition to a patient in need thereof prior to surgery.

55. 55. The composition of claim 54, wherein the surgery is a tumor resection.

56. 46. ​​The composition of claim 45, wherein imaging of the pH of the tumor environment provides information for patient management.

57. 42. A pharmaceutical composition according to any one of claims 22 to 41 for use in a method for removing a tumor in a patient in need thereof, said method comprising: a) detecting one or more optical signals from a tumor or a sample thereof obtained from a patient to whom an effective dose of the pharmaceutical composition has been administered, wherein the detected optical signals indicate the presence of a tumor; and b) removing said tumor via surgery The pharmaceutical composition comprising:

58. 58. The composition of claim 57, wherein the one or more optical signals are fluorescent signals.

59. 58. The composition of claim 57, wherein the tumor is at least 90% removed.

60. 58. The composition of claim 57, wherein the tumor is at least 95% removed.

61. 58. The composition of claim 57, wherein the tumor is at least 99% removed.

62. The composition of any one of claims 57 to 61, wherein the tumor is a solid tumor.

63. The composition of any one of claims 57 to 61, wherein the tumor is a non-solid tumor.

64. 64. The composition of claim 62 or 63, wherein the solid or non-solid tumor is of cancerous origin.

65. 65. The composition of claim 64, wherein the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, esophageal cancer, brain cancer, pancreatic cancer, skin cancer, melanoma, sarcoma, pleural metastasis, kidney cancer, lymph node cancer, cervical cancer, or colorectal cancer.

66. 65. The composition of claim 64, wherein the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), esophageal cancer, ovarian cancer, prostate cancer, or colorectal cancer.

67. The composition of any one of claims 57 to 66, wherein the pharmaceutical composition is administered as an injection or infusion.

68. 68. The composition of any one of claims 57 to 67, wherein the pharmaceutical composition is administered as a single dose or multiple doses.

69. 69. The composition of any one of claims 57 to 68, wherein the pharmaceutical composition is administered at least 1 hour before surgery.

70. 69. The composition of any one of claims 57 to 68, wherein the pharmaceutical composition is administered from about 1 hour to about 32 hours before surgery.

71. 42. A pharmaceutical composition according to any one of claims 22 to 41 for use in a method for treating cancer, said method comprising: a) detecting one or more optical signals in a cancer patient in need of cancer treatment who has been administered an effective dose of the pharmaceutical composition, wherein the detected optical signals indicate the presence of a cancerous tumor; and b) removing said cancerous tumor, thereby treating said cancer. The pharmaceutical composition comprising:

72. The composition of claim 71, wherein the method further comprises imaging a body cavity of a cancer patient, or imaging a cancerous tumor or a slice or sample thereof (e.g., fresh or formalin-fixed), optionally by back-table fluorescence-guided imaging after removal from the patient.

73. 42. The pharmaceutical composition according to any one of claims 22 to 41 for use in a method for detecting a cancerous tumor, said method comprising: a) detecting one or more optical signals in a cancer patient in need of cancerous tumor detection, to whom an effective dose of the pharmaceutical composition has been administered, wherein the detected optical signals indicate the presence of the cancerous tumor. The pharmaceutical composition comprising:

74. 42. A pharmaceutical composition according to any one of claims 22 to 41 for use in a method for minimising cancer recurrence for at least 5 years, said method comprising: a) detecting one or more optical signals in a cancer patient in need of minimizing cancer recurrence, wherein the cancer patient has been administered an effective dose of the pharmaceutical composition, wherein the detected optical signals indicate the presence of a cancerous tumor, and the presence of the tumor indicates the recurrence of the cancer; and b) treating said cancer if said one or more optical signals are detected to minimize said recurrence. The pharmaceutical composition comprising:

75. The composition of claim 74, wherein the method further comprises removing the tumor.

76. 76. The composition of any one of claims 71 to 75, wherein the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, esophageal cancer, brain cancer, pancreatic cancer, skin cancer, melanoma, sarcoma, pleural metastasis, kidney cancer, lymph node cancer, cervical cancer, or colorectal cancer.

77. 76. The composition of any one of claims 71 to 75, wherein the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), esophageal cancer, ovarian cancer, prostate cancer, or colorectal cancer.

78. 78. The composition of any one of claims 71 to 77, wherein the pharmaceutical composition is administered at least 1 hour before imaging the patient.

79. 78. The composition of any one of claims 71 to 77, wherein the pharmaceutical composition is administered from about 1 hour to about 32 hours before imaging the patient.

80. The composition of any one of claims 71 to 79, wherein the pharmaceutical composition is administered as an injection or infusion.

81. The composition of any one of claims 71 to 80, wherein the pharmaceutical composition is administered as a single dose or multiple doses.

82. A composition described in any one of claims 71 to 81, wherein the method further includes imaging of a cancer patient, including an intraoperative camera, a near-infrared camera, or an endoscopic camera.

83. The composition of any one of claims 57 to 82, wherein the patient in need thereof is a human patient.

84. 83. The composition of any one of claims 57 to 82, wherein the patient in need thereof is a canine, feline, equine, bovine, lagomorph, or porcine patient.

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