Fluorescence imaging agent for lung cancer resection

Abstract

Provided herein are methods for the surgical removal of lung cancer tissue using CXL, a near-infrared fluorescent imaging agent. This approach involves administering a pharmaceutical composition containing CXL intravenously to a patient, detecting a fluorescence signal emitted from lung cancer tissues, and surgically excising the identified cancerous tissue. Administration doses may range from about 0.001 mg / kg to about 0.10 mg / kg, occurring between up to 4 days and about 1 hour before imaging. The method supports endoscopic imaging and surgeries, including thoracoscopic lung tissue resection, intraoperative visualization, and histopathological evaluation to confirm tissue removal. Exclusion criteria cover hypersensitivity history, renal impairment, and specific laboratory markers exceeding defined limits.

Description

Fluorescence Imaging Agent For Lung Cancer Resection

[0001] This application claims the benefit of priority of the United States Provisional Patent Application Serial No. 63 / 725,695 filed on November 27, 2024, and also claims the benefit of priority of the United States Provisional Patent Application Serial No. 63 / 761,949 filed on February 22, 2025, the disclosures of which are each incorporated by reference in their entirety for all purposes.

[0002] The present disclosure generally relates to medical imaging in a patient in need thereof using near-infrared (“NIR”) fluorescence.

[0003] Each year, hundreds of thousands of people are discovered to have a pulmonary nodule or mass on radiological exams in the US, including non-small cell lung cancer (“NSCLC”). Eighty thousand patients ultimately undergo surgery for removal. Currently, the only method to determine whether the nodule or mass is malignant is histological examination. As an alternative, targeted molecular optical imaging probes have been introduced to specifically bind and identify malignant cells.

[0004] Pulmonary resection is recommended for most patients who have Stage I-II NSCLC. The goals of this procedure are complete resection of suspected lung cancer nodules, minimization of unnecessary resections, and accurate staging. Challenges include identifying small nodules, recognizing synchronous cancers or advanced cancers, and confirming negative margins. These challenges are magnified because of the increasing use of minimally invasive surgery (“MIS”), where tactile feedback is reduced, and chest assessment is compromised.

[0005] Molecular imaging is a technique by which contrast agents identify a target on tumor cells. For example, positron emission tomography (“PET”) scanning is one type of molecular imaging. It is used to evaluate lung nodules using 2-deoxy-2-(18F) fluoro-D-glucose (“18FDG”), the only FDA-approved molecular contrast agent for PET imaging.18FDG crosses into the cell via the GLUT1 transporter. However, despite the common use of18FDG- PET scanning, it is not selective for malignant cells.

[0006] Intraoperative fluorescence imaging using cronexitide lanocianine (“LS301” or “CXL”) during surgery may address these opportunities and assist the surgeon in distinguishing malignant from non-malignant tissue, allowing the fluorescence image to guide surgeons in excising tumor tissue margins and potentially aid in identifying and surgically excising suspected cancer. Several United States (“US”) Food and DrugAdministration (“FDA”)-cleared imaging devices can detect CXL and its investigational formulation, CXL-IT. For this disclosure, commercial and prototype imaging systems visualize CXL intraoperatively and / or ex vivo (e.g., intact, bread-loafed, and fixed tissue specimens).

[0007] The present disclosure provides a method for surgically removing lung cancer tissue in a patient in need thereof, comprising administering an effective amount of a pharmaceutical composition comprising CXL to the patient; detecting a signal emitted from the CXL in lung cancer tissue of the patient; and surgically excising a portion of the lung cancer tissue identified from the emitted signal.

[0008] Other objects and features will be in part apparent and in part pointed out below.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 shows an endoscopic in-situ white light image of a lung containing the primary tumor of an adenocarcinoma. The location of the primary tumor is unclear on the white-light image. FIG. 2 shows a near-infrared (“NIR”) CXL fluorescence overlay on FIG. 1, which visualizes the primary tumor.

[0010] FIG. 3 shows an endoscopic in-situ white light image of a lung containing the primary tumor of a metastatic melanoma. The location of the primary tumor is unclear on the whitelight image.

[0011] FIG. 4 shows a NIR CXL fluorescence overlay on FIG. 3, which visualizes the primary tumor.

[0012] FIG. 5 shows an endoscopic in-situ white light image of a lung containing the primary tumor of an adenocarcinoma. The location of the primary tumor is unclear on the white-light image.

[0013] FIG. 6 shows a NIR CXL fluorescence overlay on FIG. 5, which visualizes the primary tumor.

[0014] FIG. 7 shows an endoscopic in-situ white light image of a lung containing the primary tumor of an adenocarcinoma. The location of the primary tumor is unclear on the white-light image.

[0015] FIG. 8 shows a NIR CXL fluorescence overlay on FIG. 7, which visualizes the primary tumor.

[0016] FIG. 9 identifies a lesion within 10 mm of the staple line that white light alone could not detect.

[0017] FIG. 10 shows the NIR CXL fluorescence overlay on FIG. 9, which visualizes the lesion.

[0018] FIG. 11 identifies a lesion within 10 mm of the staple line that white light alone could not detect.

[0019] FIG. 12 shows the NIR CXL fluorescence overlay on FIG. 11, which visualizes the lesion.

[0020] FIG. 13 shows the in-situ tumor mean fluorescence intensity (“MFI”) as a function of dose level.

[0021] FIG. 14 shows the in-situ tumor-to-background ratio (“TBR”) of fluorescence as a function of dose level.DETAILED DESCRIPTION

[0022] In embodiments of the present disclosure, the method and pharmaceutical compositions for near-infrared imaging using CXL can be applied to identify and surgically resect malignant tissue in a variety of cancers, regardless of their anatomical location. For example, the disclosed methods equally apply to lung cancer resection, including non-small cell lung cancer (“NSCLC”), lung adenocarcinoma, lung squamous cell carcinoma, neuroendocrine carcinoma, and other malignancies. During clinical investigations, one case of metastatic squamous cell carcinoma was observed. Another case of metastatic adenocarcinoma in the pleura (parietal and visceral) was also observed. Thus, CXL fluoresced and visualized metastatic adenocarcinoma in non-lung tissue as well as in lung tissue.

[0023] The disclosed imaging parameters, dosing regimens, and delivery modes are broadly suitable for guiding surgical resection procedures, such as minimally invasive thoracoscopic (e.g., “VATS”) or robotic-assisted (“RATS”) lung resections. In some embodiments, CXL is administered to a patient scheduled for lung cancer resection via minimally invasive thoracic surgery. The near-infrared fluorescence imaging of CXL provides enhanced delineation of pulmonary tumor margins, thereby guiding the surgeon in performing targeted resection of malignant lung tissue and associated lymph nodes

[0024] Pafolacianine is a folic acid analog conjugated to a fluorescent dye, which can bind to folate receptor-positive cells. Certain cancers, such as ovarian cancer, overexpress high- affinity folate receptors, which increase folate uptake and support tumor growth. The ELUCIDATE trial, a Phase 3, twelve-center study of 112 patients with suspected or biopsy-confirmed lung cancer for sublobar resection, randomized patients to surgery with or without intraoperative molecular imaging (“IMI”) with pafolacianine administered intravenously within 24 hours before surgery. The study concluded that IMI with pafolacianine improved surgical outcomes by identifying occult tumors and close surgical margins.

[0025] This study, however, had several major shortcomings. First, the tumor had to express folate receptor alpha, so the non-adenocarcinomas did not fluoresce. Second, the agent could not be detected by any indocyanine green (“ICG”)- compatible camera, limiting its clinical application in the US and worldwide. Thus, other molecular imaging agents are needed to aid in the resection of non-folate receptor lung cancers and improve on the safety and tolerability profile of pafolacianine.

[0026] Such a molecular imaging agent is CXL, which comprises (1) a cyclic peptide that binds phosphorylated Annexin A2 (“pANXA2”), which is found on the surface of malignant cells typically located in solid tumors and in malignant lymph nodes, and (2) cypate, a NIR molecule conjugated to the peptide, which fluoresces when illuminated using NIR light. In addition to preclinical data demonstrating pANXA2 binding, the clinical pharmacodynamic fluorescence data and literature are consistent with the active agent binding albumin (a receptor for a variety of lipophilic drugs and dyes).

[0027] At a clinical dose of 0.025 mg / kg investigational formulation of CXL (“CXL-IT”), the targeted highest dose (20 mg / kg) from the rat toxicity study would be 129-fold higher than the clinical dose. Because the actual highest dose in that study was 17.6 mg / kg (a correction factor of 0.88 was applied based on results from the formulation analysis), the conservative safety margin would be 114-fold higher than the proposed clinical dose of 0.025 mg / kg. Furthermore, the lowest effective dose tested (0.1 mg / kg) in the mouse efficacy study was 0.32-fold the proposed clinical dose. The CXL-IT 0.025 mg / kg dose is also lower than the highest CXL-human specific antigen (“HSA”) dose of 0.1 mg / kg used in the Washington University (“WU”) Phase 1 clinical study, which was well within the estimated range for the maximum recommended starting dose (“MRSD”) estimated from the rat toxicity study.

[0028] In certain embodiments, the method comprises administering the pharmaceutical composition via intravenous delivery.

[0029] In certain embodiments, the method comprises an effective amount ranging from about 0.001 mg / kg to about 0.2 mg / kg, such as from about 0.001 mg / kg to about 0.15 mg / kg, from about 0.001 mg / kg to about 0.1 mg / kg, or from about 0.001 mg / kg to about 0.050mg / kg. In certain embodiments, the effective amount is chosen from about 0.1 mg / kg, about 0.05 mg / kg, about 0.075 mg / kg, about 0.025 mg / kg, about 0.0125 mg / kg, about 0.00625 mg / kg, and about 0.003125 mg / kg. In certain embodiments, the dose ranges from about 0.001 mg / kg to about 0.025 mg / kg. In certain embodiments, the dose is about 0.025 mg / kg. In certain embodiments, the dose is about 0.0125 mg / kg. In certain embodiments, the dose is about 0.00625 mg / kg. In certain embodiments, the dose is about 0.003125 mg / kg.

[0030] In certain embodiments, the administration occurs up to about 7 days before the emitted signal is detected; that is, up to about 7 days before the patient undergoes surgery to remove lung cancer tissue. In certain embodiments, the administration occurs about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day before detecting the emitted signal. In certain embodiments, the administration occurs between about 4 days and about 3 days before the signal is detected. In certain embodiments, the administration occurs between about 2 days (48 hours) and about 1 day (24 hours) before detecting the emitted signal. In certain embodiments, the administration occurs between about 24 hours to about 1 hour before detecting the emitted signal.

[0031] One of skill in the art would understand that dose adjustments may be needed to account for a longer interval between dosing and surgery. For example, if a patient is dosed about 3 or 4 days before the signal is detected, the patient can be dosed on a Thursday or Friday for a Monday surgery. In some embodiments, a higher dose is used when the patient is dosed a few days before surgery rather than one day before surgery. The increased dose is calculated to account for the patient’s mass and metabolism, the interval between dosing and surgery, safety, and efficacy. At longer intervals between dosing and imaging, a higher dose can provide a brighter fluorescence to aid cancer visualization.

[0032] One of skill in the art would understand how to adjust the imaging parameters based on the patient, dosage level, interval, allergies, lesion size, and other factors. For example, one of skill in the art would also recognize that patients with known allergies to dyes or contrast agents, such as CXL, can be administered compositions formulated in a larger volume and administered over a longer period to avoid a suspected reaction. In other instances, the pre-op estimated size of a lesion may affect the recommended dosing regimen ( / .< ., dose level and / or dosing time interval). For example, smaller lesions (< 1 cm) may have a better detection rate at a higher dose and / or a longer dosing interval to improve the signal- to-noise ratio between tumor and background, or a lower dose and / or a shorter dosinginterval. In other embodiments, the pre-op diagnosis or characterization may be used to calibrate the detection with certain dose and / or dosing time interval ranges.

[0033] In certain embodiments, the signal emitted from the CXL comprises a signal from a CXL metabolite formed in the patient after administration. As used herein, “metabolite” refers to a modified form of a drug (such as CXL) produced in the patient after the drug undergoes metabolic breakdown through specialized enzymatic systems. In certain embodiments, the metabolite is formed via Phase I processes such as oxidation, reduction, or hydrolysis. In certain embodiments, the metabolite is formed from Phase II conjugation reactions with endogenous substances to increase water solubility. In certain embodiments, metabolites exhibit varying pharmacological activity, comprising active or inactive forms. In certain embodiments, the metabolite is readily excreted in urine or bile, often via cytochrome P450 enzymes in the liver. In certain embodiments, the metabolite remains present in the patient longer than the parent drug.

[0034] In certain embodiments, medical imaging is performed using a 510(k)-cleared imaging system capable of simultaneous fluorescence and white light imaging. In certain embodiments, the FDA-cleared imaging system has been cleared under § 510(k), for example, a device listed in Table 1. Clinical results consistently showed that, regardless of the imaging device, CXL-IT generated adequate fluorescence intensity to discern tumor margins in both in situ and ex vivo settings, even when standard white-light imaging failed to detect the primary tumor or lesions (See FIGS. 1-12.)Table 1: 510(k)-cleared imaging system capable of simultaneous fluorescence and white light imaging

[0035] In certain embodiments, the signal identifies potentially cancerous tissue, including within a primary tumor site, other undiagnosed tumors, tumor margins, surgical cavity, or lymph nodes draining the primary tumor site in the patient.

[0036] In certain embodiments, the patient is suspected of having lung adenocarcinoma, lung squamous cell carcinoma, or neuroendocrine carcinoma. In certain embodiments, the lung cancer is not detectable with pafolacianine.

[0037] In certain embodiments, the surgical excision is performed via endoscopy. In certain embodiments, the surgical excision comprises thoracoscopic lung resection.

[0038] In certain embodiments, each patient serves as their own control for imaging by comparing captured signals with baseline anatomical images.

[0039] In certain embodiments, after surgically excising the portion of lung cancer tissue, the method further comprises one or more of the following steps: detecting a signal emitted from the CXL to visualize surgical margins; assessing resected specimens for fluorescence signals indicating residual cancerous tissues; and / or confirming tissue status using histopathological evaluation post-surgery. In certain embodiments, this further detection comprises endoscopy. In certain embodiments, surgical margins are visualized in situ (before excision) or ex vivo (during resection).

[0040] In certain embodiments, histopathological confirmation comprises standard staining and fluorescence microscopy.

[0041] In certain embodiments, the method further comprises surgically excising a further portion of the lung cancer tissue.

[0042] In certain embodiments, findings of fluorescence imaging predict pathological outcomes confirmed by histological analysis.

[0043] In certain embodiments, the intraoperative fluorescence visualization of lung tissue types is determined based on a semi-quantitative assessment, for example, by a surgeon or an artificial intelligence (“Al”) model trained on a relevant database of medical imaging data.

[0044] In certain embodiments, the fluorescence imaging characteristics are evaluated based on the cancer type, histological grade, anatomical location, folate receptor status, and other potential receptor statuses.

[0045] In certain embodiments, the correlation between CXL fluorescence and central pathology review of lung cancer samples is assessed using pANXA2 immunohistochemistry.

[0046] In certain embodiments, additional suspicious nodules or lymph nodes visualized instill during imaging are validated by excised tissue specimens.

[0047] In certain embodiments, fluorescence signal intensity and signal-to-background ratio are evaluated in defined regions of interest using a different imaging device or different imaging modes.

[0048] In certain embodiments, the patient is excluded if they have a history of drug-related hypersensitivity or anaphylactic reactions, including sensitivities to indocyanine green or shellfish.

[0049] In certain embodiments, the patient is excluded if known to be sensitive to fluorescent light (e.g., NIR).

[0050] In certain embodiments, the patient is excluded if they have impaired renal function, characterized by a creatinine clearance (“CrCl”) of less than about 60 mL / min.

[0051] In certain embodiments, the patient is excluded if they show clinically significant abnormalities in their echocardiogram, such as a QT interval corrected for heart rate by Fridericia’s formula (“QTcF”) exceeding about 470 ms.

[0052] In certain embodiments, the patient is excluded if they have a total bilirubin level greater than about 1.5 times the upper limit of normal at screening.

[0053] In certain embodiments, the patient is excluded if their aspartate aminotransferase or alanine transaminase levels exceed 2.5 times the upper limit of normal at screening.PHARMACEUTICAL COMPOSITIONS

[0054] In certain embodiments, the pharmaceutical composition comprises 1-4 wt.% a dye- peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro-Cys)-Lys-OH (cronexitide lanocianine, CXL, LS301), or a salt thereof, wherein each amino acid residue is independently in a D or L configuration, 1-4 wt.% calcium salt, 4-6 wt.% polysorbate, 8-12 wt.% Z>eto-cyclodextrin, 0.2-2 wt.% sodium acetate, 1-10 wt.% dextrose, 1-100 mM histidine, and 62-86 wt.% water.

[0055] In certain embodiments, the cypate is chosen from LS288, LS798, LS276, LS843, Cypate 2, Cypate 3, Cypate 4, Cy5, Cy5.5, Cy7, and Cy9. In certain embodiments, the cypate is Cy5.5. In certain embodiments, the cypate is Cy7. Cypates are known in the art. In many embodiments, the cypate is cypate 4.

[0056] In certain embodiments, the cypate is

[0057] When depicted as a zwitterion, as in the structure above, a person of skill in the art would understand that the carboxylate anion will be protonated under the appropriate pH conditions and exist in equilibrium with the depicted anion.

[0058] In certain embodiments, at least one of the Cys amino acid residues is D-Cys.

[0059] In certain embodiments, the dye-peptide conjugate is CXL. In certain embodiments, the dye-peptide conjugate comprises the structurewherein R is OH or O'.

[0060] In certain embodiments, the dye-peptide conjugate comprises the structurewherein R is OH or O'.

[0061] In certain embodiments, the dye-peptide conjugate is CXL and comprises the structural formula

[0062] In certain embodiments, the dye-peptide conjugate is CXL, comprising the structural formula

[0063] In certain embodiments, the pharmaceutical composition has been adjusted to a target pH of about 3.0 with acetic acid.

[0064] In certain embodiments, the pharmaceutical composition comprises about 1-4 wt.% of CXL, such as about 1 wt.%, about 2 wt.%, about 3 wt.%, or about 4 wt.% dye CXL. In certain embodiments, the pharmaceutical composition comprises at least 1 wt.% CXL.

[0065] In certain embodiments, LS838 may be substituted for CXL in any of the lyophilized products or pharmaceutical compositions described herein.

[0066] In certain embodiments, the pharmaceutical composition comprises about 2 wt.% CXL, about 2 wt.% calcium salt, about 5 wt.% polysorbate, about 10 wt.% Z>eta-cyclodextrin, between about 1.2 and 1.4 wt.% sodium acetate, about 4 wt.% dextrose, about 10 mM histidine, and about 75 wt.% water. In certain embodiments, the pharmaceutical composition has a pH between about 5.5 and about 6.5. In certain embodiments, the pharmaceutical composition has an osmolality of about 330 mOsm / kg.

[0067] The agents and compositions described herein can be formulated in any conventional manner using one or more pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions contain a therapeutically effective amount of a biologicallyactive agent described herein, which can be in purified form, together with a suitable amount of carrier to provide the form for proper administration to the subject.

[0068] The pharmaceutical composition can be formulated to suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes, which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, rectal, and intra-lymphatic. In certain embodiments, the administration is intravenous. The individual agents may also be administered with one or more additional agents or other biologically active or inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0069] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as further described below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treating the disease, disorder, or condition.

[0070] In certain embodiments, intraoperative imaging of CXL during surgery assists the surgeon in distinguishing tumor tissue from noncancerous tissue. In certain embodiments, the fluorescence image generated from CXL guides surgeons in the excision of tumor tissue margins and the biopsy of cancerous lymph nodes. In some embodiments, successful imaging may be assessed by measuring one or more clinically significant events (CSEs). Suitable CSEs for cancer resection including, but are not limited to, (1) localizing a pulmonary nodule using CXL guided NIR imaging when white light or palpation failed to identify a nodule; (2) identifying a histologically confirmed synchronous or occult lesion using CXL NIR imaging when not identified by white light or palpation, and (3) identifying a positive margin (< 10 mm) with only CXL NIR imaging when deemed negative under white light or palpation.

[0071] In certain embodiments, the CXL dose is between about 0.16 and about 0.26 mg / kg. In certain embodiments, the CXL dose in humans is about 0.006 mg / kg. In certain embodiments, the CXL dose in humans is about 0.1 mg / kg. In certain embodiments, the 114 NOAEL corresponds to a starting dose of about 0.025 mg / kg, up to a target maximum dose of about 0.1 mg / kg. “NOAEL” refers to the “No-Observed-Adverse-Effect Level”. It is thehighest tested dose or exposure level of a substance, such as CXL, at which no adverse effects are observed in a specific test population, usually animals or humans. The NOAEL is used to establish safe exposure levels for humans and to determine the dosage levels for the drug product.

[0072] The actual dosage amount of a compound of the present disclosure or composition comprising a compound of the present disclosure administered to a subject may be determined by physical and physiological factors, such as type of animal treated, age, sex, body weight, the severity of the condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the subject and on the route of administration. A skilled artisan may determine these factors. The practitioner responsible for administration will typically determine the concentration of active ingredient(s) in a composition and the appropriate dose(s) for the individual subject. The individual physician may adjust the dosage if any complications arise.

[0073] Agents and compositions described herein can be administered according to the methods described herein in various means known to the art. The agents and composition can be used therapeutically as exogenous or endogenous materials. Exogenous agents are substances produced or manufactured outside the body that are administered to the body. Endogenous agents are those produced or manufactured inside the body by some device (biological or other) for delivery within or to other organs in the body.DEFINITIONS

[0074] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0075] The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” are also open-ended. For example, any method that “comprises,” “has,” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition ordevice that “comprises,” “has,” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0076] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” indicates that a value includes the standard deviation of the mean for the device or method employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and the attached claims are approximations that may vary depending on the desired properties of a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0077] “Formulation” refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a “formulation” can include pharmaceutically acceptable excipients, including diluents or carriers. As used herein, “formulation” can refer to a lyophilized product, a pharmaceutical composition, or a reconstitution diluent.

[0078] As used herein, “lyophilized product” refers to a product that has undergone freeze- drying, known as lyophilization, to remove water and preserve its structure and activity. In these embodiments, the lyophilized product is a dry and stable form of the drug, providing, for example, increased shelf-life and reduced storage costs.

[0079] A “stable” formulation or pharmaceutical composition or lyophilized product can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about -20 °C and about 60 °C, for a commercially reasonable time, such as atleast about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0080] As used herein, “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, z.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. When the term “pharmaceutically acceptable” refers to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient has met the required toxicological and manufacturing testing standards or is included in the Inactive Ingredient Guide prepared by the US FDA. “Pharmacologically active” (or “active”) as in a “pharmacologically active” (or “active”) derivative or analog refers to a derivative or analog having the same type of pharmacological activity as the parent compound and about equivalent in degree.

[0081] The term “pharmaceutically-acceptable carrier” is art-recognized. It refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ or portion of the body to another organ or portion of the body. Each carrier must be “acceptable” in terms of compatibility with the subject composition and its components, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other nontoxic compatible substances employed in pharmaceutical compositions.

[0082] “Water for injection” or “WFI” refers to a type of purified water used for pharmaceutical applications, particularly in the preparation of drugs and medical devices. It isa highly purified form of water that meets the standards of the United States Pharmacopeia (“USP”), Chapter <1231>. WFI is substantially free of any substances that could potentially harm patients. WFI is produced via distillation or reverse osmosis, which removes impurities and other contaminants from the water, resulting in water substantially free of dissolved solids, organic matter, and microorganisms.

[0083] In some embodiments, “pharmaceutically acceptable salt” refers to acid addition salts with an inorganic or organic acid. Lists of suitable salts are found in WO 87 / 05297, Johnston et al., published September 11, 1987; Remington’s Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., 1985, p. 1418; and J. Pharm. Sci., 66, 2 (1977), each incorporated herein by reference in its entirety. A reference for the preparation and selection of pharmaceutical salts of the present disclosure is P. H. Stahl & C. G. Wermuth “Handbook of Pharmaceutical Salts,” Verlag Helvetica Chimica Acta, Zurich, 2002, which is incorporated herein by reference in its entirety. The organic or inorganic acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic, 2-hydroxy ethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic, camphoric, camphorsulfonic, 4-methylbicyclo[2.2.2]-oct-2-ene-l-carboxylic, glucoheptonic, 3 -phenylpropionic, trimethylacetic, tert-butyl acetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, muconic acid, and the like. In some embodiments, “pharmaceutically acceptable salt” refers to base addition salts with an inorganic or an organic base. Inorganic bases which may be used to prepare salts include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, manganese, aluminum hydroxides, carbonates, bicarbonates, phosphates, and the like; particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium hydroxides, carbonates, bicarbonates, or phosphates. Organic bases from which may be used to prepare salts include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

[0084] As used herein, “medical imaging” or “medically imaging” refers to the use of various imaging technologies to produce visual representations of the internal or external structures and functions of a patient for diagnosis, treatment planning, or monitoring of medical conditions. Medical imaging includes, but is not limited to, X-ray, magnetic resonance imaging (“MRI”), computed tomography (“CT”), Positron Emission Tomography (“PET”), ultrasound, and fluorescence imaging. In certain embodiments, medical imaging refers to the use of fluorescence imaging to visualize and analyze the distribution and behavior of fluorescent molecules, such as CXL, in tissues and cells in a subject. In certain embodiments, medical imaging comprises detecting a signal emitted from a fluorescent molecule, such as CXL.

[0085] As used herein, “treating,” “treatment,” and the like mean ameliorating a disease, to reduce or eliminate its cause, its progression, its severity, or one or more of its symptoms, or otherwise beneficially alter the disease in a subject. In certain embodiments, “treating” or “treatment” refers to a subject at risk of developing a disease or of disease progression to a worse state. Prevention of a disease may involve complete protection from disease, for example, in the case of prevention of infection with a pathogen, or may involve prevention of disease progression, for example, from prediabetes to diabetes. For example, preventing a disease may not mean complete elimination of its effects at any level. Instead, it may mean preventing the symptoms of a disease to a clinically significant or detectable level. Preventing disease may also mean preventing its progression to a later stage.

[0086] The term “therapeutic agent” is art-recognized and refers to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance acting locally or systemically in a subject. Examples of therapeutic agents, also referred to as “drugs,” are described in well-known literature references such as the Merck Index (14thedition), the Physicians’ Desk Reference (64thedition), and The Pharmacological Basis of Therapeutics (12thedition). These therapeutic agents include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure, or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.

[0087] “Weight percent” or “wt.%” or “% (w / w)” refers to the ratio of the weight of the specified component in relation to the weight of the total composition, unless specified otherwise.

[0088] An “adverse event” is any untoward medical occurrence associated with treatment with a pharmaceutical composition described herein. A “mild adverse event” is easily tolerated by the subject, causes minimal discomfort, and does not interfere with everyday activities. A “moderate adverse event” is sufficiently discomforting to interfere with everyday activities; intervention may be needed. A “severe adverse event” prevents everyday activities; treatment or other intervention is usually needed. A “serious adverse event” results in death; is life-threatening (immediate risk of death from the event as it occurred); requires or prolongs inpatient hospitalization; results in persistent or significant disability / incapacity; or results in a congenital anomaly / disability, cancer, or drug overdose. An adverse event is incapacitating or disabling if it results in a substantial or permanent disruption of the subject’s ability to conduct normal life functions.

[0089] A patient is said to “tolerate” a dose of a compound if administering that dose to that patient does not result in an unacceptable adverse event or an unacceptable combination of adverse events. One skill in the art will appreciate that tolerance is a subjective measure and that what may be tolerable to one patient may not be tolerable to a different patient. For example, one patient may not be able to tolerate a headache. In contrast, a second patient may find headaches tolerable but cannot tolerate vomiting. For a third patient, either headache alone or vomiting alone is tolerable, but the patient cannot tolerate the combination of headache and vomiting, even when each is less severe than when experienced alone.

[0090] “Maximum tolerated dose” refers to the highest drug dose or therapeutic dose a patient can tolerate without experiencing intolerable side effects. The maximum tolerated dose is typically determined empirically in clinical trials.

[0091] “Effective amount” and “therapeutically effective amount” of an agent, compound, drug, composition, or combination is an amount that is nontoxic and effective for producing some desired therapeutic effect upon administration to a subject or patient (e.g., a human subject or patient). The precise therapeutically effective amount for a subject may depend upon, e.g., the subject’s size and health, the nature and extent of the condition, the therapeutics or combination of therapeutics selected for administration, and other variablesknown to those of skill in the art. The effective amount for a given situation is determined by routine experimentation and is within the clinician’s judgment.

[0092] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of all examples or exemplary language (e.g., “such as”) provided concerning certain embodiments herein is intended merely to better illuminate the present disclosure and does not limit the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0093] Groupings of alternative elements or embodiments of the present disclosure are not construed as limitations. Each group member can be referred to and claimed individually or combined with other group members or elements found herein. One or more group members can be included or deleted from a group for convenience or patentability reasons. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified, thus fulfilling the written description of all Markush groups used in the appended claims.

[0094] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each publication, patent, patent application, or other reference were specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0095] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.EXAMPLES

[0096] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure and thus can constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate thatmany changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1 - Lyophilized product (CXL-IT)

[0097] A lyophilized product was formed by dissolving CXL in a solution of calcium gluconate, polysorbate 80, hydroxypropyl-Z»eto-cyclodextrin, and sodium acetate in water at a ratio of 1 gram of dye-peptide conjugate per 10 liters of the solution, filtering the CXL solution, and lyophilizing the filtered CXL solution to form the lyophilized product.

[0098] The amounts of each component in the lyophilized product are shown in Table 2.Table 3 compares the prior lyophilized product CXL-HSA (disclosed in copending US patent application no. 17 / 119,305) and the lyophilized product CXL-IT shown in Table 2 (disclosed in copending PCT patent application nos. PCT / US24 / 28383 and PCT / US24 / 28386). Table 4 shows the drug product release specifications for CXL, including test items, test methods, and acceptance criteria.Table 2: Composition of CXL Lyophilized Product (CXL-IT)API = active pharmaceutical ingredient; JP = Japanese Pharmacopoeia; NF = NationalFormulary; Ph Eur = European Pharmacopoeia; qs = quantum sufficit (sufficient quantity);USP = United States Pharmacopeia.a% w / w was calculated based on the density value of 1.0099 g / mL at 5 °C.bNot listed in FDA’s Inactive Ingredient Database. Approved as a prescription and over-the- counter (OTC) drug as a sterile injection at 100 mg / mL (10%) and in tablet dosage forms.Table 3: Comparison of Pharmaceutical CompositionsAPI = active pharmaceutical ingredient, HSA = human serum albumin; NA = not applicable; qs = quantum sufficil WFI = water for injection.Table 4: Drug Product Release SpecificationEU = endotoxin unit; ID = identification; NMT = not more than; USP = United States Pharmacopeia; UV = ultraviolet; HPLC = high-pressure liquid chromatography; UPLC = ultrahigh-pressure liquid chromatography

[0099] Based on these results, the recommended long-term storage conditions for CXL-IT and CXL-HSA are -20 °C in a lyophilized powder form, protected from light. Under these conditions, both CXL lyophilized products are stable. However, many preclinical andnonclinical studies require the reconstituted pharmaceutical compositions to be prepared and maintained in solution for extended periods.

[0100] In certain embodiments, the lyophilized product is formulated to be reconstituted with water for injection or 5% Dextrose solution, as shown in Table 5.Table 5: CXL-IT formulated for reconstitution with water for injection

[0101] Generally, fresh preparation of the lyophilized CXL drug product in diluent ensures the highest stability and fluorescence signal. However, based on these results, serial dilutions of either pharmaceutical composition can be prepared, stored, and shipped frozen (e.g., -20 °C) with minimal loss of fluorescent signal. The CXL-IT lyophilized product is more stable than the CXL-HSA drug product when reconstituted with the diluent, subjected to serial dilution, or stored for long-term use.Example 2 - Phase lb clinical study of CXL-IT for lung cancer

[0102] This Phase lb, open-label study investigated CXL-IT (Example 1), a fluorescence imaging agent for visualizing tumor margins and other suspicious nodules in patients with a primary diagnosis or a high clinical suspicion of lung cancer based on CT, PET, biopsy, or other imaging. Up to 24 patients (four dose cohorts, up to six patients each) for Phase lb study (total of 24 patients maximum). Dosing interval or lung cancer type (e.g., lungadenocarcinoma, lung squamous cell carcinoma) are not stratified, but the evaluation of the timing of dose both the day before surgery and the day of surgery is attempted.

[0103] Patient participation in the study has the following durations:• A Screening period (2 to 60 days before the start of treatment).• Day 1, consisting of an initial period to collect baseline data, and a treatment period consisting of a single dose of CXL-IT administered on up to 4 days before surgery.• An interim visit between Days 2 to 10. The Interim Visit should occur between Day 2 and 10 (inclusive) after CXL-IT administration. This visit may occur up to 10 days after CXL-IT administration to accommodate the patient’s and site’s schedules, holidays, etc. An end-of-study (“EOS”) visit between Days 20 and 40 after surgery.

[0104] Toxicity is evaluated per National Cancer Institute Common Terminology Criteria for Adverse Events (“NCI-CTCAE”). The dose-limiting toxicity (DLT) observation period for CXL-IT is between dosing and the Interim Visit. A DLT is defined as an adverse event (AE) that is at least possibly related to CXL-IT and not reasonably attributed to the patient’s underlying disease, other medical conditions, or concomitant medications or procedures. Any Grade 2, 3, 4, or 5 AE at least possibly related to CXL-IT is considered a DLT. If no more than two DLTs have been observed in three patients, selected cohorts may enroll up to six patients to define a dose regimen. The CXL-IT dose administered to the first cohort of patients is 0.025 mg / kg. Depending on the results, subsequent doses may be increased or decreased.

[0105] Each dose is administered as a single intravenous injection up to four days before scheduled surgery, starting at the time of the first incision. See Table 6. Patients are assessed for local tolerance at the point CXL-IT is administered and for about one hour afterward.Table 6: CXL-IT Dose-FindingIV = intravenous; SRC = Safety Review Committee

[0106] The study used a variation of a Modified Toxicity Probability Interval (“mTPI”) design to inform decisions of whether to escalate or de-escalate a dose. The algorithm for this is presented in Table 7, showing the predicted number of patients treated (columns) versus the predicted number of patients who experience a DLT (rows), with the dosing decision at the intercept as follows: de-escalate to the next lower dose (D); de-escalate to a lower dose and never test this dose again (DU; / .< ., unacceptably toxic dose); escalate to the next higher dose (E); stay at the current dose (S); or stay at current dose if at maximum dose per Table 7 or change to a higher or lower dose (SC).Table 7: Dose-Finding Rules for CXL-IT per the mTPI PrincipleD = de-escalate to the next lower dose; DLT = dose-limiting toxicity; DU = unacceptably toxic dose; E = escalate to the next higher dose; mTPI = Modified Toxicity Probability Interval; S = stay at the current dose; S / C = stay at current if at maximum dose or escalate to the next higher dose if at a lower dose.Fluorescence Imaging Criteria

[0107] For each dose of CXL-IT during Period 1, fluorescence intensity and / or ratio imaging results for in vivo and ex vivo tissues were assessed by analyzing videos recorded during surgery. Per the standard of care (“SOC”), tissue was collected during surgery for histopathologic analysis. Clinical observations demonstrated that CXL performed reliably across a flexible range of dosing intervals. When administered one day before surgery, theagent consistently produced clear fluorescence signals, while dosing three days prior still yielded robust imaging outcomes with sufficient tumor-to-background contrast. Even same-day dosing has been shown to generate effective fluorescence, thus providing multiple valid windows for administration.

[0108] Patients were assessed for local tolerance at the CXL-IT administration site for about one hour afterward. If the administration of CXL-IT was interrupted due to administration- related reactions, the patient received no more CXL-IT and underwent the procedures and evaluations required at the EOS visit. The patient was followed as clinically indicated to assess the resolution of any adverse reactions related to administration. Based on the results from the rat and minipig toxicity studies, a starting dose of 0.025 mg / kg provided appropriate safety margins for the clinical trial.Table 8: Trial Schedulea. CXL administration occurred up to 4 days before surgery. b. The surgery and the surgeons used imaging devices the day after CXL administration. c. A 12-lead ECG was obtained at Screening, on Day 1 at any time before CXL-IT administration, on Day 1 within 60 minutes after CXL-IT administration, Interim Visit, and at EOS (Days 25 to 35) or ET. d. Patients received a single dose of CXL-IT by IV up to 4 days before scheduled surgery. CXL administration occurs before other pre-operative medications (e.g., antibiotics). Before administration, the patient could receive pre-medication for IV contrast agents per standard of care. e. Height (cm) should be measured at Screening only, but no later than on Day 1 before CXL-IT administration. The weight collected at the Screening Visit was used for the dose level on the Day of Administration.f. Medical history included confirmation of lung cancer diagnosis, significant concomitant illnesses / diseases, prior therapy (for lung cancer and other prior cancer), and current symptoms. g. A complete physical examination was performed at Screening or on Day 1 before CXL- IT administration. If a physical examination occurred during Screening, an abbreviated physical exam could be conducted on Day 1. All subsequent physical examinations, / .< ., at the Interim Visit, and at EOS or ET, are abbreviated examinations. h. ECOG performance status was assessed at Screening, on Day 1 before CXL-IT administration, and at EOS or ET. i. A serum pregnancy test (human chorionic gonadotropin (“hCG”)) should be done for all female patients of childbearing potential at Screening. A urine or serum pregnancy test should be done on Day 1 before CXL-IT administration, with results reviewed before CXL-IT administration to confirm patient eligibility. A serum pregnancy test should be repeated at EOS or ET. j . Hematology included red blood cell (“RBC”) count, hematocrit, hemoglobin concentration, mean corpuscular hemoglobin (“MCH”), mean corpuscular volume (“MCV”), % reticulocytes; white blood cell (“WBC”) count with differential: basophils, eosinophils, lymphocytes, monocytes, neutrophils, platelet count. k. Clinical chemistry included alkaline phosphatase (“ALP”), alanine aminotransferase (“ALT”), aspartate aminotransferase (“AST”), lactate dehydrogenase (“LDH”), albumin, blood urea nitrogen (“BUN”), creatinine, glucose, total bilirubin, total protein, bicarbonate, calcium, chloride, potassium, and sodium. Calculated CrCl was also required. Blood samples for clinical chemistry were drawn at Screening, on Day 1 before CXL-IT administration, Interim Visit, and at EOS or ET. All events of ALT [or AST] > 3 x ULN and total bilirubin > 1.5 x ULN (> 35% direct bilirubin) or ALT [or AST] > 3 * ULN, and INR > 1.5 (if INR measured), which may indicate severe liver injury (possible Hy’s law), must be reported expeditiously. l. Coagulation tests included activated partial thromboplastin time (“aPTT”), international normalized ratio (“INR”), and prothrombin time (“PT”). Coagulation tests were done on blood samples drawn at Screening and on Day 1 within 4 hours post-dosing. Next day blood samples were not collected from patients dosed more than one day (24 hours) before surgery.m. Urinalysis with microscopic examination included an assessment of blood, glucose, ketones, leukocytes, protein, pH, and specific gravity by dipstick. Microscopic examination was included if blood or protein was abnormal. Urine was inspected for appearance at collection, including color. n. Clinical laboratory tests were repeated before dosing if the Screening Visit occurred between 60 and 31 days before dosing. o. Times of blood sample collection for PK analysis were provided for participating patients. p. The surgeon removed tissue for pathology review per standard of care. Tissue and images may be sent to a central laboratory for additional review and analysis per the Exploratory Objective. q. Patients were assessed for AEs when CXL-IT was administered. Patients should be observed for at least one hour after CXL-IT administration for potential administration- related reactions. r. If a study participant experienced a potential hypersensitivity reaction, a sample for tryptase level should be collected within 3 hours of the suspected reaction and 24 to 28 hours after the onset of the suspected reaction. s. In case of hypersensitivity reaction, surgery may be delayed as clinically indicated at the discretion of the investigator.

[0109] This study enrolled patients scheduled to undergo MIS for known or suspected lung cancer via thoracoscopy (i.e., VATS or RATS). To be eligible for participation, each patient must meet all the following inclusion criteria and none of the following exclusion criteria.

[0110] Patients were eligible to be included in the study only if all the following criteria applied:1. Male or Female patients greater than 18 years of age, inclusive, at the time of signing the informed consent.2. ECOG performance status of 0 to 2.3. Have a primary diagnosis, or a high clinical suspicion, for cancer in the lung based on CT, biopsy, or other imaging.4. Are scheduled to undergo surgical thoracoscopy and resection of the lung.5. If of childbearing potential, the patient must have a negative serum pregnancy test at screening, on Day 1 before CXL-IT administration, and use a medically acceptable form of contraception (e.g., hormonal birth control, double-barrier method) or abstinence.6. Ability to understand the requirements of the study, provide written informed consent and authorization of use and disclosure of protected health information, and agree to abide by the study restrictions and to return for the required assessments.[OHl] Patients were excluded from the study if any of the following criteria applied:1. Contraindications for surgery or any medical condition that could jeopardize the subject’s safety.2. No planned surgical use of any other fluorescent optical imaging agent, or exposure to another optical imaging agent within 8 weeks before surgery (e.g., Cytalux™).3. History of any drug-related hypersensitivity or anaphylactic reactions, including those attributed to ICG or other agents used in the study, or known shellfish allergies.4. Known sensitivity to fluorescent light.5. Patients with impaired renal function as defined by a CrCl <60 mL / min at Screening, according to the Cockcroft-Gault formula ((140 - age) x body weight / plasma creatinine x 72 (x 0.85 for female).6. History, or presence in the ECG at Screening, of any clinically significant abnormalities, including cardiac conduction abnormalities such as a QTcF > 470 ms.7. History of radiation therapy to the chest.8. Received a systemic investigational drug of any kind for any indication within 4 weeks or 5 half-lives before administration of CXL-IT, and / or the patient has received CXL-IT previously.9. Total bilirubin level >1.5 times the upper limit of normal at screening.10. Aspartate aminotransferase (“AST”) / serum glutamic-oxaloacetic transaminase (“SGOT”) and alanine aminotransferase (“ ALT”) / serum glutamic-pyruvic transaminase (“SGPT”) > 2.5 times the upper limit of normal (“ULN”) at Screening.11. The patient is pregnant, breastfeeding, or plans to become pregnant within 6 months of the administration of CXL-IT or has a positive serum pregnancy test at Screening or a positive urine pregnancy test on the day of surgery or day of admission for female subjects of childbearing potential.

[0112] All fluorescence imaging endpoints in which fluorescence was compared were assessed with fluorescence assessed intraoperatively by the surgeon and postoperatively upon subsequent review of the fluorescence image files. Fluorescence imaging of the surgical fields was performed by the surgeon or other qualified medical personnel using commercialor prototype fluorescence imaging systems. The fluorescence imaging data from each patient were evaluated using fluorescence analysis tools to score each image per a series of parameters.

[0113] For the primary assessments, the surgeon used one or more NIR fluorescence imaging systems to visualize the CXL fluorescent signal, video-record, and score the fluorescence signal from the following tissues: the primary tumor viewed endoscopically in situ, and any additional potential foci of malignant tissue viewed endoscopically in situ that were excised. CXL fluorescence image data from these tissues (e.g., fluorescence signal intensity, tumor to non-tumor ratio, etc.) were evaluated. Additional imaging systems can be used to evaluate resected tissue ex vivo per the exploratory endpoints, such as an Al system trained on relevant medical images. Imaging systems can be used to evaluate resected tissue ex vivo (“back table”) per the exploratory endpoints. The specimens were imaged before transport to pathology.

[0114] Before using NIR fluorescence imaging, the surgeon followed SOC protocols to detect and count potential tumors, nodules, and positive lymph nodes. In certain embodiments, this information was documented in real time. The surgeon then used a NIR fluorescence imaging in situ in the endoscopic surgical field to detect and count potential tumors, nodules, and positive lymph nodes, which may also be documented in real time. This prolonged the surgery by about 8 to 10 minutes. After NIR imaging in situ, the surgeon followed SOC techniques to excise potential tumors, nodules, and positive lymph nodes. If the fluorescence signal drew attention to areas suspicious for cancer, and if the resection of this tissue did not significantly change the scope of the surgical procedure or the safety outcome to the patient, then, at the surgeon’s discretion, the tissue was excised. Information on the removed specimens was documented, including type of specimen(s), number of specimens, specimen location, method of identification (SOC or fluorescent imaging or both), etc. Also, the margins (e.g., distance of lesion to nearest staple lines) were imaged to determine if the lesion margin is within 10 mm of the nearest staple line.

[0115] For the exploratory ex vivo assessments, the surgeon may visualize and score excised specimens using a NIR fluorescent imaging mode to determine positive and negative tumor margins of the primary specimen, positive and negative tumor margins in any additional specimens from the area of the primary tumor, and potential foci of additional cancer excised, such as secondary pulmonary nodules or potential lymph nodes. The fluorescence image datafrom these tissues e.g., fluorescence signal intensity, tumor-to-non-tumor ratio, etc.) were evaluated.

[0116] The assessments for the exploratory endpoints included the same tissue specimens or sectioning of the same blocks used by a pathology laboratory for histological staining with a mouse monoclonal antibody against phosphorylated Tyr(23) ANXA2, or fluorescence microscopy for CXL in the tissue sections. These staining results were compared with standard histopathology results to determine specificity and sensitivity.Safety Assessments

[0117] Relevant results were reviewed before CXL-IT administration to confirm patient eligibility. (See Tables 9 and 10.) Patients underwent surgical thoracoscopy, VATS, and RATS lung resection, a minimally invasive thoracic surgery performed using a small video camera mounted to a fiberoptic thoracoscope (e.g., 5-mm or 10-mm caliber), with or without angulated visualization (e.g. 0°, 30°, 45°, and 90°), and flexibility (rigid versus flexible scopes), which allowed the surgeon to see inside the chest by viewing the video images relayed onto a television screen, and perform procedures using elongated surgical instruments, such as endoscopes. The camera and instruments were inserted into the patient’s chest cavity through small incisions in the chest wall, usually via specially designed guiding tubes known as “ports.”Table 9: Safety Laboratory Tests

[0118] Table 10: Patient and cancer characteristics

[0119] VATS and RATS procedures were performed with either conventional or laparoscopic instruments. Unlike in laparoscopy, carbon dioxide insufflation was optional in VATS and RATS because of the thoracic cage’s inherent rigidity. However, lung deflation on the side of the operated chest enables the surgeon to visualize and pass instruments into the thorax. Potential complications from VATS and RATS lung resection procedures may include conversion to an open procedure due to massive bleeding, airway injury, and / or the presence of problematic adhesions. The most common complications after VATS and RATS lung resection were an air leak from the lung in the chest cavity that requires a chest tube, bleeding, infection, blood clots, arrhythmia, and / or pain.

[0120] Primary pharmacokinetic (“PK”) endpoints include concentration-time profiles of CXL and its main metabolite in plasma, standard non-compartmental PK parameters of CXL, including the maximum observed plasma concentration (Cmax), area under the plasma concentration time curve from time 0 extrapolated to infinite time (AUCo- / [AUCinf]), area under the plasma concentration-time curve from time 0 to the last quantifiable, plasma concentration (AUCo-t [AUCiast]), time to reach maximal plasma concentration (Tmax), elimination / apparent terminal elimination half- life (ti / 2), terminal elimination slope (Az), and apparent clearance (CL).

[0121] Although a prior Phase la breast cancer study was conducted on a similar formulation (CXL-HSA) to CXL-IT, no PK data were generated. However, prior nonclinical studies using both CXL formulations, CXL-HSA and CXL-IT, were used to model a blood-collection sampling scheme in humans using allometric scaling. These studies included separate GLP rat toxicity / toxicokinetic studies for each CXL formulation and a non-GLP rat PK study that compared both formulations. The preliminary PK modeling suggested that blood collection should begin within minutes of administration because the Cmax occurred immediately after administration, and the estimated plasma half-life for the formulations ranged from about 0.6 hours to about 5 hours.

[0122] All treatment-emergent events of ALT [or AST] > 3 x upper limit of normal (ULN) and total bilirubin > 1.5 x ULN (> 35% direct bilirubin) or ALT [or AST] > 3 x ULN and international normalized ratio (“INR”) > 1.5 (if INR measured), which may indicate severe liver injury (possible Hy’s law).

[0123] FIGS. 1 to 12 show images of lung cancer resections performed in this clinical study. The most common procedures in this Phase lb study involved wedge resections, segmentectomies, and lobectomies. Due to the patients presenting as Stage 1 or 2, full lung resections (pneumonectomy) did not take place.

[0124] FIG. 1 shows an endoscopic in-situ white light image of a lung containing the primary tumor of an adenocarcinoma. This image was captured on a Karl Storz Rubina in a patient dosed with CXL at 0.1 mg / kg on the day before surgery. The location of the primary tumor is unclear on the white-light image. FIG. 2 shows a near-infrared (“NIR”) CXL fluorescence overlay on FIG. 1, which visualizes the primary tumor.

[0125] FIG. 3 shows an endoscopic in-situ white light image of a lung containing the primary tumor of a metastatic melanoma. This image was captured on a Karl Storz Rubina in a patientdosed with CXL at 0.1 mg / kg on the day before surgery. The location of the primary tumor is unclear on the white-light image. FIG. 4 shows a NIR CXL fluorescence overlay on FIG. 3, which visualizes the primary tumor.

[0126] FIG. 5 shows an endoscopic in-situ white light image of a lung containing the primary tumor of an adenocarcinoma. The image was captured on a Medtronic VisionSense in a patient dosed with CXL at 0.5 mg / kg on the day before surgery. The location of the primary tumor is unclear on the white-light image. FIG. 6 shows a NIR CXL fluorescence overlay on FIG. 5, which visualizes the primary tumor.

[0127] FIG. 7 shows an endoscopic in-situ white light image of a lung containing the primary tumor of an adenocarcinoma. The image was captured on an Intuitive Surgical da Vinci Sensitive Firefly in a patient dosed with CXL at 0.5 mg / kg on the day before surgery. The location of the primary tumor is unclear on the white-light image. FIG. 8 shows a NIR CXL fluorescence overlay on FIG. 7, which visualizes the primary tumor.

[0128] FIG. 9 identifies a lesion within 10 mm of the staple line that white light alone could not detect. The image was captured on Medtronic VisionSense of an adenocarcinoma in a patient dosed with CXL at 0.075 mg / kg on the day before surgery. FIG. 10 shows the NIR CXL fluorescence overlay on FIG. 9, which visualizes the lesion.

[0129] FIG. 11 identifies a lesion within 10 mm of the staple line that white light alone could not detect. The image was captured on Medtronic VisionSense of an adenocarcinoma in a patient dosed with CXL at 0.1 mg / kg on the day before surgery. FIG. 12 shows the NIR CXL fluorescence overlay on FIG. 11, which visualizes the lesion.

[0130] In nearly half of the cases, lesions or resection margins were identified solely by fluorescence imaging with CXL, whereas these findings were not observed under conventional white light. For instance, multiple surgeries documented that primary tumors and synchronous nodules were detected exclusively through the fluorescence modality, and margins were evaluated to within 10 mm — a threshold not achieved with standard imaging. These clinically significant events, verified by subsequent histopathological analysis, quantitatively demonstrate that CXL enhances surgical visualization and tumor delineation in scenarios where traditional methods may overlook tissue boundaries.

[0131] Furthermore, quantitative analysis of intraoperative fluorescence images using CXL objectively supports its utility in differentiating malignant tissue from normal background tissue. Measured mean fluorescence intensity values (FIG. 13), together with calculatedtumor-to-background ratios (FIG. 14), consistently demonstrated significant contrast between cancerous regions and adjacent non-malignant areas. These reproducible metrics confirm that CXL consistently enhances tumor delineation.

[0132] Adjustments during surgery can improve fluorescence detection. For instance, in subject 001-005, the surgeon adjusted the intensity and blackpoint sliders in Sensitive Firefly mode. This modification allowed a fluorescent signal from a lesion in the left upper lobe to become clear, even though the lesion was not easily seen under the default settings. In another subject (001-010), the intact resected tissue did not exhibit a distinct fluorescence signal. However, once the specimen was sectioned, a small lesion of about 4-5 mm was clearly visible under fluorescence. Thus, adjusting imaging device settings or sectioning tissue can improve the visual contrast between malignant tissue and the background.

[0133] CXL was well tolerated and simple to administer via a 15-minute IV infusion. The CSEs are summarized in Table 11 below. There were no serious adverse events. One patient had a transient Grade 1 elevation in blood pressure, possibly related to CXL, that resolved without sequelae. Dose escalation proceeded over four cohorts without a dose-limiting toxicity to a dose of 0.1 mg / kg

[0134] Table 11 : Clinically significant events

[0135] Twenty-four patients received CXL under varied dosing regimens and time intervals without any serious adverse events, despite the inherent risks associated with thoracoscopic and robotic-assisted lung surgeries. Comprehensive intraoperative and postoperative monitoring confirmed that no patient experienced complications attributable to the agent, even when it was administered on the day of surgery or up to 3 days beforehand.

[0136] Moreover, the study data show that CXL performs well in both thoracoscopic (VATS) and robotic (RATS) approaches, demonstrating its broad clinical application. Surgeons have mentioned that when they switch from video-assisted surgery to robotic surgery, they lose the ability to palpate or feel the lesion. So, using a fluorescent imaging agent is an added benefit in robotic surgeries where palpation is not an option. For example, in a VATS procedure using Medtronic VisionSense and Karl Storz Rubina, surgeons reliably detected fluorescence that clearly outlined tumor margins in patients with adenocarcinoma. In robotic procedures using the da Vinci Firefly system, cases, such as subjects 001-027 and 001-030, showed that even when white-light imaging did not reveal a clear tumor, fluorescence imaging with CXL identified lesions that were later confirmed by histopathology.

[0137] Surgeon’s visual assessment of fluorescence signal improved by increasing dose; 0.1 mg / kg consistently yielded the highest visible signal. CXL has been visualized across a wide dosing interval from the day of surgery through three days before surgery. The dosing interval will continue to be evaluated in the next phase of development. Mean fluorescence signal intensity improved without altering the tumor-to-background ratio in both VATS and RATS procedures, with the highest signal in situ observed at a dose of 0.1 mg / kg. (See FIGS. 13 &14.) CSEs were reported for all 3 CSE categories: 48% of surgeries had at least one CSE. (See Table 11 for details.)

Claims

AMENDED CLAIMS received by the International Bureau on 14 APR 2026 (14.04.2026)What is claimed is:

1. A method for surgically removing lung cancer tissue in a patient in need thereof, comprising: administering an effective amount of a pharmaceutical composition comprising CXL to a patient in need thereof; detecting a signal emitted from the CXL in lung cancer tissue of the patient; and suigically excising a portion of the lung cancer tissue identified from the emitted signal, wherein the signal emitted from the CXL comprises a signal emitted from a metabolite of CXL formed in the patient after administration.

2. The method of claim 1, wherein the signal emitted from the CXL or the metabolite thereof is bound to a receptor.

3. The method of claim 1 or 2, where detecting and / or surgical excision are endoscopic.

4. The method of any one of claims 1 to 3, wherein the administration is via an intravenous delivery method.

5. The method of any one of claims 1 to 4, wherein the effective amount ranges from about 0.001 mg / kg to about 0.2 mg / kg, or optionally from about 0.001 mg / kg to about 0.015 mg / kg, or optionally from about 0.001 mg / kg to about 0.050 mg / kg, or optionally from about 0.001 mg / kg to about 0.100 mg / kg.

6. The method of claim 5, wherein the effective amount is chosen from about 0.1 mg / kg, 0.05 mg / kg, 0.075 mg / kg, from about 0.025 mg / kg, about 0.0125 mg / kg, about 0.00625 mg / kg, and about 0.003125 mg / kg.

7. The method of any one of claims 1 to 6, wherein the administration occurs up to about 4 days before detecting the signal, optionally between about 4 days to about 3 days before detecting the signal, optionally between about 2 days to about 1 day before detecting the signal, or optionally between about 24 hours to about 1 hour before detecting the signal.

8. The method of any one of claims 1 to 7, wherein the medical imaging is performed using a 510(k)-cleared imaging system capable of simultaneous fluorescence and white light imaging.

9. The method of any one of claims 1 to 8, wherein the signal identifies potentially cancerous tissue, including within a primary tumor site, other undiagnosed tumors, tumor margins, surgical cavity, or lymph nodes draining the primary tumor site in the patient.

10. The method of any one of claims 1 to 9, wherein the patient is suspected of having lung adenocarcinoma, lung squamous cell carcinoma, or neuroendocrine carcinoma.

11. The method of any one of claims 1 to 10, wherein the surgical excision comprises thoracoscopic lung resection.

12. The method of any one of claims 1 to 11, wherein each patient serves as their own control for imaging by comparing captured signals with baseline anatomical images.

13. The method of any one of claims 1 to 12, after surgically excising the portion of lung cancer tissue, further comprising one or more of the following steps: detecting a signal emitted from the CXL or a metabolite thereof to visualize surgical margins; or assessing resected specimens for fluorescence signals indicating residual cancerous tissues; or confirming tissue status using histopathological evaluation post-surgery.

14. The method of claim 13, wherein histopathological confirmation comprises standard staining and fluorescence microscopy.

15. The method of either claim 13 or 14, further comprising surgically excising a further portion of the lung cancer tissue.

16. The method of any one of claims 1 to 15, wherein findings of fluorescence imaging predict pathological outcomes confirmed by histological analysis.

17. The method of any one of claims 1 to 16, further comprising determining intraoperative fluorescence visualization of lung tissue types based on a semi-quantitative assessment.

18. The method of any one of claims 1 to 17, wherein fluorescence imaging characteristics are evaluated based on cancer type, histological grade, anatomical location, folate receptor status, and other potential receptor statuses.

19. The method of any one of claims 1 to 18, further comprising correlating CXL fluorescence with central pathology review of lung cancer samples using pANXA2 immunohistochemistry staining.

20. The method of any one of claims 1 to 19, wherein additional suspicious nodules or lymph nodes visualized in situ during imaging are validated by excised tissue specimens.

21. The method of any one of claims 1 to 20, further comprising evaluating fluorescence signal intensity and signal-to-background ratio in defined regions of interest using a differing imaging device.

22. The method of any one of claims 1 to 21, wherein the patient is excluded if they have a history of drug-related hypersensitivity or anaphylactic reactions, including sensitivities to indocyanine green or shellfish.

23. The method of any one of claims 1 to 22, wherein the patient is excluded if they are known to have sensitivity to fluorescent light.

24. The method of any one of claims 1 to 23, wherein the patient is excluded if they have impaired renal function, characterized by a creatinine clearance of less than about 60 mL / min.

25. The method of any one of claims 1 to 24, wherein the patient is excluded if they show clinically significant abnormalities in their echocardiogram, such as a QT interval corrected for heart rate by Fridericia’s formula exceeding about 470 ms.

26. The method of any one of claims 1 to 25, wherein the patient is excluded if they have a total bilirubin level greater than about 1.5 times the upper limit of normal at screening.

27. The method of any one of claims 1 to 26, wherein the patient is excluded if their aspartate aminotransferase or alanine transaminase levels are more than about 2.5 times the upper limit of normal at screening.

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