Composition comprising pafrazonin for identifying malignant

Abstract

The compositions of the invention are configured to be administered to a subject undergoing a malignant lesion resection procedure. The composition comprises a pharmaceutically effective amount of pallaxinine or a pharmaceutically acceptable salt thereof. Parelaxinine, or a pharmaceutically acceptable salt thereof, is configured to bind to one or more malignant lesions and emit an optical signal.

Description

[0001] Related Applications

[0002] This patent application claims priority to U.S. patent application serial number 18 / 111,704 filed on February 20, 2023 and U.S. provisional patent application serial number 63 / 312,965 filed on February 23, 2022, the contents of each of which are incorporated by reference into this disclosure in their entirety. Background Art

[0003] Many treatment plans for cancer, such as ovarian cancer and lung cancer, include surgery. For example, a patient with cancer may undergo surgery with the goal of removing cancer cells. In some aspects, a method or system can be used to help surgeons visualize malignant lesions that are intended to be removed by surgery. In some such cases, an imaging agent can be provided to the patient, and a light source or imaging technology can be used to capture the imaging agent in the patient's body. For example, the patient can receive the imaging agent by ingestion, intravenous injection, local or other means. Such visualization techniques can produce better surgical results (e.g., removing a higher percentage of cancer cells) than surgery without the use of visualization techniques.

[0004] Although conventional visualization compositions, methods, and techniques can provide better visualization of cancerous lesions in a patient's body than surgery without any visualization compositions, methods, or techniques, such conventional compositions, methods, and techniques may not be completely effective in identifying cancerous parts of the body. Therefore, there is a need for compositions and methods that more effectively identify cancerous lesions in a patient's body.

[0005] Embodiments of the present invention address these and other challenges individually and collectively.

[0006] Additional limitations and disadvantages of conventional and traditional approaches will become apparent to those skilled in the art by comparing such systems with certain aspects of the present disclosure as described in the remainder of this application and with reference to the accompanying figures. Summary of the invention

[0007] The inventors have recognized the need for improved visualization techniques and compositions to identify cancerous lesions in a patient. Therefore, the compositions, methods, and techniques described herein may enable visualization of cancerous tumors, cells, and / or lesions that are not identifiable by conventional compositions and techniques.

[0008] It is believed that the present technology targets folate receptors (FRs) that can be overexpressed in certain cancer cells, such as ovarian cancer cells and lung cancer cells. The disclosed compositions, methods and techniques comprising pafolacianine are believed to bind to cancer cells expressing FRs and fluoresce when exposed to light, allowing visualization of cancer cells expressing FRs bound to pafolacianine. The present technology can improve the detection of cancer cells and the specificity of the detection.

[0009] In one aspect, a composition is configured for administration to a subject undergoing surgery to remove a malignant lesion, the composition comprising a pharmaceutically effective amount of pafolacinin, wherein the pafolacinin is configured to fluoresce to identify one or more malignant lesions.

[0010] In some aspects, the one or more malignancies include ovarian cancer. In other aspects, the one or more malignancies include lung cancer. In still other aspects, the pharmaceutically effective amount of pafolacinin comprises at least about 0.25 mg of pafolacinin per about 1 kg of subject body weight. In additional aspects, the composition is configured to be administered intravenously to a subject.

[0011] In some aspects, the composition comprises an injectable solution. In other aspects, the solution is blue-green. In other aspects, the solution is transparent or translucent. In other aspects, the composition comprises between about 1 mg and about 20 mg of pavlacine per about 1.6 mL volume of solution. In other aspects, the composition comprises about 3.2 mg of pavlacine per about 1.6 mL volume of solution.

[0012] In some aspects, about 3.2 mg of pavlacine per about 1.6 mL volume of solution is equivalent to about 3.4 mg of pavlacine sodium per about 1.6 mL volume of solution. In other aspects, pavlacine sodium has the following chemical structure:

[0013]

[0014] In some aspects, the composition comprises at least one of sodium chloride, potassium dihydrogen phosphate or sodium phosphate dibasic heptahydrate. In other aspects, the composition comprises sodium chloride between about 1 mg and about 50 mg per about 1.6 mL volume of solution. In other aspects, the composition comprises about 14.4 mg sodium chloride per about 1.6 mL volume of solution. In other aspects, the composition comprises potassium dihydrogen phosphate between about 0.01 mg and about 5 mg per about 1.6 mL volume of solution.

[0015] In some aspects, the composition comprises about 0.23 mg potassium dihydrogen phosphate per about 1.6 mL volume of solution. In other aspects, the composition comprises sodium dihydrogen phosphate heptahydrate between about 0.1 mg and about 10 mg per about 1.6 mL volume of solution. In other aspects, the composition comprises about 1.27 mg sodium dihydrogen phosphate heptahydrate per about 1.6 mL volume of solution. In other aspects, the composition comprises about 3.2 mg pafolacinin, about 14.4 mg sodium chloride, about 0.23 mg potassium dihydrogen phosphate and about 1.27 mg sodium dihydrogen phosphate heptahydrate per about 1.6 mL volume of solution.

[0016] In some aspects, the pH of the composition is between about 6 and about 8. In other aspects, the composition comprises at least one of sodium hydroxide or hydrochloric acid. In still other aspects, at least one of sodium hydroxide or hydrochloric acid is used to adjust the pH of the composition. In other aspects, the solution comprising the composition is diluted before the composition is administered to a subject.

[0017] In some aspects, the solution is diluted with 5% glucose solution. In other aspects, the solution is diluted with about 220mL to about 250mL of 5% glucose solution. In other aspects, the composition is configured to be applied to the subject about 1 to about 24 hours before the malignant lesion resection operation. In other aspects, the composition is configured to be applied to the subject over a period of time.

[0018] In some aspects, the period of time includes about 1 to about 3 hours. In other aspects, pavlacinin fluoresces when exposed to light in the near infrared range. In still other aspects, pavlacinin fluoresces when exposed to light with a wavelength between about 760 nm and about 785 nm. In additional aspects, pavlacinin emits fluorescence between about 790 nm and about 815 nm.

[0019] In some aspects, the composition is contained in a vial. In other aspects, the vial is a single-dose vial. In other aspects, a carton contains at least 10 vials. In other aspects, at least 10 vials are individually packaged.

[0020] In some aspects, the subject is an animal. In other aspects, the subject is a mammal. In still other aspects, the subject is a human. In other aspects, the subject is an adult.

[0021] In one aspect, a method of treating a subject undergoing surgical resection of a malignant lesion comprises administering to the subject a pharmaceutically effective amount of a composition comprising pafolacinin; and causing the pafolacinin to fluoresce.

[0022] In some aspects, the method includes identifying one or more malignant lesions based on the fluorescence of pafolacinin. In other aspects, the method includes resection of the one or more malignant lesions. In still other aspects, the one or more malignant lesions include ovarian cancer. In other aspects, the one or more malignant lesions include lung cancer.

[0023] In some aspects, the pharmaceutically effective amount comprises at least about 0.25 mg of pavlacin per about 1 kg of subject body weight. In other aspects, the composition is administered to the subject intravenously. In still other aspects, the composition comprises an injectable solution. In additional aspects, the solution is blue-green.

[0024] In some aspects, the solution is transparent or translucent. In other aspects, the solution comprises between about 1 mg and about 20 mg of pafolacin per about 1.6 mL volume of solution. In still other aspects, the composition comprises about 3.2 mg of pafolacin per about 1.6 mL volume of solution. From other aspects, about 3.2 mg of pafolacin per about 1.6 mL volume of solution is equivalent to about 3.4 mg of pafolacin sodium per about 1.6 mL volume of solution.

[0025] In some aspects, pavlacidine sodium has the following chemical structure:

[0026]

[0027] In some aspects, the solution further comprises at least one of sodium chloride, potassium dihydrogen phosphate or sodium phosphate dibasic heptahydrate. In other aspects, the solution comprises sodium chloride between about 1 mg and about 50 mg per about 1.6 mL volume of solution. In other aspects, the solution comprises about 14.4 mg sodium chloride per about 1.6 mL volume of solution. In other aspects, the solution comprises potassium dihydrogen phosphate between about 0.01 mg and about 5 mg per about 1.6 mL volume of solution.

[0028] In some aspects, the solution comprises about 0.23 mg potassium dihydrogen phosphate per about 1.6 mL volume of solution. In other aspects, the solution comprises about 0.1 mg and about 10 mg disodium hydrogen phosphate heptahydrate per about 1.6 mL volume of solution. In other aspects, the solution comprises about 1.27 mg disodium hydrogen phosphate heptahydrate per about 1.6 mL volume of solution. In other aspects, the solution comprises about 3.2 mg pafolacinin, about 14.4 mg sodium chloride, about 0.23 mg potassium dihydrogen phosphate and about 1.27 mg disodium hydrogen phosphate heptahydrate per about 1.6 mL volume of solution.

[0029] In some aspects, the pH of the solution is between about 6 and about 8. In other aspects, the solution further comprises at least one of sodium hydroxide or hydrochloric acid. In still other aspects, at least one of sodium hydroxide or hydrochloric acid is used to adjust the pH of the solution. In other aspects, the method includes diluting the solution before administering the composition to a subject.

[0030] In some aspects, the dilution solution comprises diluting the solution with a 5% glucose solution. In other aspects, the dilution solution comprises diluting the solution with about 220 mL to about 250 mL of a 5% glucose solution. In other aspects, administering a pharmaceutically effective amount of a composition comprises administering a pharmaceutically effective amount of a composition about 1 to about 24 hours before a malignant lesion resection operation. In other aspects, administering a pharmaceutically effective amount of a composition comprises administering a pharmaceutically effective amount of a composition over a period of time.

[0031] In some aspects, the period of time comprises about 1 to about 3 hours. In other aspects, causing pfolacinin to fluoresce comprises exposing pfolacinin to light in the near infrared range. In still other aspects, causing pfolacinin to fluoresce comprises exposing pfolacinin to light with a wavelength between about 760 nm and about 785 nm. In further aspects, pfolacinin emits fluorescence between about 790 nm and about 815 nm.

[0032] In some aspects, the composition is contained in a vial. In other aspects, the vial is a single-dose vial. In other aspects, a carton contains at least 10 vials. In other aspects, at least 10 vials are individually packaged.

[0033] In some aspects, the subject is an animal. In other aspects, the subject is a mammal. In still other aspects, the subject is a human. In other aspects, the subject is an adult.

[0034] In some aspects, the fluorescence of pavlascinin is detected using an imaging system or imaging software. In another aspect, the imaging system or imaging software is selected from the group consisting of: imaging system FAST (fiber array scanning technology), flow cytometry, confocal microscopy, two-photon microscopy, epifluorescence microscopy, fluorescence microscopy, fluorescence goggles, and innovative wearable devices. In some aspects, the fluorescence of pavlascinin is detected in vivo.

[0035] In some aspects, the method also includes guiding the flexible probe to the malignant lesion after applying the composition comprising pafolacinin. In some aspects, the flexible probe is a flexible endoscope, a fluorescent endoscopic imaging probe, a fiberscope, a videoscope, a gastroscope, a colonoscope, a bronchoscope, a laryngoscope, a cystoscope, a duodenoscope, an enteroscope, a ureteroscope, a sigmoidoscope, an enteroscope, a choledochoscope, a nasolaryngoscope, an angioscope or a hysteroscope. In other aspects, the flexible probe is equipped to detect the wavelength with maximum absorption and emission between about 400nm and about 900nm.

[0036] In some aspects, the resection procedure is performed using an iBiopsy, iKnife, iLaser, iBurner, a powered cutting loop, a rotating blade, a curved blade, an expandable blade, a dissector with a cutting blade, a blunt dissector, forceps, an electrolytic element, a biopsy needle, a microwave ablation probe, a radiofrequency ablation probe, a cryoablation probe, or a laser.

[0037] In some aspects, the resection procedure is non-invasive.

[0038] In some aspects, the resection procedure is performed manually or using robotic-assisted techniques. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is a graph illustrating the relative binding of pavlacinim, the L-isomer of pavlacinim (OTL0039), and folic acid to FR.

[0040] Figure 2A is a whole-body fluorescent image of a FR-positive KB tumor-bearing mouse injected with pafolacinin. Figure 2B Whole-body fluorescence images of FR-positive KB tumor-bearing mice injected with the L-isomer of pavlazone (OTL0039). Figure 2C This is a fluorescence image of tissue distribution of pafolacinin 2.5 hours after injection. Figure 2D The fluorescence images of tissue distribution of L-isomer of palforacenine (OTL0039) 2.5 hours after injection.

[0041] Figure 3A is a whole-body fluorescent image of a FR-negative A549 tumor-bearing mouse injected with pafolacinin. Figure 3B yes Figure 3A Fluorescence images of tissue distribution of FR-negative A549 tumor-bearing mice injected with pafolacin.

[0042] Figure 4 is a graph illustrating mean concentrations of pafolacin in male and female rats and dogs following intravenous administration of pafolacin. DETAILED DESCRIPTION

[0043] The present technology provides compositions comprising palascocin and methods of using such compositions, such as methods of identifying or visualizing cancer cells in a patient.

[0044] As used herein, the following conventional unit abbreviations and terms are used as follows: "pg" means picogram, "ng" means nanogram, "μg" means microgram, "mg" means milligram, "g" means gram, "kg" means kilogram, "mL" means milliliter, "h" means hour, and "t" means time.

[0045] As used herein, "patient" and / or "subject" refers to a human or an animal. In some aspects, a human is an adult. In other aspects, a human can be a child. In some aspects, an animal is a mammal. In other aspects, an animal is an animal other than a mammal. "Patient" and "subject" can be used interchangeably throughout the disclosure.

[0046] As used herein, "pharmaceutically effective amount" refers to an effective dose or effective concentration that produces a biological response.

[0047] As used herein, "and / or" means any one or more items in a list connected by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y, and / or z" means "one or more of x, y, and z".

[0048] The term "exemplary" means serving as a non-limiting example, case or illustration. As used herein, the terms "eg and for example" list one or more non-limiting examples, cases or illustrations.

[0049] As used herein, the words "about" and "approximately" when used to modify or describe a value (or range of values) mean reasonably close to the value or range of values ​​(e.g., + / - 10% of the value or range of values). Therefore, the aspects described herein are not limited to only the recited values ​​and ranges of values, but should also include reasonably feasible deviations.

[0050] In this specification, the use of the singular includes the plural unless otherwise specified.

[0051] As described above, malignant lesion resection surgery, or surgical removal of cancer cells or other malignant diseases, is part of many treatment plans for such cancers or malignant diseases. In some cases, surgical removal can be one of the most effective methods for treating cancer. For example, removal of all detectable malignant lesions can result in no detectable disease recurrence in approximately 50% of all cancer patients. In cases where the disease does recur, malignant lesion resection surgery can still extend the patient's life expectancy or reduce the patient's morbidity. Therefore, in order to obtain the best possible results for malignant lesion resection surgery, it is important to remove as many cancer cells as possible during surgery.

[0052] In many cases, visualization methods are used to identify malignant lesions. Conventional visualization methods include identification based on the color, texture and / or morphology of the tumor. Additionally or alternatively, tumors can be identified based on differences in plasticity, elasticity or solidity with adjacent healthy tissues. In some aspects, these methods can be performed by surgeons without using additional tools or compositions, or using tools and compositions commonly used during surgery. For example, surgeons can use their own senses to identify malignant lesions based on color, texture, morphology, plasticity, elasticity and / or solidity. Another example of conventional visualization technology is the use of fluorescent dyes, which can passively flow from tumors into draining lymph nodes. Such conventional dyes can fluoresce in the visible light range. Fluorescent lymph nodes (e.g., due to dyes) can then be visually identified and removed to determine whether cancer cells have metastasized to lymph nodes. However, in many cases, at least a portion of malignant lesions may not be identified using these conventional visualization methods and therefore cannot be removed. In addition, some conventional dyes can spontaneously fluoresce in the visible light range, have a relatively short shelf life, are relatively unstable, and have poor tissue permeability.

[0053] Therefore, there is a need for improved visualization techniques and compositions to better identify malignant lesions in patients. Disclosed are compositions, formulations and methods for identifying cancer cells. The disclosed compositions, formulations and methods include pavlacin. Without being bound by theory, it is believed that pavlacin has an increased binding affinity to folate receptor (FR) positive cancers. Compositions and formulations including pavlacin bind to such FR of malignant lesions expressing FR, and can then be used to identify malignant lesions. Compared to the conventional visualization methods described above, such compositions, formulations and methods can lead to increased visualization and identification of malignant lesions expressing FR. In this way, the use of the compositions, formulations and methods disclosed herein can lead to improved outcomes for cancer patients (e.g., removal of a higher percentage of malignant lesions, increased life expectancy, greater chance of cancer remission and / or reduced mortality). In addition, compared to conventional dyes, the disclosed technology can have reduced autofluorescence, extended shelf life, improved stability and / or more effective tissue permeability.

[0054] As described above, compositions, formulations and methods comprising pavlacinim are believed to have increased binding affinity to FR. In this way, the disclosed compositions and methods can lead to improved visualization of cancerous lesions in cancers that express or overexpress FR. Such cancers include, for example, ovarian cancer, kidney cancer, lung cancer, endometrial cancer, breast cancer and colon cancer. Other cancers not explicitly disclosed herein may also express or overexpress FR. In such cases, the compositions, formulations and methods disclosed herein may also lead to improved identification of those cancers. It is contemplated that the compositions, formulations and methods disclosed herein are used for any type of cancer or other malignant disease.

[0055] Thus, the present disclosure allows identification of a biological tissue expressing FR by contacting the tissue with a composition of the present disclosure containing pavlacin and allowing a period of time for the composition to distribute within the tissue and interact with the site of the FR. After sufficient time for such interaction, the tissue is illuminated with excitation light to cause the composition to fluoresce. The fluorescence is then observed to visualize the malignant lesions. In a similar manner, the composition of the present disclosure is used to identify a target cell type in a biological sample by contacting the biological sample with such a composition for a period of time under conditions that allow the composition to bind to at least one cell of the target cell type. The combined composition is then caused to fluoresce so that the tissue to which the composition is bound can be identified.

[0056] The compositions disclosed herein comprise an optical imaging agent. In some aspects, the optical imaging agent is pafolacin. In some aspects, the composition comprises pafolacin as a pharmaceutically acceptable salt. In some aspects, the composition comprises pafolacin as a tetrasodium salt. For example, the composition can comprise pafolacin sodium. The chemical formula of sodium pavlazone is (S)-2-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-3-(4-(((E)-2-(3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-3H-indol-1-ium-2-yl)vinyl)-6-((E)-2-(3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)indol-2-ylidene)ethylylidene)cyclohex-1-en-1-yl)oxy)phenyl)propionic acid tetrasodium hydrate, with a molar mass of 1414.42 g / mol. The general structure of sodium pavlazone of the present technology is:

[0057]

[0058] Without wishing to be bound by theory, it is believed that pavlacinin binds to cancer cells expressing FR. In some aspects, pavlacinin can bind to cancer cells expressing FR with at least about 1 nanomolar (nM) affinity, at least about 0.75 nM affinity, at least about 0.5 nanomolar (nM) affinity, at least about 0.25 nanomolar (nM) affinity, at least about 0.1 nanomolar (nM) affinity, at least about 0.05 nanomolar (nM) affinity, at least about 0.01 nanomolar (nM) affinity, or at least about 0.001 nanomolar (nM) affinity. Pavlacinin can then be internalized into cancer cells by receptor-mediated endocytosis, thereby concentrating in FR-positive cancer tissues.

[0059] Once absorbed into the tissue, the composition of the present disclosure can be used to identify cancer cells by causing pavlacin to fluoresce. Pavlacin is excited using light with at least one excitation wavelength in the near infrared range (e.g., between about 600nm and about 1000nm). In some aspects, pavlacin absorbs light in the near infrared range from about 600nm to about 900nm, from about 700nm to about 900nm, from about 700nm to about 800nm, from about 750nm to about 800nm, or from about 760nm to about 785nm. In some aspects, the peak absorption of pavlacin can occur at about 776nm. In some aspects, pavlacin emits fluorescence between about 600nm to about 900nm, about 700nm to about 900nm, about 700nm to about 850nm, about 750nm to about 850nm, or about 790nm to about 815nm. In some aspects, the peak emission of pavlacin can occur at about 796 nm. Fluorescence emitted by pavlacin bound to cancer cell FR can visualize the cancer cells. In some aspects, visualization using pavlacin may be improved over conventional visualization methods (e.g., identifying more cancer cells). In addition, due to the use of near-infrared light rather than light in the visible range, the compositions and formulations discussed herein may be more stable, have a longer shelf life, result in less autofluorescence, and better tissue permeability than conventional dyes.

[0060] In some aspects, an operating room for a surgical procedure can be equipped with an overhead light that produces a wavelength of light within the range discussed above. For example, an operating room can include at least one lamp that produces light of an appropriate wavelength. In this manner, at least one lamp that produces light of an appropriate wavelength can be directed toward a body cavity, a surgical opening, or the like, so that pafolacinin bound to cancer cells fluoresces. In some aspects, a surgeon (or other professional) can be able to directly observe the fluorescence without the need for additional tools, accessories, or special equipment to identify malignant lesions. In other examples, additional tools, accessories, or special equipment, or alternatively, can be used to visualize the fluorescence of pafolacinin and identify malignant lesions. For example, an endoscopic device can be used to deliver excitation light to a site, receive fluorescence emitted from the site, and / or help form a fluorescence image from a malignant lesion. Additionally or alternatively, in some aspects, an image processing device, such as a CCD camera, a display, a photon collection device, or the like, can be used to help visualize cancer cells.

[0061] The compositions and formulations disclosed herein can be administered by any route known to those skilled in the art, e.g., intravenously, topically, intraarticularly, intracisternal, intraocularly, intraventricularly, intrathecally, intramuscularly, intraperitoneally, transdermally, intratracheally, or intracavitary. In some aspects, the most appropriate route of administration can vary depending on the disease state to be treated or the location of the suspected condition or tumor to be identified.

[0062] In some aspects, the disclosed compositions can be administered intravenously. For example, pavlacin can be administered intravenously as a solution. The solution can contain an effective amount (e.g., an amount of pavlacin that is capable of identifying a malignant lesion in a patient) of pavlacin and any other composition, ingredient, reagent, solvent, buffer, preservative, antioxidant, diluent, etc.

[0063] Can be before operation begins, before incision is made during the operation process, after operation begins, after surgical cavity and tumor site have been exposed by operation, or any other suitable time to apply composition.In some aspects, composition can be intravenously applied to patient at least about one hour before starting fluorescence imaging.For example, can be between about 1 hour and about 24 hours before operation, between about 1 hour and about 12 hours before operation, between about 1 hour and about 9 hours before operation, between about 1 hour and about 7 hours before operation, between about 1 hour and about 5 hours before operation, between about 1 hour and about 3 hours before operation, or between about 1 hour and 2 hours before operation to apply pafolacin.In some such examples, provide at least 1 hour before operation can make composition and cancer cell expressing FR fully combined, to allow to emit fluorescence, and therefore make cancer cell visualization.In other aspects, can be provided to patient less than about 1 hour before operation.

[0064] In some aspects, the pavlacin composition can be provided to the patient (e.g., intravenously) over a period of time. For example, in some aspects, pavlacin can be administered to the patient over a period of about 1 hour or longer. In some such aspects, the composition can be administered to the patient using an infusion tube over a period of about 1 hour, about 1.5 hours, about 2 hours, or 3 hours. For example, the composition can be administered to the patient over a period of time between about 1 hour and about 3 hours. In other aspects, the composition can be administered over different time periods.

[0065] In some aspects, the compositions disclosed herein may include an amount of pafolacinine from about 0.5 mg or more, from about 2.5 mg or more, from about 5.0 mg or more, from about 7.5 mg or more, from about 10 mg or more, from about 20 mg or more, from about 30 mg or more, from about 40 mg or more, about 50 mg or more, about 60 mg or more, about 70 mg or more, about 80 mg or more, about 90 mg or more, or about 100 mg or more, and including any additional increments thereof, for example, 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9 or 1.0 mg and multipliers thereof (e.g., x1, x2, x2.5, x5, x10, x100, etc.). In some aspects, the formulation includes between about 1 mg and about 20 mg, between about 1 mg and about 10 mg, between about 1 mg and about 5 mg, between about 1 mg and about 4 mg, or between about 2 mg and about 4 mg. For example, some formulations may include about 3.2 mg of pavlacine. In aspects where the formulation includes pavlacine sodium, the formulation may include between about 1 mg and about 20 mg, between about 1 mg and about 10 mg, between about 1 mg and about 5 mg, between about 1 mg and about 4 mg, between about 2 mg and about 4 mg, or about 3.4 mg of pavlacine sodium.

[0066] Pafolacin can be administered to a patient at any suitable dose. In some aspects, suitable doses of Pafolacin include, but are not limited to, between about 0.01 mg and about 1 mg per 1 kg of patient weight, between about 0.01 mg and about 0.5 mg per 1 kg of patient weight, between about 0.01 mg and about 0.25 mg per 1 kg of patient weight, between about 0.01 mg and about 0.1 mg per 1 kg of patient weight, between about 0.01 mg and about 0.09 mg per 1 kg of patient weight, between about 0.01 mg and about 0.07 mg per 1 kg of patient weight, between about 0.25 mg and about 0.07 mg per 1 kg of patient weight, or between about 0.25 mg and about 0.05 mg per 1 kg of patient weight. In some aspects, about 0.25 mg of Pafolacin can be administered per 1 kg of patient weight. In other aspects, different amounts of pafolacin per 1 kg of patient body weight can be administered. Similar doses of compositions containing pafolacin sodium can be administered to patients.

[0067] In some aspects, the present technology provides a combination formulation comprising pavracine and at least one other composition, ingredient, reagent, solvent, buffer, preservative, antioxidant, diluent, etc. For example, the at least one other composition, ingredient, reagent, solvent, buffer, preservative, antioxidant or diluent may be a salt. In some such aspects, the salt may include sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate heptahydrate, sodium bisulfate, calcium chloride, potassium iodide or another salt. As a non-limiting aspect, the combination formulation may include pavracine (and / or pavracine sodium), sodium chloride, potassium dihydrogen phosphate and disodium hydrogen phosphate heptahydrate. In some aspects, the combination formulation may additionally or alternatively include an acid, a base and / or a buffer. For example, in some such aspects, the combination formulation may include sodium hydroxide, hydrochloric acid, perchloric acid, hydroiodic acid, nitric acid, sulfuric acid, potassium hydroxide and / or calcium hydroxide. As a non-limiting aspect, the combination formulation may include sodium hydroxide and / or hydrochloric acid.

[0068] As another non-limiting example, the combination formulation may include pafolacin (and / or pafolacin sodium), sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate heptahydrate, sodium hydroxide, and / or hydrochloric acid. In some aspects, the combination formulation may include between about 1 mg and about 50 mg, between about 1 mg and about 25 mg, between about 1 mg and about 20 mg, between about 1 mg and about 15 mg, between about 5 mg and about 50 mg, between about 5 mg and about 25 mg, between about 5 mg and about 20 mg, between about 5 mg and about 15 mg, between about 10 mg and about 15 mg, or about 14.4 mg of sodium chloride. In some aspects, the combination formulation may contain between about 0.01 mg and about 5 mg, between about 0.01 mg and about 1 mg, between about 0.01 mg and about 0.5 mg, between about 0.01 mg and about 0.25 mg, between about 0.05 mg and about 5 mg, between about 0.05 mg and about 1 mg, between about 0.05 mg and about 0.5 mg, between about 0.05 mg and about 0.25 mg, between about 0.1 mg and about 0.25 mg, or about 0.23 mg of potassium dihydrogen phosphate. In some aspects, the combination formulation may include between 0.1 mg and about 10 mg, between about 0.1 mg and about 5 mg, between about 0.1 mg and about 2.5 mg, between about 0.5 mg and about 10 mg, between about 0.5 mg and about 5 mg, between about 0.5 mg and about 2.5 mg, between about 1 mg and about 10 mg, between about 1 mg and about 5 mg, between about 1 mg and about 2.5 mg, or about 1.27 mg of monobasic potassium phosphate. In some such aspects, the combination formulation may include about 3.2 mg of pafolacin (equivalent to about 3.4 mg of pafolacin sodium), 14.4 mg of sodium chloride, 0.23 mg of monobasic potassium phosphate, 1.27 mg of disodium hydrogen phosphate heptahydrate in a volume of about 1.6 mL of solution. Thus, in some aspects, 3.2 mg of pavlacin (equivalent to about 3.4 mg of pavlacin sodium) can be administered to a patient per about 1.6 mL volume of solution of the combined formulation. In other aspects, the combined formulation can include additional or alternative compositions, ingredients, agents, solvents, buffers, preservatives, antioxidants, diluents, etc. Furthermore, in other aspects, the combined formulation can include different amounts of any composition, ingredient, agent, solvent, buffer, preservative, antioxidant, diluent, etc.

[0069] In some aspects, the pH of the composition can be between about 6 and about 9, between about 6 and about 8, between about 7 and about 8, or between about 7.1 and about 7.8. In other aspects, the pH can be different. In some aspects, a buffer or another composition can be used to adjust the pH of the preparation to between about 6 and about 9, between about 6 and about 8, between about 7 and about 8, or between about 7.1 and about 7.8. For example, sodium hydroxide and / or hydrochloric acid can be used to adjust the pH of the combined formulation. In other aspects, different buffers, compositions, materials or solvents, such as perchloric acid, hydroiodic acid, nitric acid, sulfuric acid, potassium hydroxide and / or calcium hydroxide can be used to adjust the pH.

[0070] In some aspects, the dosage discussed above can be supplied in the form of a single-dose bottle. For example, a single-dose bottle can include 3.2mg of pafolacin (or 3.4mg of pafolacin sodium) in the above-mentioned combined formulation solution of about 1.6mL. In other aspects, the preparation can include between about 1mg and about 20mg, between about 1mg and about 10mg, between about 1mg and about 5mg, between about 1mg and about 4mg, or between about 2mg and about 4mg pafolacin or between about 1mg and about 10mg, between about 1mg and about 5mg, between about 1mg and about 4mg, between about 2mg and about 4mg pafolacin sodium or about 3.4mg. Such a combined formulation can be a dark, blue-green, transparent, aqueous solution. In other aspects, the dosage can not be provided in a single-dose bottle form. In such aspect, bottle (or other packaging) can comprise any suitable dosage number, for example about 20 doses, about 10 doses, about 5 doses or about 2 doses.In some aspects, bottle can be sealed, amber, glass bottle.In some aspects, bottle can be individually packaged and supplied in carton, and this carton comprises at least about 5 bottles, at least about 10 bottles, at least about 20 bottles, at least about 50 bottles or at least about 100 bottles.In other respects, composition can occur in alternative form, has different amounts, is packaged in different ways and / or has different outward appearance.

[0071] In some aspects, the compositions disclosed herein can be diluted prior to administration to a patient. For example, pavlacin can be diluted in a glucose solution, e.g., a 5% glucose solution. In some such aspects, about 0.25 mg of pavlacin per 1 kg of patient body weight can be diluted in about 220 mL to about 250 mL of a 5% glucose solution. In other examples, additional or alternative solutions and / or solution concentrates can be used to dilute the disclosed compositions.

[0072] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific aspects of the present technology. By providing these specific examples, it is not intended to limit the scope and spirit of the present technology. Those skilled in the art will understand that the full scope of the presently described technology includes the subject matter defined by the claims attached to this specification, as well as any changes, modifications or equivalents to these claims.

[0073] Example

[0074] Example 1: Detection of malignant lesions using a composition comprising pafolacinin

[0075] The safety and efficacy of the compositions disclosed herein were evaluated in a randomized, multicenter, open-label study. The study recruited 178 women diagnosed with ovarian cancer or with clinically highly suspected ovarian cancer who were scheduled to undergo primary surgical cytoreduction, interval cytoreduction, or recurrent ovarian cancer surgery. 150 women with highly suspected or confirmed ovarian cancer received a composition containing pafolacinin (administered at 0.25 mg / kg at least 1 hour before the start of fluorescence imaging). Among them, 134 women with an average age of 60 years (range 33 to 81 years) received both normal optical imaging evaluation and fluorescence imaging evaluation (Intent-to-Image Set).

[0076] This study evaluated the proportion of patients who had at least one evaluable ovarian cancer lesion that was confirmed by central pathology and detected under fluorescent light with a composition containing palascoline but not detected under normal light or palpation, and who had not otherwise been identified for resection prior to surgery. The estimated detection proportions in women who underwent both normal light and fluorescence (intention-to-image group) are shown in Table 1.

[0077] Table 1

[0078]

[0079] Using near-infrared fluorescence to detect centrally pathologically confirmed ovarian cancer lesions, the patient-level false positive rate for the composition containing palascoline was 20.2%, with a confidence interval of 95% (13.7%, 28.0%).

[0080] Example 2: Pharmacodynamics

[0081] The tumor to background ratio with different mass doses of pafolacinim was studied.At a dose of 0.25 mg pafolacinim per 1 kg patient body weight, a high tumor to background ratio was observed.

[0082] Example 3: In vitro pharmacodynamics

[0083] KB cells (a derivative of HeLa cells) overexpressing FR were combined with [3H]-folate in the presence of increasing concentrations (0.1 nM-1 μM) of pavlazone (L-isomer), OTL0039 (D-isomer), or folic acid. Figure 1 As shown in , pavlacinib exhibits high affinity for FR (KD = 10.4 nM), which is roughly equivalent to the binding affinity of folic acid (KD = 7.4 nM). In contrast, the D-isomer of pavlacinib (OTL0039) exhibits a relatively low affinity for the receptor (KD = 81.8 nM). Pavlacinib competes well with [3H]-folic acid for the receptor.

[0084] Example 4: In vivo pharmacodynamics

[0085] Whole-body imaging and tissue distribution of pafolacin and OTL0039 were performed in female nu / nu mice bearing FR-positive KB cell tumor xenografts. Results showed that 2.5 hours after intravenous (IV) administration of 10 nmol pafolacin, fluorescence was mainly in the tumor, and fluorescent signals were also observed in the kidney ( Figure 2A and 2C ). Pafosinib uptake in the kidney was expected, as the apical membrane of the renal proximal tubules has been shown to express high levels of FR. All other normal tissues showed little or no fluorescent signal, resulting in an excellent tumor to normal tissue fluorescence ratio. Tissue distribution pattern of OTL0039 ( Figure 2D ) Similar to the tissue distribution pattern of pavlazone ( Figure 2C ), although in tumors ( Figure 2B The fluorescence signal intensity in both the folate and kidney was weaker than that of pavlacin. This observation is consistent with the lower binding affinity of the D-isomer to FR than the L-isomer. Tumors in mice receiving pavlacin fluoresce more brightly compared to other folate-conjugated near-infrared dyes such as LS288, IR800, and ZW800.

[0086] In FR-positive tumor-bearing mice, the optimal dose range of pavlazone relative to tumor-background ratio was 1 to 30 nmol / mouse, with lower fluorescence intensity observed in tumors at lower doses (0.3 nmol) and higher fluorescence intensity observed in other tissues at doses above 30 nmol. In a second experiment, whole-body imaging and tissue distribution of pavlazone were again examined; however, this experiment was performed in female nu / nu mice bearing FR-negative A549 cell tumor xenografts. In this study, fluorescence was observed primarily in the kidneys, and no fluorescence was observed in FR-negative tumors (Figure 3). Therefore, pavlazone does not target FR-negative tumors in vivo, demonstrating the specificity of the imaging agent for FR-positive tumors.

[0087] Example 5: Secondary Pharmacodynamics

[0088] Pafolacenin (5 μM) was evaluated in an in vitro ligand binding screen (ExpresSProfile; Cerep 100010238) that assessed the potential for "off-target" binding of pfolacenin to 55 broad, pharmacodynamically relevant receptors, ion channels, and transporters. Results from the binding experiments showed that pfolacenin specific binding % ranged from 0.2% to 29.7% for positive values ​​and from -0.3% to -16.8% for negative values. These data indicate that pfolacenin has no significant affinity for a comprehensive panel of off-target proteins, supporting the specificity of pfolacenin for FR binding.

[0089] Example 6: Pharmacokinetics

[0090] The mean maximum plasma concentration of pafolacin max ) was 59.1±5.94 ng / mL, and the area under the plasma concentration-time curve reached infinity (AUC inf ) is 63.6±12.6ng.hr / mL.

[0091] eliminate

[0092] The elimination half-life of palafoxitin is 0.44±0.23 hours, and the mean plasma clearance is 28.6±4.97 L / hr.

[0093] metabolism

[0094] Pafosin sodium is not metabolized by cytochrome P450 (CYP) enzymes.

[0095] excretion

[0096] Following a single intravenous injection of radiolabeled pafolacin sodium, approximately 35% of the dose is recovered in the urine (19.1%) and in the feces (15.8%) after approximately 3-5 weeks.

[0097] Specific populations

[0098] No clinically significant differences in the pharmacokinetics of palafoxitin were identified based on age 18-89 years, body weight 41.6-133.6 kg, mild to moderate renal impairment (creatine clearance (CLcr) 30 to 89 mL / min), or mild to moderate hepatic impairment (total bilirubin <3 times upper limit of normal (ULN) and aspartate aminotransferase (AST) >ULN).

[0099] absorb

[0100] Following a single intravenous dose of pafolacin in male and female rats, systemic exposure to pafolacin (AUClast) appeared to be similar in males and females at the 46.3 mg / kg dose level (Table 2). At the two lower dose levels, there was a slight trend toward increased AUClast values ​​in females compared with males; however, the significance of this slight trend could not be evaluated due to sparse blood sampling. AUClast values ​​were calculated, but the time associated with the last measurable concentration was variable (Tlast; 5 min to 24 hr), which confounded the ability to assess dose proportionality. Terminal data were often sparse and / or the terminal phase was not well defined ( Figure 4 ). Therefore, the parameters that rely on accurate determination of the terminal phase (AUCinf, T1 / 2, CL, Vz) are not reported.

[0101] Following a single intravenous dose of pafolacin in male and female dogs, systemic exposure to pafolacin (C0, AUClast) appeared to be similar in males and females at all dose levels (Table 3). Similar to rats, AUClast values ​​in dogs were calculated from a range of concentration-time data, with Tlast values ​​ranging from 5 minutes to 4 hours. In addition, only limited data were reported for dogs at a TK of 0.9 mg / kg (N=1-2). Therefore, dose proportionality was not assessed in dogs. Terminal phase data were generally sparse (i.e., 2 or fewer measurable concentrations at the terminal phase) ( Figure 4 ). Similar to rats, parameters that rely on accurate determination of the terminal phase (AUCinf, T1 / 2, CL, Vz) were not reported.

[0102] Table 2

[0103]

[0104] DN = dose normalized.

[0105] * =The degree of extrapolation does not meet the acceptance criteria.

[0106] NC = not calculable; NR: not reported.

[0107] Table 3

[0108]

[0109] SD: standard deviation; DN = dose normalized; NR = not reported (extended extent did not meet acceptance criteria); NC = not calculable.

[0110] Repeated dosing

[0111] Male and female rats received weekly intravenous injections of pavlacin (0.9, 9.3, and 13.9 mg / kg) for 14 days. The TK parameters are summarized in Table 4. On days 1 and 13, the increase in pavlacin exposure (AUCinf) trended slightly greater than dose proportional, and it was noted that sparse blood sampling was used, so the variability of AUC could not be assessed. After the dose was increased to 14.9-fold, AUCinf increased to ~18-fold on day 1 and ~20-fold on day 13 (sex combined). There were no obvious sex-related differences, and there was no significant accumulation or change in TK parameters after 3 weeks of repeated weekly dosing. CL was slow (relative to hepatic blood flow) and Vss was moderate. The mean T1 / 2 ranged from approximately 6 to 11 hours.

[0112] Table 4

[0113]

[0114] In addition, male and female dogs also received weekly intravenous injections of pavlacin (3.1, 30.8 and 46.3 mg / kg) for 14 days. Overall, drug concentrations increased with increasing doses, and it was noted that the average curves for the two highest dose levels overlapped. Within the dose range (0.9 to 13.9 mg / kg), the systemic exposure (AUClast) of pavlacin was roughly proportional to the dose and within the observed variability (Table 5). There were no obvious or consistent gender differences, and there was no significant accumulation or change in TK after repeated weekly dosing for 3 weeks. On days 0 and 13, CL was slow (relative to hepatic blood flow), while Vss was moderate. The average T1 / 2 ranged from approximately 13 to 18 hours.

[0115] Table 5

[0116]

[0117]

[0118] Overall, in both species, mean CL was found to be slow relative to hepatic blood flow, and mean Vss values ​​were higher than total body water, 668 and 693 mL / kg in rats and monkeys, respectively. This suggests that pafolacin is distributed in tissues. There were no observable changes in TK after repeated dosing, and PK was similar in both sexes.

[0119] distributed

[0120] The average volume of distribution (V z ) was 17.1±5.99 L, indicating distribution into tissues.

[0121] The plasma protein binding rate of palforacenim is 93.7%. No significant partitioning into erythrocytes was observed.

[0122] Tissue distribution was evaluated in male and female rats after receiving a single 2 mg / kg (~25 μCi / kg) intravenous injection of [14C]pafolacinine. The changes in radioactivity levels (pafolacinine equivalents) in tissues over time were determined using QWBA. Radioactivity was rapidly (within 15 minutes) and widely distributed to most tissues. Tissue to plasma radioactivity ratios were >1.0 for all tissues in male and female animals, except for bone (femur), brain, eyes, and fat, indicating that [14C]pafolacinine-derived radioactivity was distributed to tissues. The overall highest radioactivity concentration was measured in the kidneys of male and female rats 24 hours after dosing. Approximately 11% of the dose remained in the kidneys 96 hr after dosing. The lowest radioactivity concentrations were found in the brain and eyes. Concentrations of [14C]pafoline-derived radioactivity in the eyes, uvea, and skin of male pigmented (Long Evans) rats were roughly similar to those found in albino Sprague Dawley rats, indicating that [14C]pafoline-derived radioactivity has no affinity for melanin-containing tissues.

[0123] Plasma protein binding

[0124] Plasma protein binding of pavlacinim was studied in vitro in fresh human, Sprague Dawley rat and beagle dog plasma using equilibrium dialysis. Pavlacinim was introduced into the plasma samples in duplicate to give a final concentration of 5 μM. The mean (±SD) percentage of plasma protein bound pavlacinim was 99.1±0.1%, 98.6±0.1% and 93.7±1.5% for rats, dogs and humans, respectively. Pavlacinim was highly bound to plasma proteins in all species examined.

[0125] Human PK parameters

[0126] A total of 73 potentially eligible healthy volunteers signed informed consent to participate in a Phase 1a study to evaluate the safety and pharmacokinetics of pavlazone versus placebo. Fifty-four were found suitable to participate, of whom 30 were recruited for the study. Twenty-four subjects were white, 1 subject was black / African American, 1 was Asian, 1 was mixed race, and 3 were of other races. Table 6 shows the characteristics of the study population.

[0127] Table 6

[0128] feature Pafolasinin (N=23) Placebo (N=7) Age, mean (range) 30.3(18-64) 20(20-29) Male (n, %) 10(43) 2(29) <![CDATA[BMI(kg / m 2 )]]> 23.7 21.4

[0129] The pharmacokinetic (PK) analysis population was defined as all subjects who were randomized and received study treatment. Of note, one subject stopped the drug infusion prematurely and another subject interrupted the drug infusion. The pharmacokinetics of palforacenimide following a single intravenous administration of 0.25, 0.05, 0.1, or 0.2 mg / kg were determined using noncompartmental analysis; key PK parameters are summarized in Table 7.

[0130] Table 7

[0131]

[0132] a OTL38 was dissolved in 20 ml of 0.9% sodium chloride and infused for 10 and 60 minutes.

[0133] b OTL38 was dissolved in 220 ml of 5% glucose solution and infused for 60 min.

[0134] c OTL38 was pretreated with an antihistamine (clemastine)

[0135] The maximum concentration (Cmax) for each dose was obtained near the end of the infusion. After the infusion was completed, the plasma concentration decreased rapidly. The drug concentration-time curves obtained at higher doses showed that the decline in plasma concentration was (at least) biphasic. Exposure (measured by Cmax and AUClast) increased with increasing doses of pafolacinim. With an 8-fold increase in dose (0.25 to 0.2 mg / kg), Cmax values ​​increased to ~12-fold, while AUClast values ​​increased to ~22-fold, indicating that the increase in exposure was greater than dose proportional. Clearance values ​​(CL) decreased with increasing dose, indicating that clearance may be saturable at higher doses, which is consistent with an increase in exposure that is greater than dose proportional. Overall, clearance was moderate to low relative to hepatic blood flow (approximately 84 L / hr for a 70 kg subject) and varied from ~29 to 10 L / hr across the dose range. Drug half-life values ​​were short, ranging from ~0.4 to 2.7 hr.

[0136] Example 7: Safety evaluation of compositions comprising pavlacin

[0137] The safety of the disclosed composition was evaluated in three open-label clinical studies, two studies (N=150 and N=44) in patients with ovarian cancer and one study (N=100) in patients with cancer in the lung. A total of 294 patients received 0.25 mg / kg of the disclosed composition by intravenous administration. The average age of the patients was 63.5 years; 51% of the patients were 65 years or older; 89% of the patients were female; and 84% of the patients were white.

[0138] Adverse reactions occurring in >1% of patients were: nausea (15%), vomiting (5.8%), abdominal pain (2.7%), flushing (1.7%), dyspepsia (1%), chest discomfort (1%), pruritus (1%), and hypersensitivity (1%). In 2.4% of patients, these adverse reactions occurred during administration of the composition. Reactions usually occurred within 15 minutes of the start of the infusion.

[0139] Example 8: In vitro evaluation of camera sensitivity and specificity using Pafolacin

[0140] Pafoclacin sodium (2 mg / mL dissolved in 1.6 mL PBS, 1414.2 g / mol (or Da), λex=774-776 nm, λem=794-796 nm) was diluted 100 times in series from 100 μM to 1 pM using PBS or 5% glucose solution. The diluent was used as a negative control, and ICG (10 uM, with the same diluent) was used as a positive control. The serially diluted samples were transferred to a black 96-well plate and imaged using an evaluation camera to determine the sensitivity of the camera.

[0141] Penetration Depth

[0142] Purchase fresh pork from a slaughterhouse and cut into 1 cm thick slices. Dispense 100 μL of 10 μM sodium pfosfoline solution into a glass tube and place under the pork. Take images using an evaluation camera. Increase the number of pork slices and image them to determine the penetration depth of the camera.

[0143] Initial phantom studies were performed using an agarose-based gel phantom to construct an agar phantom with the desired organ geometry. Glass tubes ranging in size from 3 mm to 10 mm with 10 μM sodium pforacenine (100 μL) were placed in the agar phantom gel at different measurement distances and imaged to determine the penetration depth of the camera.

[0144] Field of view

[0145] 100 μL of 10 μM sodium pfosfoline solution was dispensed into 9 glass tubes and placed in the center and on the 8 arrows as shown in Figure 5. Images were taken using an evaluation camera to determine a uniform field of view.

[0146] Example 9: In vivo evaluation of camera sensitivity and specificity using pafolacin

[0147] KB cells (expressing ~2x10 6Folate receptor / cells (a derivative of the HeLa / human cervical carcinoma cell line) and A549 cells (an alveolar basal epithelial carcinoma cell line that does not express the folate receptor) were obtained from ATCC (Rockville, MD) and cultured in monolayers using folate-free or normal 1640 RPMI-1640 medium (Gibco, NY) containing 10% heat-inactivated fetal bovine serum (Atlanta Biological, Atlanta Biological, GA) and 1% penicillin-streptomycin (Gibco, NY) at 37°C in a 5% carbon dioxide:95% air humidity atmosphere before they were used in the study for at least six passages.

[0148] Athymic female nude (nu / nu) mice (5 to 6 weeks old, 18-20 g) were purchased from Envigo (Indianapolis, IN) and maintained on a gamma-irradiated, folate-deficient special diet (Envigo, IN) for 2 weeks prior to the start of the study. Animals were housed 5 per cage in barrier, pathogen-free isolators. Autoclaved tap water and food were given as needed. Animals were housed in a sterile environment with a standard 12 h light-dark cycle for the duration of the study.

[0149] Seven-week-old female nu / nu mice were inoculated subcutaneously in the shoulder or neck with 1.0×10 6 KB cells or A549 cells / mouse. A549 xenograft model was used as a negative control to determine the receptor specificity of palascocillin sodium. Tumor growth was measured in the vertical direction using calipers every 2 days (body weight was monitored on the same schedule), and tumor volume was calculated as 0.5×L×W 2 (L = longest axis and W = axis perpendicular to L, in millimeters). Once the tumor reaches approximately 300-400 mm 3 Animals (5 mice / group) were injected intravenously with an appropriate dose of pafolacin sodium (2-4 nmol, i.e., HDE of 0.0125-0.25 mg / kg) in PBS or 5% glucose solution at a volume of 100 μl. For whole-body imaging and biodistribution studies, animals were euthanized by CO2 asphyxiation 2 to 3 hours after the administration of pafolacin sodium. For time-dependent studies, animals were imaged under anesthesia using isoflurane. After whole-body imaging, animals were dissected, the fluorescence activity of the selected tissues of interest was analyzed using a camera, and the ROI of the tissue was calculated using appropriate software.

[0150] Tumors are additionally dissected into small pieces (e.g., 1 / 2, 1 / 4, 1 / 8, 1 / 16, etc.) and imaged directly or surgically placed in organs of interest (e.g., lung, liver, kidney, etc.) in larger animals such as pigs, dogs, human cadavers, or other animals to determine camera sensitivity (i.e., see the camera's ability to determine the smallest tumor under the appropriate circumstances).

[0151] Example 10: Embryo-Fetal Development (EFD) Animal Studies

[0152] During organogenesis, pavlazone was administered intravenously from gestational day (GD) 6 to GD17 in rats at doses of 0.015, 0.15, and 1.5 mg / kg / d (human equivalent dose (HED) 0.002, 0.24, and 0.242 mg / kg / d) and from GD7 to GD20 in rabbits at 0.3, 1, and 3 mg / kg / d (HED 0.97, 0.323, and 0.968 mg / kg / d). No significant drug-related maternal toxicity and embryo-fetal developmental toxicity were observed. No observed adverse reaction levels were 1.5 mg / kg / d in rats and 3 mg / kg / d in rabbits. Based on comparisons of the area under the plasma concentration-time curve (AUC), the estimated systemic exposure at a human dose of 0.25 mg pavlazone per 1 kg of patient body weight was 158 times (rats) and 570 times (rabbits) the human exposure.

[0153] Although the present disclosure has been described with reference to certain embodiments, it will be appreciated by those skilled in the art that various changes and equivalent substitutions may be made without departing from the scope of the present disclosure or the appended claims. In addition, many modifications may be made to adapt specific circumstances or materials to the teachings of the present disclosure without departing from the scope thereof. Therefore, the present disclosure is not intended to be limited to the disclosed specific embodiments, but the present disclosure will include all aspects falling within the scope of the appended claims.

[0154] All patents, patent applications, publications, and descriptions mentioned above are incorporated herein by reference in their entirety.

Claims

1. A composition configured to be administered to a subject undergoing surgery to remove a malignant lesion, the composition comprising a pharmaceutically effective amount of pafolacianine or a pharmaceutically acceptable salt thereof, wherein pafolacianine or a pharmaceutically acceptable salt thereof is configured to bind to at least one malignant lesion and emit an optical signal.

2. The composition according to claim 1, in, The at least one malignant lesion is an ovarian cancer malignant lesion or a lung cancer malignant lesion.

3. The composition according to claim 1, in, The pharmaceutically effective amount of pavlacin comprises at least about 0.25 mg of pavlacin per about 1 kg of subject body weight.

4. The composition according to claim 1, in, The composition is configured for intravenous administration to a subject.

5. The composition of claim 1, comprising between about 1 mg and about 20 mg of pafolacinine per about 1.6 mL volume of solution.

6. The composition according to claim 1, in, The pharmaceutically acceptable salt of pavlacinim has the following chemical structure:

7. The composition of claim 1, further comprising at least one of sodium chloride, potassium dihydrogen phosphate, or disodium hydrogen phosphate heptahydrate.

8. The composition of claim 7, comprising between about 1 mg and about 50 mg of sodium chloride per about 1.6 mL volume of solution, or between about 0.01 mg and about 5 mg of potassium dihydrogen phosphate per about 1.6 mL volume of solution, or between about 0.1 mg and about 10 mg of disodium hydrogen phosphate heptahydrate per about 1.6 mL volume of solution.

9. The composition according to claim 1, in, The pH of the composition is between about 6 and about 8.

10. The composition according to claim 1, in, The composition is configured to be administered to a subject from about 1 to about 24 hours prior to malignant lesion resection surgery.

11. The composition according to claim 1, in, The pafolacinimide or a pharmaceutically acceptable salt thereof fluoresces when exposed to light in the near infrared range.

12. A method for treating a subject undergoing surgical resection of a malignant lesion, the method comprising: include: administering to a subject a pharmaceutically effective amount of the composition comprising pavlacinim or a pharmaceutically acceptable salt thereof, wherein the pavlacinim or a pharmaceutically acceptable salt thereof is distributed in at least a portion of one or more malignant lesions; irradiating one or more malignant lesions with excitation light of a wavelength absorbable by pafolacinim or a pharmaceutically acceptable salt thereof; and detecting an optical signal emitted by pafolacinim or a pharmaceutically acceptable salt thereof.

13. The method of claim 12, further comprising identifying one or more malignant lesions based on the emitted optical signals.

14. The method of claim 12, further comprising resecting one or more malignant lesions.

15. The method according to claim 14, in, The resection procedure is non-invasive and is performed manually or using robotic-assisted technology and / or using an iBiopsy, iKnife, iLaser, iBurner, a powered cutting loop, a rotating blade, a curved blade, an expandable blade, a dissector with a cutting blade, a blunt dissector, forceps, an electrolytic element, a biopsy needle, a microwave ablation probe, a radiofrequency ablation probe, a cryoablation probe, or a laser.

16. The method according to claim 12, in, The one or more malignant lesions are ovarian cancer malignant lesions or lung cancer malignant lesions.

17. The method according to claim 12, in, The optical signal is detected using an imaging system or imaging software.

18. The method according to claim 17, in, The imaging system or imaging software is selected from the group consisting of: imaging system FAST (Fiber Array Scanning Technology), flow cytometry, confocal microscopy, two-photon microscopy, epifluorescence microscopy, fluorescence microscopy methods, fluorescent glasses and innovative wearable devices.

19. The method according to claim 12, in, The method further comprises guiding a flexible probe to the at least one malignant lesion after administering the composition.

20. The method according to claim 18, in, The flexible probe is a flexible endoscope, a fluorescent endoscope imaging probe, a fiberscope, a videoscope, a gastroscope, a colonoscope, a bronchoscope, a laryngoscope, a cystoscope, a duodenoscope, a colonoscope, a ureteroscope, a sigmoidoscope, an enteroscope, a choledochoscope, a nasolaryngoscope, an angioscope or a hysteroscope.

21. A composition configured to be administered to a subject undergoing surgery to remove a malignant lesion, the composition comprising a pharmaceutically effective amount of pafolacinin, wherein the pafolacinin is configured to fluoresce to identify one or more malignant lesions.

22. The composition according to claim 21, in, The one or more malignancies include ovarian cancer.

23. The composition according to claim 21, in, The one or more malignant lesions include lung cancer.

24. The composition of any one of claims 21 to 24, wherein the pharmaceutically effective amount of pafolacinim comprises at least about 0.25 mg of pafolacinim per about 1 kg of subject body weight.

25. A composition according to any one of the preceding claims, in, The composition is configured for intravenous administration to a subject.

26. A composition according to any one of the preceding claims, in, The compositions include injectable solutions.

27. The composition according to claim 26, in, The solution was blue-green in color.

28. A composition according to claim 26 or claim 27, in, The solution is transparent or translucent.

29. The composition of any one of claims 21 to 28, comprising between about 1 mg and about 20 mg of pafolacinine per about 1.6 mL volume of solution.

30. The composition of any one of claims 21 to 29, comprising about 3.2 mg of pafolacinine per about 1.6 mL volume of solution.

31. A composition according to any one of claims 21 to 30, in, About 3.2 mg of pavlacidine per about 1.6 mL volume of solution is equivalent to about 3.4 mg of pavlacidine sodium per about 1.6 mL volume of solution.

32. A composition according to any one of claims 21 to 31, in, The pavlazone sodium has the following chemical structure:

33. A composition according to any one of claims 21 to 32, comprising at least one of sodium chloride, potassium dihydrogen phosphate or disodium hydrogen phosphate heptahydrate.

34. The composition of any one of claims 21 to 33, comprising between about 1 mg and about 50 mg of sodium chloride per about 1.6 mL volume of solution.

35. The composition of any one of claims 21 to 34, comprising about 14.4 mg sodium chloride per about 1.6 mL volume of solution.

36. The composition of any one of claims 21 to 35, comprising between about 0.01 mg and about 5 mg of monopotassium phosphate per about 1.6 mL volume of solution.

37. The composition of any one of claims 21 to 36, comprising about 0.23 mg of potassium dihydrogen phosphate per about 1.6 mL volume of solution.

38. The composition of any one of claims 21 to 37, comprising between about 0.1 mg and about 10 mg of disodium hydrogen phosphate heptahydrate per about 1.6 mL volume of solution.

39. The composition of any one of claims 21 to 38, comprising about 1.27 mg of disodium hydrogen phosphate heptahydrate per about 1.6 mL volume of solution.

40. The composition of any one of claims 21 to 39, comprising about 3.2 mg of pafolacinimide, about 14.4 mg of sodium chloride, about 0.23 mg of potassium dihydrogen phosphate, and about 1.27 mg of disodium hydrogen phosphate heptahydrate per about 1.6 mL volume of solution.

41. A composition according to any one of claims 21 to 40, in, The pH of the composition is between about 6 and about 8.

42. The composition of any one of claims 21 to 41, further comprising at least one of sodium hydroxide or hydrochloric acid.

43. The composition according to claim 42, in, At least one of sodium hydroxide or hydrochloric acid is used to adjust the pH of the composition.

44. A composition according to any one of claims 21 to 43, in, The solution containing the composition is diluted prior to administering the composition to a subject.

45. The composition according to claim 44, in, The solution was diluted with 5% glucose solution.

46. ​​The composition according to claim 45, in, The solution is diluted with about 220 mL to about 250 mL of a 5% dextrose solution.

47. A composition according to any one of claims 21 to 46, in, The composition is configured to be administered to a subject from about 1 to about 24 hours prior to malignant lesion resection surgery.

48. A composition according to any one of claims 21 to 47, in, The composition is configured to be administered to a subject over a period of time.

49. The composition according to claim 48, in, The period of time includes from about 1 to about 3 hours.

50. A composition according to any one of claims 21 to 49, in, The paflashnin fluoresces when exposed to light in the near-infrared range.

51. A composition according to any one of claims 21 to 50, in, The pafolacinin fluoresces when exposed to light of a wavelength between about 760 nm and about 785 nm.

52. A composition according to any one of claims 21 to 51, in, The palascocinin emits fluorescence between about 790 nm and about 815 nm.

53. A composition according to any one of claims 21 to 52, in, The composition is contained in a vial.

54. The composition according to claim 53, in, The vials are single-dose vials.

55. A composition according to claim 53 or claim 54, in, One carton contains at least 10 vials.

56. The composition of claim 55, wherein the at least 10 vials are individually packaged.

57. A composition according to any one of claims 21 to 56, in, The subject is an animal.

58. A composition according to any one of claims 21 to 57, in, The subject is a mammal.

59. A composition according to any one of claims 21 to 58, in, The subject is a human.

60. A composition according to any one of claims 21 to 59, in, The subject is an adult.

61. A method of treating a subject undergoing surgical resection of a malignant lesion, the method include: administering a pharmaceutically effective amount of a composition comprising pafolacinin to a subject; and causing the pafolacinin to fluoresce.

62. The method of claim 61, further comprising identifying one or more malignant lesions based on the fluorescence of the palascocin.

63. The method of claim 61 or claim 62, further comprising resecting one or more malignant lesions.

64. The method of claim 62 or claim 63, in, The one or more malignancies include ovarian cancer.

65. The method according to any one of claims 61 to 64, in, The one or more malignant lesions include lung cancer.

66. The method according to any one of claims 61 to 65, in, The pharmaceutically effective amount comprises at least about 0.25 mg of pafolacinine per about 1 kg of subject body weight.

67. The method according to any one of claims 61 to 66, in, The composition is administered intravenously to the subject.

68. The method according to any one of claims 61 to 67, in, The compositions include injectable solutions.

69. The method according to claim 68, in, The solution was blue-green in color.

70. The method of claim 68 or claim 69, in, The solution is transparent or translucent.

71. The method according to any one of claims 61 to 70, in, The solution contains between about 1 mg and about 20 mg of pafolacinine per about 1.6 mL volume of solution.

72. The method according to any one of claims 61 to 71, in, The composition comprises about 3.2 mg of pafolacinine per about 1.6 mL volume of solution.

73. The method according to any one of claims 61 to 72, in, About 3.2 mg of pavlacidine per about 1.6 mL volume of solution is equivalent to about 3.4 mg of pavlacidine sodium per about 1.6 mL volume of solution.

74. The method according to any one of claims 61 to 73, in, The pavlazone sodium has the following chemical structure:

75. The method according to any one of claims 61 to 74, in, The solution additionally comprises at least one of sodium chloride, potassium dihydrogen phosphate, or disodium hydrogen phosphate heptahydrate.

76. The method according to any one of claims 61 to 75, in, The solution comprises between about 1 mg and about 50 mg of sodium chloride per about 1.6 mL volume of solution.

77. The method according to any one of claims 61 to 76, in, The solution contained about 14.4 mg of sodium chloride per about 1.6 mL volume of solution.

78. The method according to any one of claims 61 to 77, in, The solution contains between about 0.01 mg and about 5 mg of monopotassium phosphate per about 1.6 mL volume of solution.

79. The method according to any one of claims 61 to 78, in, The solution contained about 0.23 mg of potassium dihydrogen phosphate per about 1.6 mL volume of solution.

80. The method according to any one of claims 61 to 79, in, The solution contains between about 0.1 mg and about 10 mg of disodium hydrogen phosphate heptahydrate per about 1.6 mL volume of solution.

81. The method according to any one of claims 61 to 80, in, The solution contained about 1.27 mg of disodium hydrogen phosphate heptahydrate per about 1.6 mL volume of solution.

82. The method according to any one of claims 61 to 81, in, The solution contains about 3.2 mg of pafolacinine, about 14.4 mg of sodium chloride, about 0.23 mg of potassium dihydrogen phosphate, and about 1.27 mg of disodium hydrogen phosphate heptahydrate per about 1.6 mL volume of solution.

83. The method according to any one of claims 61 to 82, in, The pH of the solution is between about 6 and about 8.

84. The method according to any one of claims 61 to 83, in, The solution additionally comprises at least one of sodium hydroxide or hydrochloric acid.

85. The method according to claim 84, in, At least one of sodium hydroxide or hydrochloric acid is used to adjust the pH of the solution.

86. The method of any one of claims 61-85, further comprising diluting the solution prior to administering the composition to a subject.

87. The method according to claim 86, in, Diluting the solution includes diluting the solution with a 5% glucose solution.

88. The method according to claim 87, in, Diluting the solution includes diluting the solution with about 220 mL to about 250 mL of a 5% dextrose solution.

89. The method according to any one of claims 61 to 88, in, Administering the pharmaceutically effective amount of the composition includes administering the pharmaceutically effective amount of the composition from about 1 to about 24 hours prior to surgery to remove the malignant lesion.

90. The method according to any one of claims 61 to 89, in, Administering a pharmaceutically effective amount of the composition includes administering the pharmaceutically effective amount of the composition over a period of time.

91. The method according to claim 90, in, The period of time includes from about 1 to about 3 hours.

92. The method according to any one of claims 61 to 91, in, Causing the paflashinine to fluoresce includes exposing the paflashinine to light in the near infrared range.

93. The method of any one of claims 61-92, wherein causing pfolacin to fluoresce comprises exposing pfolacin to light having a wavelength between about 760 nm and about 785 nm.

94. The method according to any one of claims 61 to 93, in, The palascocinin emits fluorescence between about 790 nm and about 815 nm.

95. The method according to any one of claims 61 to 94, in, The composition is contained in a vial.

96. The method according to claim 95, in, The vials are single-dose vials.

97. The method of claim 95 or claim 96, in, One carton contains 10 vials.

98. The method according to claim 97, in, The 10 vials are individually packaged.

99. The method according to any one of claims 61 to 98, in, The subject is an animal.

100. The method according to any one of claims 61 to 99, in, The subject is a mammal.

101. The method according to any one of claims 61-100, in, The subject is a human.

102. The method according to any one of claims 61-101, in, The subject is an adult.

103. The method according to any one of claims 61-102, in, Detect the fluorescence of the paflashnin using an imaging system or imaging software.

104. The method according to claim 103, in, The imaging system or imaging software is selected from the group consisting of: imaging system FAST (Fiber Array Scanning Technology), flow cytometry, confocal microscopy, two-photon microscopy, epifluorescence microscopy, fluorescence microscopy methods, fluorescent glasses and innovative wearable devices.

105. The method according to any one of claims 61 to 104, in, The fluorescence of the palascoline was detected in vivo.

106. The method according to any one of claims 61 to 105, in, The method further comprises guiding the flexible probe to the malignant lesion after administering the composition comprising pafolacinin.

107. The method according to claim 106, in, The flexible probe is a flexible endoscope, a fluorescent endoscope imaging probe, a fiberscope, a videoscope, a gastroscope, a colonoscope, a bronchoscope, a laryngoscope, a cystoscope, a duodenoscope, a colonoscope, a ureteroscope, a sigmoidoscope, an enteroscope, a choledochoscope, a nasolaryngoscope, an angioscope or a hysteroscope.

108. The method of claim 106 or claim 107, in, The flexible probe is equipped to detect wavelengths having absorption and emission maxima between about 400 nm and about 900 nm.

109. The method according to any one of claims 61 to 108, in, The resection procedure is performed using an iBiopsy, iKnife, iLaser, iBurner, a powered cutting loop, a rotating blade, a curved blade, an expandable blade, a dissector with a cutting blade, a blunt dissector, forceps, an electrolytic element, a biopsy needle, a microwave ablation probe, a radiofrequency ablation probe, a cryoablation probe, or a laser.

110. The method according to any one of claims 61 to 109, in, The resection procedure is non-invasive.

111. The method according to any one of claims 61 to 110, in, The resection procedure is performed manually or using robotic-assisted techniques.

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