Method and composition for visualizing the ureter in surgical procedures

Abstract

A method for visualizing the ureter, including fluorescent visualization of the ureter and other target tissues during a surgical procedure, comprising administering to a subject a fluorescent conjugate comprising a peptide and a fluorescent moiety.

Description

Technical Field

[0001] Description of the Sequence Listing A sequence listing related to the present application is provided in text format instead of in writing and is incorporated herein by reference. The name of the text file containing the sequence listing is 110267_405WO_SEQUENCE_LISTING.txt. The text file is 142 KB, was created on January 13, 2022, and was provided electronically via EFS-Web.

[0002] Field The present invention generally relates to intraoperative imaging methods in surgical procedures, and more specifically to the fluorescence visualization of ureters and nerves.

Background Art

[0003] Background The ureters are a pair of multi-layered muscular tubes - each about 10-12 inches long in the average adult - that transport urine from the kidneys to the bladder. The transport utilizes peristaltic movements caused by contractions of the muscular layer of the ureter wall initiated by a pacemaker. As the wall rhythmically tenses and relaxes, urine pulsates from the ureter to the bladder.

[0004] Because it extends from the abdomen to the pelvis, the ureters are proximal to many structures in the abdominal-pelvic cavity, including the gonadal and uterine blood vessels, the iliac arteries, the inferior mesenteric and sigmoid colon blood vessels, as well as the cervix, colon, and rectum. Furthermore, the ureters are thin-walled tubes that are often in a state of collapse and are difficult to distinguish from surrounding tissues. Given their location and physical condition, the ureters are at considerable risk of injury in many surgical procedures, including gynecological, urological, colorectal, and cardiovascular procedures. See, for example, Engelsgjerd and LaGrange, 2020, StatPearls, Treasure Island (FL): StatPearls Publishing; 2020 Jul 10; Ferrara and Kann, 2019, Clin. Colon Rectal Surg. 32, 196-203; Ahn et al., 2019, Quant. Imaging Med. Surg. 9, 1056-1065; Gild et al., 2018, Asian J. Urol. 5, 101-106. In fact, ureteral injuries often go unnoticed during surgery, leading to delayed diagnosis and treatment, which in some cases can result in long-term complications such as renal failure. Tan-Kim et al. 2015, J. Minimally Invasive Gynecol. 22, 1278-1286; Al-Awadi et al. 2005, Int. Urol. Nephrol. 37, 235-241.

[0005] As a result, ureteral injury has a significant impact on patient morbidity and associated healthcare costs. In the United States, with approximately 600,000 hysterectomies and 300,000 colon surgeries performed annually, ureteral injury rates are high, at 2.5% and 7.6%, respectively. (See Gild et al. 2018, Asian J. Urol. 5, 101-106; Doll et al. 2016, JAMA Surg. 151, 876-877; Teeluckdharry et al. 2015, Obstet. Gynecol. 126, 1161-1169; Briggs and Goldberg, 2011, Clin. Colon. Rectal Surg. 30, 130-135.) In lower abdominal surgery, the estimated incidence of iatrogenic ureteral injury (IUI) is 0.5–1% in cancer surgery, 0.3–2.5% in laparoscopic gynecological surgery, and up to 10% in gynecological tumor surgery. For example, de Valk et al. 2019, Nat. Commun. 16, 3118; Anderson et al. 2015, Surg. Endosc. 29, 1406-1412; Engel et al. 2015, Curr. Opin. Urol. 25, 331-335; Minas et al. 2014, Obstet. Gynecol. 16, See 19-28; Zafar et al. 2014, JSLS. 18, e2014.00158; Silva et al. 2012, Asian J. Endosc. Surg. 5, 1050110; Delacroix and Winters, 2010, Clin. Colon Rectal Surg. 23,104-112. The combined economic impact of ureteral injuries exceeds $1 billion in the United States alone, resulting in an average of four days of hospitalization and costs exceeding $30,000 per injury.

[0006] Several approaches are being explored to reduce the risk of ureteral injury during surgery. These include urological surgical procedures involving the insertion of stents or illumination catheters into the ureter, but these may disrupt the surgical workflow and cause unwanted delays, for example, by requiring intraoperative consultation with a urologist or other specialist. See, for example, Wood et al. 1996, J. Am. Assoc. Gynecol. Laparosc, 3, 393-397; Chahin et al. 2002, JSLS 6, 49-52. Other approaches include the administration or injection of radioactive dyes or fluorophores (or certain conjugates) to visualize the ureter. However, such procedures may involve additional risks and complexities, and the sensitivity, strength, and duration of action of the agents may be limited. For example, Ikeda et al., 2017, Am. J. Physiol. Renal Physiol. 312, F629-F639; Al-Taher et al., 2016, J. Laparoendosc. Adv. Surg. Tech. A. 26, 870-875; Verbeek et al., 2014, Ann. Surg. Oncol. 21, S528-S537; Hyun et al. 2012, Contrast Media Mol. Imaging 7, 516-524; Choi et al., 2011, Angew Chem. Int. Ed. Engl. 50, 6258-6263; Tanaka, et al. 2007, J. Urol. 178, 2197-2201; Cadeddu et al. al., 2001, See J. Endourology 15, 111-116. [Overview of the project] [Problems that the invention aims to solve]

[0007] In this field, there remains a need to identify compositions with desirable properties, including visualization of the ureter during surgical procedures. The present invention satisfies these and other needs by providing peptide compositions, particularly fluorescent peptide conjugates, that enable sensitive and long-term ureteral visualization after administration and can further support neuro-visualization, including simultaneous neuro- and ureteral visualization. [Means for solving the problem]

[0008] overview Disclosed herein are methods for visualizing the ureter, including a method for fluorescence visualization of the ureter during a surgical procedure.

[0009] In one embodiment, the present invention relates to a method for visualizing the ureter in a subject, comprising (a) administering an effective amount of a fluorescent conjugate containing a peptide and a fluorescent moiety to the subject, and (b) detecting the fluorescence of the fluorescent moiety in the ureter after administration. In one embodiment, the subject undergoes a surgical procedure.

[0010] In another embodiment, the present invention relates to a method comprising (a) administering an effective amount of a fluorescent conjugate comprising a peptide and a fluorescent moiety to a subject undergoing a surgical procedure; and (b) visualizing the ureter in the subject by detecting the fluorescence of the fluorescent moiety in the ureter.

[0011] In another embodiment, the present invention relates to a method for reducing ureteral injury, comprising: (a) identifying subjects at risk of ureteral injury before a surgical procedure; (b) administering an effective dose of a fluorescent conjugate containing a peptide and a fluorescent moiety to subjects undergoing the surgical procedure; and (c) visualizing the ureter by detecting the fluorescence of the fluorescent moiety in the ureter. In one embodiment, ureteral injury is caused by ligation, angulation, amputation, laceration, crushing, imaginary Caused by bleeding or excision.

[0012] In one embodiment of the method, the peptide may be a neuronal targeting peptide. In any of the methods, the peptide may consist of or include any of the amino acid sequences disclosed herein, including those shown in international patent applications PCT / US2018 / 045054 and PCT / US21 / 61821. In one embodiment, the fluorescently conjugated peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 16, 18-82 and 84-748.

[0013] In any embodiment of the method, the fluorescent moiety may be conjugated directly or indirectly via a linker to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the peptide. In any embodiment of the method, the fluorescent moiety is selected from the group consisting of fluorescent proteins, fluorescent peptides, fluorescent dyes, or combinations thereof disclosed herein.

[0014] In some embodiments, any of the methods may further include detecting the fluorescence of the fluorescent moiety at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours after administration. In some embodiments, the method further includes detecting the fluorescence of the fluorescent moiety at times more than 1 hour, more than 2 hours, more than 3 hours, more than 4 hours, more than 5 hours, more than 6 hours, more than 7 hours, or more than 8 hours after administration.

[0015] In one embodiment, the fluorescent conjugate in any of the methods is not administered to the subject again after the initial dose. In any of the methods, detection of fluorescence of the fluorescent portion in the ureter may include detection of urine flow in the ureter, more specifically detection of peristaltic flow of urine in the ureter. In any of the methods, the fluorescent conjugate may be administered intravenously. In any of the methods, the fluorescent conjugate may be administered orally. In any of the methods, the fluorescent conjugate may be administered to the subject before a surgical procedure.

[0016] In one embodiment, the surgical procedure in any of the methods may include gynecological, urological, colorectal, or cardiovascular surgical procedures; the abdomen or pelvis may be involved; it may be performed on the kidneys, bladder, prostate, uterus, male or female reproductive system, rectum, colon, small intestine, or large intestine; it may be an open surgery, a laparoscopic surgery, a microscopic or endoscopic procedure; or it may be a cancer surgery, more specifically, a prostate cancer surgery or a colorectal cancer surgery.

[0017] In one embodiment, the fluorescent conjugate in any of the methods comprises a fluorescent moiety selected from the group consisting of fluorescent proteins, fluorescent peptides, fluorescent dyes, or combinations thereof described herein.

[0018] In one embodiment, each method involves administering a fluorescent conjugate in a pharmaceutical composition which may contain pharmaceutically acceptable additives.

[0019] In one embodiment, the fluorescence conjugate may enable the visualization of nerves in the target, as well as associated (or simultaneous) nerve and ureteral visualization. In another embodiment, the fluorescence conjugate may result in the visualization of nerves and ureters in the target at various points in time during a surgical procedure. [Brief explanation of the drawing]

[0020] [Figure 1] Bright-field and fluorescence overlay images of the ureters in living wild-type mice after intravenous administration of approximately 2000 nanomoles of a fluorescence conjugate (SEQ ID NO: 747). Images were obtained 2 hours after administration using a customized fluorescence detection microscope. Left panel: White reflectance image. Right panel: Fluorescence overlay. [Modes for carrying out the invention]

[0021] Detailed description Disclosed herein is a method of visualizing the ureter, more specifically a method of fluorescently visualizing the ureter, by administration of a fluorescent conjugate (also referred to herein as a "conjugate") comprising a peptide and a fluorescent moiety. It has been discovered by the present invention that the metabolism and excretion of certain fluorescent conjugates, including those containing a nerve targeting peptide, support the sensitivity and long-term detection of fluorescence during urination from the kidney to the bladder in the ureter. Advantageously, the use of the fluorescent conjugate herein enables detection of the ureter both early and late after administration. These properties highlight the broad utility of such conjugates, which result in short-term and long-term visualization of the ureter after administration.

[0022] Thus, in one embodiment, the method includes visualization of the ureter in a subject by detection of the fluorescent moiety in the urinary ureteral flow, more specifically the peristaltic ureteral flow of urine. The fluorescent conjugate can be administered intravenously and can be administered to the subject prior to a surgical procedure.

[0023] In one embodiment, the peptide in the fluorescent conjugate includes a nerve targeting peptide, more specifically a nerve targeting peptide disclosed in International Patent Application PCT / US2018 / 045054 or International Patent Application PCT / US21 / 61821, both of which are hereby incorporated by reference in their entirety. Thus, in one embodiment, the fluorescent conjugate disclosed herein may enable simultaneous detection of the ureter and nerves.

[0024] In one embodiment, the peptide in the fluorescent conjugate includes the amino acid sequence QVPWEEPYYVVKKSSGG (HNP401-N-2 with a GG linker; SEQ ID NO: 21) disclosed in International Patent Publication WO2019028281A2. In one embodiment, the fluorescent conjugate is 5FAM-QVPWEEPYYVVKKSSGG-NH2 (HNP401-N-2 with a GG linker; SEQ ID NO: 747).

[0025] The present invention may be more fully understood by referring to the following description, including examples. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Methods and materials similar to or equivalent to those described herein, which may be used in carrying out or testing the present invention, are described herein. Furthermore, materials, methods and examples are illustrative and not intended to be limiting.

[0026] For the sake of brevity, all documents or parts of documents cited herein, including patents, patent applications, articles, books, manuals, and professional texts, are expressly incorporated herein by reference in their entirety for any purpose. However, no citation of any such publication shall be construed as an admission that it is prior art of the present invention.

[0027] The titles and subtitles in the sections of this specification are for structural purposes only and should not be construed as limiting the subject matter described herein, which should be interpreted by referring to this specification as a whole. For example, those skilled in the art will recognize that various combinations of aspects from different titles and sections are useful as appropriate in accordance with the spirit and scope of the invention described herein.

[0028] As will be apparent to those skilled in the art from this specification, the embodiments described and their various alternatives can be carried out without further detail in the described examples.

[0029] I. Definition The singular expression used herein refers to "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. Furthermore, individual compounds or groups of compounds resulting from various combinations of structures and substituents described herein are disclosed to the same extent as each compound or group of compounds is shown individually. Thus, the selection of a particular structure or particular substituent is within the scope of the present invention.

[0030] As used herein, the terms “approximately” or “about” mean a range of values ​​that includes the specified value, which a person skilled in the art would consider reasonably similar to the specified value. In some embodiments, “approximately” means within a standard deviation using measurements generally accepted in the art. In some embodiments, “approximately” means a range of ±10% of the specified value. In some embodiments, “approximately” means the specified value.

[0031] Whether the term “approximately” is explicitly used or not, all numerical values ​​presented herein mean approximations of such numerical values ​​that can be reasonably inferred based on the prior art of the art, including actual numerical values ​​and those that are equivalent or approximate to such numerical values ​​by experimental and / or measurement conditions. Accordingly, with respect to any embodiment of the invention in which “approximately” or “approximately” precedes a numerical value, the invention includes embodiments in which exact values ​​are mentioned. Conversely, with respect to any embodiment of the invention in which “approximately” or “approximately” does not precede a numerical value, the invention includes embodiments in which “approximately” or “approximately” precedes a value.

[0032] The terms “include” and “contain” are used herein in their open, non-restrictive sense. Other terms and expressions and their variations used herein should be interpreted open-ended and not terminally restrictive unless otherwise expressly indicated. For example, the term “example” is invoked to provide illustrative cases of the items being discussed, and not a thorough or restrictive list thereof. Adjectives and similar terms such as “conventional,” “ordinary,” and “known” should not be interpreted as limiting the items described to items available at a particular time or point in time, but rather as encompassing conventional or ordinary art that may be available or known at any point in the present or future. Similarly, where this specification describes art that is apparent or known to those skilled in the art, such art includes that which may be apparent or known to those skilled in the art at any point in the present or future.

[0033] The following terms used herein have the meanings they encompass unless otherwise specified.

[0034] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably here to refer to polymers of amino acid residues. These terms apply to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are not naturally occurring amino acids (e.g., amino acid analogs). These terms encompass amino acid chains of any length, including full-length proteins (i.e., neural targeting molecules), where amino acid residues are linked by covalent peptide bonds. As used herein, “peptide” typically refers to polymers of amino acid residues ranging from approximately 2 to about 50 residues in length. In some embodiments, peptides range from approximately 2, 3, 4, 5, 7, 9, 10, or 11 residues to approximately 50, 45, 40, 45, 30, 25, 20, or 15 residues in length. In some embodiments, peptides range from approximately 8, 9, 10, 11, or 12 residues to approximately 15, 20, or 25 residues in length. Where amino acid sequences are described herein, L-, D-, or beta-amino acid versions of the sequence, as well as reverse, inverted, and inverted-inverted isoforms, are also considered. Peptides also include amino acid polymers and naturally occurring amino acid polymers in which one or more amino acid residues are artificial analogs of corresponding naturally occurring amino acids. Furthermore, the term also applies to amino acids linked by peptide bonds or other modifying bonds (for example, when peptide bonds are replaced by α-esters, 3-esters, thioamides, phosphoamides, carbamates, hydroxylates, etc. (see, e.g., Spatola 1983, Chem. Biochem. Amino Acids and Proteins 7: 267-357), or when amides are replaced by saturated amines (see, e.g., Skiles et al., U.S. Patent 4,496,542 and Kaltenbronn et al., 1990, pp. 969-970 in Proc. 11th American Peptide Symposium, ESCOM Science Publishers, The Netherlands, etc., as incorporated herein by reference)).

[0035] Therefore, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids refer to those encoded by the genetic code as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acids can be L- or D-amino acids. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons bonded to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, and methionine sulfoxide. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes refer to chemical compounds that have a different structure from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0036] Amino acids can be represented by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUP AC-IUB Biochemical Nomenclature Commission. Nucleotides can similarly be represented by commonly accepted single-letter codes.

[0037] Those skilled in the art will recognize that individual substitutions, deletions, or additions to peptide, polypeptide, or protein sequences that alter, add, or delete one amino acid or a small percentage of an amino acid in the coding sequence are “conserved variants” that result in substitutions with amino acids that are chemically similar to the original amino acid. Tables of conserved substitutions that provide functionally similar amino acids are well known in the art. Such conserved variants are further polymorphic variants, interspecific homologs, and alleles, and these are not excluded.

[0038] The following eight groups each contain amino acids that are conservedly substituted for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)).

[0039] As used herein, "sequence identity" (or "identity") refers to the percentage of amino acid residues in a single sequence that are identical to amino acid residues in other reference polypeptide sequences, after sequence alignment and, if necessary, the introduction of gaps required to achieve maximum percentage sequence identity, and excluding any conservative substitutions. As used herein, "sequence homology" (or "homology") refers to the percentage of amino acid residues in a single sequence that have the same or similar chemical properties as amino acid residues in other reference polypeptide sequences, after sequence alignment and, if necessary, the introduction of gaps required to achieve maximum percentage sequence homology. Therefore, in contrast to sequence identity, sequence homology considers all conservative substitutions (as described above) as part of sequence alignment. Sequence identity or sequence homology can be determined using well-known methods, including commonly available computer programs such as the NCBI BLAST 2.0 software, as defined by Altschul et al. 1997, Nucl. Acids Res. 25, 3389-3402. These programs optimally align sequences using default gap weighting to crudely determine the highest level of sequence identity or sequence homology between a given sequence and a reference sequence.

[0040] The term "label" as used herein refers to a molecule that facilitates the visualization and / or detection of the targeting molecule disclosed herein. In one embodiment, the label is a fluorescent moiety.

[0041] The terms “individual,” “patient,” or “subject” are used interchangeably. As used herein, they mean any mammal (i.e., any species of any order, family, and genus within the taxonomic kingdom Animalia: Chordate: Vertebrate: Mammalia). In some embodiments, mammals are cattle, horses, sheep, pigs, cats, dogs, goats, mice, rats, rabbits, guinea pigs, non-human primates, or humans. In some embodiments, mammals are human subjects, more specifically, human subjects undergoing surgical procedures. The term herein includes, but does not include, any employee who does not require, a situation characterized by the supervision (e.g., constantly or intermittently) of a healthcare professional (e.g., physician, registered nurse, nurse practitioner, physician's assistant, general assistant, or hospice employee).

[0042] The terms “administer,” “to administer,” and “to administer” as used herein refer to methods that may be used to enable the delivery of a drug or composition to a desired biological site of action. These methods include, but are not limited to, non-enteral injections (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravitreous, infusion, or local). Administration techniques used with the drugs and methods described herein, if desired, are, for example, Gennaro: Remington: The Science and Practice of Pharmacy, 21 st This includes those described in Ed. (Lippincott Williams & Wilkins, 2005) and Goodman & Gilman's The Pharmacological Basis of Therapeutics (McGraw-Hill Professional, 2005). In one embodiment, administration is via systemic intravenous injection into a human patient.

[0043] The term “effective dose” is interchangeable with “therapeutic effective dose” and means a sufficient amount to achieve the purpose described, for example, to achieve an effect in the target of administration, such as enabling fluorescence visualization during ureteral surgery (the course of the surgery, surgical procedure, surgical method, or during the operation). An example of an “effective dose” is a sufficient amount to contribute to the treatment, prevention, or reduction of surgical or medical procedure-induced adverse events, such as iatrogenic ureteral injury (IUI) during abdominal surgery. The desired effect may, but not necessarily, occur with a single dose. It may also occur after a series of doses. Therefore, an effective dose may be administered in one or more doses. Determining the therapeutic effective dose of a composition is within the scope of the skills of those skilled in the art, particularly in light of the detailed description herein. For example, a dose for humans may be formulated to achieve a concentration that has been found to be effective in animals. Doses in humans may be adjusted by monitoring the effectiveness of the drugs or compositions disclosed herein and adjusting the dose upward or downward. The preparation of doses to achieve maximum efficacy in humans based on the methods described herein and other methods is well within the capabilities of those skilled in the art.

[0044] As used herein, "pharmaceutically acceptable" means a substance that does not inhibit the biological activity or properties of the drugs and compositions described herein and is relatively non-toxic (i.e., the toxicity of the substance does not significantly outweigh the benefits of the substance). In some cases, a pharmaceutically acceptable substance may be administered to an individual without causing significant undesirable biological effects or significant harmful interactions with any of the components of the composition.

[0045] As used herein, the term “surgery” refers to any method or technique that may be used to manipulate, alter, or cause an effect through physical intervention. Surgical techniques include, but are not limited to, open surgery, microsurgery, endoscopic surgery, laparoscopic surgery, minimally invasive surgery, and robotic surgery. In some cases, the surgical subject is a human subject or human patient. Surgical techniques may include gynecological, urological, colorectal, or cardiovascular surgical techniques. Surgical techniques may also include, but are not limited to, abdominal or pelvic procedures. In some cases, surgical techniques are performed on the kidneys, bladder, prostate, uterus, male or female reproductive system, rectum, colon, small intestine, or large intestine. Surgical techniques may include, for example, cancer surgical techniques, including prostate cancer surgical techniques or colorectal surgical techniques. Surgical procedures may include those that carry a risk of ureteral injury, and where present, may also affect nerves, as the compositions and methods described herein may support intraoperative nerve visualization, including simultaneous visualization of nerves and ureters in the subject during surgery.

[0046] II. Method The present invention provides a method for visualizing the ureter, particularly during surgery, by using a fluorescent conjugate containing a peptide conjugated to a fluorescent portion, i.e., a labeled peptide. In one embodiment, the peptide includes a neurotargeting peptide. In one embodiment, the fluorescent conjugate (and its composition) described herein is useful as a surgical aid in such a method to reduce the possibility of ureteral injury and, in particular, iatrogenic ureteral injury (IUI) during surgical procedures.

[0047] As further described here, it has been observed that nerve-targeting fluorescence conjugates can be removed by renal excretion under conditions that allow for significant and prolonged fluorescence as they pass through the ureters. Consequently, ureteral visualization can be achieved by detecting the fluorescence portion of the urine flow through the ureters, more specifically, the peristaltic flow of urine through the ureters. Ureteral visualization can persist for extended periods, sometimes for several hours or more, after administration of a particular fluorescence conjugate.

[0048] Accordingly, in one embodiment, the present invention provides a method for visualizing the ureter in a subject using any of the molecules and compositions described herein. In one embodiment, the subject is undergoing a procedure such as a diagnostic procedure, a medical procedure or a surgical procedure.

[0049] In one embodiment, the method comprises (a) administering an effective amount of a fluorescent conjugate containing a peptide and a fluorescent moiety to a subject; and (b) detecting the fluorescence of the fluorescent moiety in the ureter of the subject after administration.

[0050] In another embodiment, the method comprises (a) administering an effective amount of a fluorescent conjugate containing a peptide and a fluorescent moiety to a subject undergoing a surgical procedure; and (b) visualizing the ureter in the subject by detecting the fluorescence of the fluorescent moiety in the ureter.

[0051] In another embodiment, the method comprises (a) identifying subjects at risk of ureteral injury prior to a surgical procedure; (b) administering an effective dose of a fluorescent conjugate containing a peptide and a fluorescent moiety to subjects undergoing the surgical procedure; and (c) visualizing the ureter by detecting the fluorescence of the fluorescent moiety in the ureter. Subjects at risk of ureteral injury as used herein include subjects requiring the diagnostic, medical, or surgical procedures described herein.

[0052] In one embodiment, the fluorescent conjugate is administered first before the surgical procedure. In another embodiment, the fluorescent conjugate is not administered after the initial dose. In another embodiment, the fluorescent conjugate is administered during the surgical procedure. In yet another embodiment, the fluorescent conjugate is administered before and during the surgical procedure.

[0053] In some embodiments, the surgical procedure includes gynecological, urological, colorectal, or cardiovascular surgical procedures. In some embodiments, the surgical procedure includes abdominal or pelvic procedures. In some embodiments, the surgical procedure is performed on the kidneys, bladder, prostate, uterus, male or female reproductive system, rectum, colon, small intestine, or large intestine. In some embodiments, the surgical procedure is an open surgery, a laparoscopic surgery, a microscopic procedure, or an endoscopic procedure. In some embodiments, the surgical procedure is a cancer surgery, more specifically, a prostate cancer surgery.

[0054] In any embodiment of these methods, the fluorescent conjugate is administered to the subjects described herein, including humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In some embodiments, the subjects are mammals. In some embodiments, the subjects are primates. In some embodiments, the subjects are humans. In some embodiments, the human subjects are children (under 21 years of age), adults (22 to 65 years of age), or elderly (65 years of age and older).

[0055] The fluorescent conjugate may be administered by any route as further described herein. In one embodiment, the fluorescent conjugate is administered intravenously. In another embodiment, the fluorescent conjugate is administered orally. In another embodiment, the fluorescent conjugate administered by any route may be administered as a pharmaceutical composition as described herein, which may contain pharmaceutically acceptable additives.

[0056] The fluorescent portion of a fluorescent conjugate can be detected in the ureter by any suitable method known in the art. For example, the fluorescent portion can be detected by excitation of a fluorophore in the surgical field with light of an appropriate wavelength and detection of the resulting fluorescence (and the surgical field) using a combination of filters, lenses, and cameras.

[0057] In one embodiment of the method, the fluorescent portion is detected in the ureter by a medical device adapted for fluorescence-guided surgery (FGS). Such adapted devices may include, but are not limited to, portable or fixed devices used in open surgery; minimally invasive devices such as laparoscopes and endoscopes used in abdominal and pelvic procedures; robotic surgical systems used in prostate surgery; and other devices such as surgical magnifiers. The device may provide real-time synchronization information from bright-field illumination and fluorescence emission. One or more light sources may be used to excite and illuminate the sample or surgical field. Optical filters, imaging lenses, and digital cameras (e.g., CCD and CMOS detector systems) customized for the emission spectrum of the fluorescent portion may be used to collect the light and create a final image. Live video processing may also be performed to enhance contrast and improve sensitivity of the fluorescence signal during fluorescence detection.

[0058] In one embodiment of the method, the fluorescence of the fluorescent portion is detected in the ureter within at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours after administration.

[0059] In one embodiment of the method, the fluorescence of the fluorescent portion is detected in the ureter for more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours after administration.

[0060] In one embodiment of the method, the fluorescence of the fluorescent portion is detected in the ureter 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours after administration.

[0061] In one embodiment of the method, the fluorescence conjugate enables associated nerve detection and ureter visualization, including intraoperative nerve and ureter visualization. Such nerves may include, for example, human nerves such as motor nerves, sensory nerves, sympathetic and parasympathetic nerves, peripheral nerves and autonomic nerves, as disclosed in the '054 application.

[0062] More generally, the methods, conjugates, and compositions described herein may also encompass open surgical procedures, laparoscopic surgical procedures, microscopic procedures, and endoscopic procedures.

[0063] Furthermore, the methods, conjugates, and compositions described herein may also be used in surgical procedures where visualization of nerves and ureters is desirable.

[0064] III. Fluorescent Conjugates The fluorescent conjugate used in this method is Peptides, more specifically, neuronal targeting peptides (or peptides containing fluorescent labeling in other ways) bound to a fluorescent moiety.

[0065] peptide In one embodiment, the neuronal targeting peptide is any peptide, including any deletion or linker variant, disclosed in international patent application PCT / US2018 / 045054 ("Application 054", published as WO2019028281A2, incorporated herein by reference). Thus, such a peptide includes or consists of any of the following amino acid sequences (peptide sequences) listed in Table 1 (SEQ ID NOs: 1-17, 20-28, and 96-102). [Table 1]

[0066] In one embodiment, the peptide comprises or consists of any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102.

[0067] Furthermore, the peptides may include or consist of any variant of the amino acid sequence described in the '054 application. Such variants may include, but are not limited to, peptides containing or consisting of an amino acid sequence that shares 75%–99% identity or 75%–99% homology (or as otherwise disclosed in the '054 application) with the amino acid sequence in Table 1.

[0068] In one embodiment, the peptide contains or comprises an amino acid sequence having at least 75% identity with any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102. In one embodiment, the peptide contains or comprises an amino acid sequence having at least 80% identity with any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102. In one embodiment, the peptide contains or comprises an amino acid sequence having at least 85% identity with any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102. In one embodiment, the peptide contains or comprises an amino acid sequence having at least 90% identity with any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102. In one embodiment, the peptide contains or comprises an amino acid sequence having at least 95% identity with any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102. In one embodiment, the peptide comprises or consists of an amino acid sequence having at least 97% identity with any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102.

[0069] In one embodiment, the peptide comprises or consists of a peptide sequence that shares at least 80% homology with any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102. In one embodiment, the peptide comprises or consists of a peptide sequence that shares at least 80% homology with any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102. In one embodiment, the peptide comprises or consists of an amino acid sequence that shares at least 85% homology with any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102. In one embodiment, the peptide comprises or consists of an amino acid sequence that shares at least 90% homology with any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102. In one embodiment, the peptide comprises or consists of an amino acid sequence that shares at least 95% homology with any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102. In one embodiment, the peptide comprises or consists of an amino acid sequence that shares at least 99% homology with any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102.

[0070] In one embodiment, the peptide comprises or consists of an amino acid sequence of about 13 to about 25 amino acids, including a core-binding domain, the N-terminal sequences of PYYVVKK (SEQ ID NO: 40), and QVPWEE (SEQ ID NO: 41).

[0071] In one embodiment, the peptide comprises the amino acid core-binding domain of PYY or PYYVV (SEQ ID NO: 83) and the N-terminal sequence of QVPWEE (SEQ ID NO: 41). In another embodiment, the peptide comprises the amino acid core-binding domain of PYY and the N-terminal sequence of QVPWEE (SEQ ID NO: 41). In yet another embodiment, the peptide comprises the amino acid core-binding domain VV (SEQ ID NO: 83) of PYY and the N-terminal sequence of QVPWEE (SEQ ID NO: 41).

[0072] In one embodiment, the peptide comprises or consists of QVPWEEPYYVVKKSSGG (HNP401-N-2 having a GG linker; SEQ ID NO: 21).

[0073] In one embodiment, the peptide comprises or consists of an amino acid sequence selected from the group consisting of SGQVPWEEPYYVVKKSS(HNP401; SEQ ID NO: 1), WEYHYVDLNWTSQHPQ(HNP402; SEQ ID NO: 2), and DLPDIIWDFNWETA(HNP403; SEQ ID NO: 3).

[0074] In one embodiment, the peptide comprises or consists of an amino acid sequence selected from the group consisting of SGQVPWEEPYYVVKKSS(HNP401; SEQ ID NO: 1), Ac-QVPWEEPYYVVKKSSGGC(HNP401-N-2 with GGC linker; SEQ ID NO: 7), Ac-SGQVPWEEPYYVVKKGGC(HNP401-C-2 with GGC linker; SEQ ID NO: 11), QVPWEEPYYVVKKSS(HNP401-N-2; SEQ ID NO: 20), QVPWEEPYYVVKKSSGG(HNP401-N-2 with GG linker; SEQ ID NO: 21), SGQVPWEEPYYVVKK(HNP401-C-2; SEQ ID NO: 25), and SGQVPWEEPYYVVKKGG(HNP401-C-2 with GG linker; SEQ ID NO: 99).

[0075] In one embodiment, the peptide contains or consists of any of the amino sequences of SEQ ID NOs: 1, 4, 7-14, 20-28, and 96-102.

[0076] In one embodiment, the peptide contains or consists of any of the amino sequences of SEQ ID NOs: 1, 20, and 22-24.

[0077] In one embodiment, the peptide contains or consists of any of the amino sequences of SEQ ID NOs: 1 and 25-28.

[0078] In one embodiment, the peptide contains or consists of any of the amino sequences of SEQ ID NOs: 4 and 7-10.

[0079] In one embodiment, the peptide contains or consists of any of the amino sequences of SEQ ID NOs: 4 and 11-14.

[0080] In one embodiment, the peptide contains or consists of any of the amino sequences of SEQ ID NOs. 21 and 100-102.

[0081] In one embodiment, the peptide contains or consists of any of the amino sequences of SEQ ID NOs. 96 to 99.

[0082] In one embodiment, the peptides are neuronal targeting peptides disclosed in PCT application PCT / US21 / 61821 ("Application '821"), which is incorporated herein by reference in whole. In one embodiment, these peptides were derived from phage display library screening based on selective binding to any of four different neuronal target proteins: myelin protein zero (MPZ), myelin basic protein, myelin proteolipide protein (myelin PLP), and nidogen-2. Such peptides contain or consist of any of the amino acid sequences listed in Table 2 (SEQ ID NOs: 18, 19, 29-82, 84-95, and 103-471). In one embodiment, these peptides were derived from phage display screening based on selective binding to laminin trimers 421, 521, or both. Such peptides contain or consist of any of the amino acid sequences listed in Table 3 (SEQ ID NOs: 472-678). In one embodiment, these peptides were derived from phage display screening based on selective binding to neuronal and nerve extracts, rather than muscle extracts. Such peptides contain or consist of any of the amino acid sequences listed in Table 4 (SEQ ID NOs. 679-746). [Table 2-1] [Table 2-2] [Table 2-3] Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 2-8 Table 2-9 Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 4-1 Table 4-2

[0083] Therefore, in one embodiment, the peptide includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 29-82, 84-95, and 103-746. In one embodiment, the peptide includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 29-82, 84-95, and 103-746. In one embodiment, the peptide includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-82, 84-95, and 103-746. In one embodiment, the peptide includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-74 and 188-362. In one embodiment, the peptide includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-82, 84-95, and 103-187. In one embodiment, the peptide includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-54 and 363-446. In one embodiment, the peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 29-32, and 447-471. In another embodiment, the peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 472-678. In yet another embodiment, the peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 679-746.

[0084] In one embodiment, the peptide comprises or consists of a variant of the amino acid sequence described in international patent application PCT / US21 / 61821 ("Application 821"). Such variants include, but are not limited to, peptides comprising or consisting of amino acid sequences that share 75% to 99% identity or 75% to 99% homology with SEQ ID NOs. 18, 19, 29-82, 84-95 and 103-746.

[0085] In one embodiment, the peptide comprises or consists of an amino acid sequence having at least 75% identity with any of SEQ ID NOs. 18, 19, 29-82, 84-95, and 103-746. In another embodiment, the peptide comprises or consists of an amino acid sequence having at least 80% identity with any of SEQ ID NOs. 18, 19, 29-82, 84-95, and 103-746. In yet another embodiment, the peptide comprises or consists of an amino acid sequence having at least 85% identity with any of SEQ ID NOs. 18, 19, 29-82, 84-95, and 103-746. In yet another embodiment, the peptide comprises or consists of an amino acid sequence having at least 90% identity with any of SEQ ID NOs. 18, 19, 29-82, 84-95, and 103-746. In one embodiment, the peptide comprises or consists of an amino acid sequence having at least 95% identity with any of SEQ ID NOs: 18, 19, 29-82, 84-95, and 103-746.

[0086] In one embodiment, the peptide comprises or consists of an amino acid sequence that shares at least 80% homology with any of SEQ ID NOs. 18, 19, 29-82, 84-95, and 103-746. In one embodiment, the peptide comprises or consists of an amino acid sequence that shares at least 85% homology with any of SEQ ID NOs. 18, 19, 29-82, 84-95, and 103-746. In one embodiment, the peptide comprises or consists of an amino acid sequence that shares at least 90% homology with any of SEQ ID NOs. 18, 19, 29-82, 84-95, and 103-746. In one embodiment, the peptide comprises or consists of an amino acid sequence that shares at least 95% homology with any of SEQ ID NOs. 18, 19, 29-82, 84-95, and 103-746. In one embodiment, the peptide comprises or consists of an amino acid sequence that shares at least 99% homology with any of SEQ ID NOs: 18, 19, 29-82, 84-95, and 103-746.

[0087] The peptides of the present invention are synthesized by any suitable method known to those skilled in the art, including those disclosed in the '054 and '821 applications incorporated herein. For example, the peptides of the present invention can be chemically synthesized by solid-phase peptide synthesis. In some embodiments, the peptides of the present invention are acetylated at the N-terminus ("Ac" or "acetyl"), amidated at the C-terminus ("CONH2" or "NH2"), or both. For example, SEQ ID NO: 747 corresponds to an amidated peptide ending in a glycinamide.

[0088] Fluorescent part The term "fluorescent moiety" as used herein refers to any fluorescent molecule, including fluorescent proteins, fluorescent peptides, fluorophores (e.g., fluorescent dyes), or any other fluorescent ligand or marker.

[0089] The specific examples of fluorescent moieties described herein are for illustrative purposes only and are not intended to limit the use of fluorescent moieties in the peptides disclosed herein.

[0090] Fluorophores (or fluorescent dyes) include molecules that function in the visible and near-infrared (NIR) regions.

[0091] The general class of fluorophores includes, but is not limited to, xanthenes such as rhodamine, rhodol, and fluorescein and their derivatives; biman; coumarin and umbelliferone and their derivatives such as aminomethylcoumarin; aromatic amines such as dansyl; squalane dyes; benzofuran; fluorescent cyanines; carbazole; dicyanomethylenepyran; polymethine; oxabenzantran; pyrylium; carbostyryl; perylene; acridone; quinacridone; rubrene; anthracene; coronene; phenanthrethene; pyrene; butadiene; stilbene; porphyrin; phthalocyanines; lanthanide metal chelate complexes; rare earth metal chelate complexes; and their derivatives. Fluorescent dyes are disclosed, for example, in U.S. Patent 4,452,720; U.S. Patent 5,227,487; and U.S. Patent 5,543,295.

[0092] Typical fluorescein dyes for use with the conjugates disclosed herein include, but are not limited to, carboxyfluorescein, 5-carboxyfluorescein, 6-carboxyfluorescein, 5(6)-carboxyfluorescein, fluorescein isothiocyanate, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate, 5,6-dicarboxyfluorescein, 5-(and 6)-sulfofluorescein, sulfonefluorescein, succinylfluorescein, 5-(and 6)-carboxySNARF-1, carboxyfluorescein sulfonate, carboxyfluorescein zwitterion, carboxyfluorescein quaternary ammonium, carboxyfluorescein phosphonate, carboxyfluorescein GABA, carboxyfluorescein-cys-Cy5, and fluorescein glutathione. Examples of fluorescein dyes can also be found, for example, in U.S. Patents 6,008,379, 5,750,409, 5,066,580, and 4,439,356.

[0093] In one embodiment, the fluorescent portion includes a carboxyfluorescein molecule (FAM), i.e., a fluorescein molecule to which a carboxyl group has been added. Therefore, the term "FAM" includes fluorescein molecules having a carboxyl group at position 5 (5-FAM) or position 6 (6-FAM).

[0094] In one embodiment, the fluorescent moiety may include IR800CW, cyan fluorescent protein (CFP); EGFP; 5-FAM; 6-FAM; FAM; fluorescein; IAEDANS; EDANS; BODIPYFL; TRITC; Cy5; Cy3; YFP; LC Red 640; Alexa Fluor 546; tetramethylrhodamine; dabucil; QSY7; QSY9; QSY21; and BBQ-650 dye.

[0095] In one embodiment, the fluorescent moiety includes, for example, rhodamine dyes such as 5-(and 6)-carboxyrhodamine 110, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxyrhodol derivatives, tetramethyl and tetraethylrhodamine, diphenyldimethyl and diphenyldiethylrhodamine, dinaphthylrhodamine, rhodamine 101 sulfonyl chloride (TEXAS RED), and other rhodamine dyes. Other rhodamine dyes may be found, for example, in U.S. Patents 6,080,852; 6,025,505; 5,936,087; and 5,750,409.

[0096] In one embodiment, the fluorescent portion includes, for example, cyanine dyes such as Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, and indocyanine green.

[0097] In one embodiment, the fluorophore exhibits green fluorescence (e.g., 494 nm / 519 nm or 494 / 524 nm), orange fluorescence (e.g., 554 nm / 570 nm), red fluorescence (e.g., 590 nm / 617 nm), or far-red fluorescence (e.g., 651 nm / 672 nm) excitation / emission vectors. In one embodiment, the fluorophore is a fluorophore having excitation and emission vectors in the range of approximately 350 nm to approximately 775 nm. In one embodiment, the excitation and emission vectors are approximately 346 nm / 446 nm, approximately 494 nm / 519 nm, approximately 554 nm / 570 nm, approximately 555 nm / 572 nm, approximately 590 nm / 617 nm, approximately 651 nm / 672 nm, approximately 679 nm / 702 nm, or approximately 749 nm / 775 nm.

[0098] In one embodiment, the excitation and emission spectra of the fluorophore are in the visible spectrum region (approximately 380 nm to approximately 700 nm).

[0099] In one embodiment, the excitation and emission spectra of the fluorophore are in the near-infrared (NIR) range, including the NIR-I spectrum (approximately 700 nm to 900 nm) and the NIR-II spectrum (approximately 1000 nm to 1700 nm). See, for example, Zhu et al. 2018, Theranostics 8, 4141-4151.

[0100] In some embodiments, the fluorophore may include, but is not limited to, AlexaFluor 3, AlexaFluor 5, AlexaFluor 350, AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 514, AlexaFluor 532, AlexaFluor 546, AlexaFluor 555, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, and AlexaFluor 750 (available from Molecular Probes AlexaFluor dyes, Life Technologies, Inc. (USA)). In some embodiments, the fluorophore may include, but is not limited to, Cy dyes, including Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7 (available from GE Life Sciences or Lumiprobes). In one embodiment, the fluorophores may include, but are not limited to, DyLight 350, DyLight 405, DyLight 488, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 750, and DyLight 800 (available from Thermo Scientific (USA)). In one embodiment, the fluorophores are FluoProbes 390, FluoProbes 488, FluoProbes 532, FluoProbes 547H, FluoProbes 594, FluoProbes 647H, FluoProbes 682, FluoProbes 752 and FluoProbes 782, AMCA, DEAC (7-diethylaminocoumarin-3-carboxylic acid); 7-hydroxy-4-methylcoumarin-3; 7-hydroxycoumarin-3; MCA (7-methoxycoumarin-4-acetic acid); 7-methoxycoumarin-3; AMF (4'-(aminomethyl)fluorescein);5-DTAF(5-(4,6-dichlorotriazinyl)aminofluorescein); 6-DTAF(6-(4,6-dichlorotriazinyl)aminofluorescein); FAM(carboxyfluorescein); 6-FAM(6-carboxyfluorescein), 5(6)-FAMcadaverine; 5-FAMcadaverine; 5(6)-FAMethylenediamine; 5-FAMethylenediamine; 5-FITC(FITC isomer I; fluorescein-5-isothiocyanate); 5-FITCcadaverine; fluorescein-5-maleimide; 5-IAF(5-iodoacetamidefluorescein); 6-JOE(6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein); 5-CR1(5-carboxyrhodamine 110); 6-CR1 l0(6-carboxyrhodamine 110); 5-CR6G(5-carboxyrhodamine 6G); 6-CR6G(6-carboxyrhodamine 6G); 5(6)-carboxyrhodamine 6G cadaverine; 5(6)-Caroxyrhodamine 6G ethylenediamine; 5-ROX(5-carboxy-X-rhodamine); 6-ROX(6-carboxy-X-rhodamine); 5-TAMRA(5-carboxytetramethylrhodamine); 6-TAMRA(6-carboxytetramethylrhodamine); 5-TAMRA cadaverine; 6-TAMRA cadaverine; 5-TAMRA ethylenediamine; 6-TAMRA ethylenediamine; 5-TMR C6 maleimide; 6-TMR C6 maleimide; TR C2 maleimide; TR cadaverine; 5-TRITC; G isomer (tetramethylrhodamine-5-isothiocyanate); 6-TRITC; R isomer (tetramethylrhodamine-6-isothiocyanate); dansyl cadaverine (5-dimethylaminonaphthalene-l-(N-(5-aminopentyl)) sulfonamide); EDANS C2 maleimide; fluorescein; NBD; and pyromethenes and their derivatives may include, but are not limited to, these.

[0101] In one embodiment, the fluorescent portion comprises an environmentally sensitive fluorescent dye or fluorophore. Examples of environmentally sensitive fluorescent dyes or fluorophores include 5,6-carboxy-diethyl rhodol (pH sensitive), merocyanine (membrane potential sensitive), and Nile red carboxylic acid (lipid sensitive).

[0102] In one embodiment, the fluorescent portion is a fluorescent peptide or fluorescent protein. In one embodiment, the fluorescent protein is green fluorescent protein (GFP). In one embodiment, the fluorescent portion is, for example, EGFP, Emerald, Superfolder GFP, Thistle Green mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, EBFP, EBFP2, Azurite, mTagBFP, ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan 1, Midori-Ishi Cyan, TagCFP, mTFP1(Teal), EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, Kusabira... These are derivatives or variants of GFP, including orange, *Orange 2*, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-Y, DsRed, DsRed2, DsRed-Express(T1), DsRed-Monomer, mTangerine, mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, AQ143, mKalamal, YFP, and citrin.

[0103] In one embodiment, the fluorescent portion includes fluorescent molecules disclosed in applications '054 and '821, which are incorporated herein by reference in their entirety.

[0104] In one embodiment, the fluorescent portion is conjugated to a high molecular weight molecule such as a water-soluble polymer, including but not limited to dextran, PEG, serum albumin, or poly(amidoamine) dendrimers.

[0105] The fluorescent moieties disclosed herein may bind to the N-terminus, C-terminus, or internal position (e.g., internal amino acids) of a peptide. In some embodiments, two, three, four, or more peptides may bind directly or indirectly to the fluorescent moieties. In some embodiments, any of the peptides disclosed herein may bind to two or more fluorescent moieties, which may be the same or different.

[0106] In one embodiment, the fluorescent moiety disclosed herein is directly bound to any peptide disclosed herein.

[0107] Linker In one embodiment, the fluorescent moiety is indirectly bound to the peptide, for example, via a linker. The term "linker" as used herein is any molecule capable of binding (e.g., covalently) to the peptide disclosed herein. Linkers include, but are not limited to, linear or branched carbon-containing linkers, heterocyclic carbon-containing linkers, amino acid linkers (e.g., D- or L-amino acids), lipophilic residues, peptide linkers, peptide nucleic acid linkers, hydrazone linkers, SPDB disulfide, sulfo-SPDB, maleimidomethylcyclohexane-1-carboxylate (MCC), aminohexanoic acid linkers, and polyether linkers (e.g., PEG). Linkers include, for example, poly(ethylene glycol) linkers available from Quanta Biodesign, Powell, OH. These linkers optionally have amide bonds, sulfhydryl bonds, or heterofunctional group bonds. Linkers also include other linker molecules disclosed or described in the literature, including those described in applications '054 and '821, which are incorporated herein.

[0108] In one embodiment, the linker is covalently bonded to the targeting molecule disclosed herein. The linker can connect a cargo molecule, such as a fluorescent moiety, to a peptide by forming a covalent bond to the cargo molecule at one position and a covalent bond to the peptide at another position. Covalent bonding can also be managed by the reaction of a functional group of the linker with the functional groups of the peptide and the cargo molecule. In one embodiment, the covalent bond includes an ether bond, a thioether bond, an amine bond, an amide bond, a carbon-carbon bond, a carbon-nitrogen bond, a carbon-oxygen bond, or a carbon-sulfur bond. For example, the covalent bond can be formed by the condensation reaction of the carboxylic acid functional group at position 5 of fluorescein and the primary amine at the N-terminus of the peptide, as in the fluorescent conjugate 5-FAM-QVPWEEPYYVVKKSSGG-NH2 (HNP401-N-2 with GG linker; SEQ ID NO: 747).

[0109] In one embodiment, the linker is movable. In another embodiment, the linker is rigid. In another embodiment, the linker has a movable segment and a rigid segment.

[0110] In one embodiment, the linker includes a linear structure. In another embodiment, the linker includes a non-linear structure. In another embodiment, the linker includes a branched structure. In yet another embodiment, the linker includes an annular structure.

[0111] In one embodiment, the linker is alkyl. In another embodiment, the linker is heteroalkyl.

[0112] In one embodiment, the linker is alkylene. In another embodiment, the linker is alkenylene. In yet another embodiment, the linker is alkylylene. In yet another embodiment, the linker is heteroalkylene.

[0113] The "alkyl" group refers to an aliphatic hydrocarbon group. The alkyl portion can be either a saturated or unsaturated alkyl group. Depending on the structure, the alkyl group can be a monoradical or a diradical (i.e., an alkylene group).

[0114] The "alkyl" portion may have 1 to 10 carbon atoms (wherein used herein, numerical ranges such as "1 to 10" refer to each integer within that range; for example, "1 to 10 carbon atoms" means that the alkyl group may consist of carbon atoms up to 10 and including 10, such as 1 carbon, 2 carbon, 3 carbon, etc., but this definition also includes the term "alkyl" where no numerical range is specified). Alkyl groups are also "lower alkyls" having 1 to 6 carbon atoms. Alkyl groups in the compounds described herein may be designated as "C1-C4 alkyl" or similar designations. As merely an example, "C1-C4 alkyl" indicates that the alkyl chain has 1 to 4 carbon atoms, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, and butenyl.

[0115] In one embodiment, the linker includes a ring structure (e.g., aryl). The term "ring" as used herein refers to a ring-closed structure by any covalent bond. Rings include, for example, carbocyclic rings (e.g., aryl and cycloalkyl), heterocyclic rings (e.g., heteroaryl and non-aromatic heterocyclic rings), aromatic rings (e.g., aryl and heteroaryl), and non-aromatic rings (e.g., cycloalkyl and non-aromatic heterocyclic rings). The ring may be substituted as it is. The ring may be monocyclic or polycyclic.

[0116] The term "aryl" as used herein refers to an aromatic ring in which each of the atoms forming the ring is a carbon atom. Aryl rings can be formed from 5, 6, 7, 8, 9, or more than 9 carbon atoms. Aryl groups may be substituted. Examples of aryl groups include, but are not limited to, phenyl, naphthalenyl, phenantrenyl, anthracenyl, fluorenyl, and indenyl. Depending on the structure, an aryl group can be a monoradical or a diradical (i.e., an arylene group).

[0117] The term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic radical in which each of the ring-forming atoms (i.e., the skeletal atoms) is a carbon atom. Cycloalkyls may be saturated or partially unsaturated. Cycloalkyls include groups having 3 to 10 ring atoms. Cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0118] In one embodiment, the ring is a cycloalkane. In another embodiment, the ring is a cycloalkene.

[0119] In one embodiment, the ring is an aromatic ring. The term “aromatic” refers to a planar ring having a delocalized π-electron system containing 4n+2 π electrons (where n is an integer). Aromatic rings may be formed from 5, 6, 7, 8, 9, or more than 9 atoms. Aromatic rings may be substituted as they are. The term “aromatic” includes both carbocyclic aryl (e.g., phenyl) and heterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g., pyridine). The term includes monocyclic or fused polycyclic (i.e., rings shared by adjacent carbon atom pairs) groups.

[0120] In one embodiment, the ring is a heterocyclic ring. The term "heterocyclic ring" refers to heteroaromatic and heteroalicyclic groups containing 1 to 4 heteroatoms selected from O, S, and N, respectively, provided that each heterocyclic group has 4 to 10 atoms in its ring system and the ring of the group does not contain two adjacent O or S atoms. Nonaromatic heterocyclic groups include groups with only 3 atoms in their ring system, while aromatic heterocyclic groups must have at least 5 atoms in their ring system. Heterocyclic groups include benzo-condensed ring systems. An example of a 3-membered heterocyclic group is azilidinyl. An example of a 4-membered heterocyclic group is azetidinyl (derived from azetidine). An example of a 5-membered heterocyclic group is thiazolyl. An example of a 6-membered heterocyclic group is pyridyl, and an example of a 10-membered heterocyclic group is quinolinyl. Examples of non-aromatic heterocyclic groups include pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, and 1,2,3,6-tetrahydropi These include lysinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl, and quinolidinyl.Examples of aromatic heterocyclic groups include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, sinnolinyl, indazolyl, indolidinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, flazanyl, benzofuranyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthylidinyl, and phlopyridinyl. The groups may be C-bonded or N-bonded, if possible. For example, a pyrrole-derived group may be pyrrole-1-yl (N-bonded) or pyrrole-3-yl (C-bonded). Furthermore, imidazole-derived groups may be imidazole-1-yl or imidazole-3-yl (both N-bonded) or imidazole-2-yl, imidazole-4-yl, or imidazole-5-yl (all C-bonded). Heterocyclic groups include benzo-condensed ring systems and ring systems substituted with one or two oxo (=0) moieties, such as pyrrolidine-2-one. Depending on the structure, heterocyclic groups may be monoradicals or diradicals (i.e., heterocyclic groups).

[0121] In one embodiment, the ring is condensed. The term "condensation" refers to a structure in which two or more rings share one or more bonds. In one embodiment, the ring is a dimer. In one embodiment, the ring is a trimer. In one embodiment, the ring is substituted.

[0122] The term "carbocyclic formula" or "carbocyclic ring" refers to a ring in which each of the atoms forming the ring is a carbon atom. Carbocyclic rings include aryl and cycloalkyl groups. This term therefore distinguishes carbocyclic rings from heterocyclic rings ("heterocyclic formulas") which contain at least one atom (i.e., a heteroatom) whose ring skeleton is different from carbon. Heterocyclic rings include heteroaryl and heterocycloalkyl groups. Carbocyclic rings and heterocyclic rings may be substituted as they may be.

[0123] In one embodiment, the linker is substituted. The terms “partially substituted” or “substituted” mean that the base being referred to is C i -C e Alkyl, C3-C g This means that they may be substituted with one or more additional groups individually and independently selected from aminos and their protected derivatives, including cycloalkyl, aryl, heteroaryl, C2-C6 heteroalicyclic, hydroxy, C1-C6 alkoxy, aryloxy, C1-C6 alkylthio, arylthio, C1-C6 alkyl sulfoxide, aryl sulfoxide, C1-C6 alkyl sulfone, aryl sulfone, cyano, halo, C2-C8 acyl, C2-C8 acyloxy, nitro, C1-C6 haloalkyl, C1-C6 fluoroalkyl, and C1-C6 alkylamino. For example, the optional substituents may be LSRS, where each L is independently bonded and selected from -O-, -C(=O)-, -S-, -S(=O)-, -S(O)2-, -NH-, -NHC(O)-, -C(O)NH-, S(O)2NH-, -NHS(O)2-, -OC(O)NH-, -NHC(O)O-, -(CpC6 alkyl)-, or -(C2-C6 alkenyl)-; and each R is independently selected from H, (C1-C4 alkyl), (C3-C8 cycloalkyl), heteroaryl, aryl, and C1-C6 heteroalkyl. The optionally substituted non-aromatic group may be substituted with one or more oxo(=O) groups. Protecting groups that can form protected derivatives of the above substituents are known to those skilled in the art.

[0124] In one embodiment, a bifunctional linker having a functional group reactive with a group on a molecule (e.g., a targeting molecule) and another group reactive with another molecule (e.g., a fluorescent moiety or a drug) is used to form a desired conjugate. Alternatively, derivatization is performed to provide the functional group. Thus, for example, a method for producing a free sulfhydryl group on a peptide is also known (see U.S. Patent 4,659,839). The linker may include a heterobifunctional crosslinker comprising two or more different reactive groups that form a heterocyclic ring capable of interacting with the targeting molecule. For example, a heterobifunctional crosslinker such as cysteine ​​may include an amine-reactive group, and a thiol-reactive group that can interact with the aldehyde of the derivatized targeting molecule. Further combinations of reactive groups suitable for heterobifunctional crosslinkers include, for example, amine and sulfhydryl-reactive groups; carbonyl and sulfhydryl-reactive groups; amine and photoreactive groups; sulfhydryl and photoreactive groups; carbonyl and photoreactive groups; carboxylate and photoreactive groups; and arginine and photoreactive groups. Examples of heterobifunctional crosslinking agents include N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) and maleimidomethylcyclohexane-1-carboxylate (MCC).

[0125] In some embodiments, the linker is cleavable. In some embodiments, the linker is non-cleavable. The linker may include bonds that are chemically stable to the extracellular environment, for example, chemically stable in the bloodstream, and / or not. The linker may include bonds designed to be specifically or nonspecifically cleaved and / or immolate or otherwise destroyed within the cell. Cleavable linkers may be sensitive to enzymes at specific sites, such as extracellular proteases.

[0126] The cleavable linker may contain other peptides such as valine-citrulline peptide, valine-alanine peptide, phenylalanine-lysine, or peptides that form protease recognition and cleavage sites. Such peptide-containing linkers may contain a pentafluorophenyl group. Peptide-containing linkers may contain a succinimide or maleimide group. Peptide-containing linkers may contain a para-aminobenzoic acid (PABA) group. Peptide-containing linkers may contain an aminobenzyloxycarbonyl (PABC) group. Peptide-containing linkers may contain a PABA or PABC group and a pentafluorophenyl group. Peptide-containing linkers may contain a PABA or PABC group and a succinimide group. Peptide-containing linkers may contain a PABA or PABC group and a maleimide group.

[0127] Uncleavable linkers are generally protease-insensitive and insensitive to intracellular processes. Uncleavable linkers may contain a maleimide group. Uncleavable linkers may contain a succinimide group. Uncleavable linkers may be maleimide-alkyl-C(O)-linkers. Uncleavable linkers may be maleimide-caproyl linkers. Maleimide-caproyl linkers may be N-maleimidemethylcyclohexane-1-carboxylate. Maleimide-caproyl linkers may contain a succinimide group. Maleimide-caproyl linkers may contain a pentafluorophenyl group.

[0128] In one embodiment, a peptide linker consisting of one or more amino acids is used to conjugate a targeting molecule to a fluorescent moiety or drug. Generally, peptide linkers have no specific biological activity other than binding molecules or maintaining a certain minimum distance or other spatial relationship between them. However, the constituent amino acids of the linker may be selected to influence certain properties of the molecule, such as folding, net charge, or hydrophobicity. In one embodiment, the peptide linker is relatively short, typically less than about 10 amino acids, preferably less than about 8 amino acids, and more preferably less than 5 amino acids. Non-limiting descriptive examples include glycine and glycine-serine linkers that can be added to the C-terminus of a peptide. In one embodiment, the peptide linker includes cysteine ​​residues or non-natural amino acid residues for site-specific conjugation, e.g., selenocysteine ​​(Sec), p-acetophenylalanine (pAcF), p-azidomethyl-L-phenylalanine (pAMF), and azido-lysine (AzK). In one embodiment, the peptide linker is a glycine-glycine-glycine-cysteine ​​(GGGC) (SEQ ID NO: 15) linker, a glycine-glycine-cysteine ​​(GGC) linker, a glycine-glycine (GG) linker, or a cysteine ​​(C) linker. In one embodiment, a GGGC (SEQ ID NO: 15), GGC, GG, or C linker is added to the N-terminus or C-terminus of the peptide.

[0129] In one embodiment, the fluorescent conjugate of the present invention may be conjugated to a high molecular weight molecule that optionally increases the polyvalentity and avidity of the bond. In one embodiment, the high molecular weight molecule is a water-soluble polymer. Suitable water-soluble polymers include, but are not limited to, peptides, sugars, poly(vinyl), poly(ether), poly(amine), poly(carboxylic acid), etc. In one embodiment, the water-soluble polymer is dextran, polyethylene glycol (PEG), polyoxyalkylene, polysialic acid, starch, or hydroxyethyl starch. The peptide is conjugated to the water-soluble polymer using any suitable method (see Hermanson G., Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008).

[0130] In some embodiments, the fluorescent conjugate of the present invention may be modified to increase its solubility. Peptide modifications to increase solubility include the addition of hydrophilic amino acids, a PEG moiety, or both. In some embodiments, the PEG moiety is 8-amino-3,6-dioxaoctanoic acid (AEEA); 12-amino-4,7,10-trioxadodecanoic acid; or 15-amino-4,7,10,13-tetraoxapentadecanoic acid. In some embodiments, about 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) hydrophilic amino acids may be added to the N-terminus, C-terminus, internal position, or any combination thereof of the peptide to increase solubility. Hydrophilic amino acids include D, E, H, K, N, Q, R, S, T, and G. In some embodiments, the peptide contains K, KK, G, or GG at the N-terminus or C-terminus.

[0131] In one embodiment, the fluorescent conjugate comprises 5-FAM-QVPWEEPYYVVKKSSGG-NH2 (HNP401-N-2 with GG linker; SEQ ID NO: 747) and may be used in any of the methods described herein.

[0132] In one embodiment, the fluorescent conjugate includes FAM (Ac-SGQVPWEEPYYVVKKSSGGC-CONH2; SEQ ID NO: 748) bound to the C-terminal cysteine ​​of HNP401 having a GCC linker.

[0133] IV. Pharmaceutical Compositions In one embodiment, disclosed herein is a pharmaceutical composition comprising the fluorescent conjugate disclosed herein. The pharmaceutical composition of the present invention is formulated using one or more physiologically acceptable carriers comprising additives and adjuvants that facilitate the processing of the active agent into a pharmaceutically useful formulation. The appropriate formulation depends on the chosen route of administration. Such compositions are, for example, described in Gennaro: Remington: The Science and Practice of Pharmacy, 21 st It can be prepared using methods and additives known in the art, as found in Ed. (Lippincott Williams & Wilkins, 2005); Allen: Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 11th Ed. (Wolters Kluwer, 2018); Brunton et al.: Goodman & Gilman's The Pharmacological Basis of Therapeutics (McGraw-Hill Professional, 2005); and Rowe: Handbook of Pharmaceutical Excipients (Pharmaceutical Press, 2005).

[0134] In some embodiments of the pharmaceutical composition, the peptide in the fluorescent conjugate consists of or may include any amino acid sequence, including all variants and combinations described herein. In some embodiments, the peptide consists of or includes any amino acid sequence or any variant thereof listed in Table 1 described herein. In some embodiments, the peptide includes or consists of any of SEQ ID NOs: 1-14, 16, 20-28 and 96-102. In some embodiments, the peptide includes or consists of any amino acid sequence or any variant thereof listed in Tables 2-4. In some embodiments, the peptide includes an amino acid sequence selected from SEQ ID NOs: 75-82, 84-95 and 103-187. In some embodiments, the peptide includes an amino acid sequence selected from SEQ ID NOs: 55-74 and 188-362. In some embodiments, the peptide includes an amino acid sequence selected from SEQ ID NOs: 33-54 and 363-446. In some embodiments, the peptide includes an amino acid sequence selected from SEQ ID NOs: 18, 19 and 29-32 and 447-471. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NOs: 472-678. In another embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NOs: 679-746. In yet another embodiment, the peptide consists of or comprises the amino acid sequence of SEQ ID NO: 21.

[0135] In one embodiment, the fluorescent conjugate in the pharmaceutical composition includes SEQ ID NO: 747.

[0136] In some embodiments, the pharmaceutical compositions disclosed herein further comprise pharmaceutically acceptable diluents, additives, carriers, or solvents. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or media include water, ethanol, polyols (such as propylene glycol, polyethylene glycol, glycerol, and cremophor), suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Other commonly used surfactants such as tweene, spun, and other emulsifiers, or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes. Bioavailability enhancers may include penetration or permeability enhancers. See, for example, Muheem et al., 2016, Saudi Pharm. J. 24, 413-428; Brayden et al. 2020, Adv. Drug Deliv. Rev. 2020 May 29:S0169-409X(20)30040-5; Ibrahim et al. 2020, J. Pham. Sci. 28, 403-416. In some formulations, adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0137] In one embodiment, the pharmaceutical composition comprises other agents or pharmaceuticals, carriers, adjuvants, such as preservatives, stabilizers, wetting or emulsifiers, dissolution accelerators, osmotic salts, and / or buffers. Furthermore, the pharmaceutical composition also comprises other therapeutically valuable substances.

[0138] In one embodiment, the peptide conjugate disclosed herein is delivered to a target by a drug delivery medium or carrier. In one embodiment, the delivery medium is manufactured from natural or synthetic materials or both. In one embodiment, the delivery medium is nanoparticles, microparticles, polymer micelles, nanocapsules, dendrimers, large PEGs, nanogels, liposomes, fullerenes, nanostructured lipid carriers, nanoshells, quantum dots, protein-based nanocarriers (e.g., albumin, elastin, gliadin, legumin, zein, soy protein, milk protein, whey-based nanocarriers), organic nanocarriers (e.g., gelatin, dextran, guar gum, chitosan, collagen), polysaccharide-based carriers (e.g., dextran, chitosan, pectin), lipid emulsions, or combinations thereof.

[0139] In one embodiment, the pharmaceutical composition disclosed herein is administered to a subject by any suitable route of administration, including but not limited to topical, oral, rectal, vaginal, nasal, inhalation, and non-enteral (intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, and intravitreous injection) administration. In one embodiment, the pharmaceutical composition disclosed herein is administered to a subject topically or systemically. In one embodiment, the pharmaceutical composition disclosed herein is administered intravenously.

[0140] Formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Formulations suitable for subcutaneous injection also include optional additives such as preservatives, wetting agents, emulsifiers, and dispersants, as known in the art. In some embodiments, formulations suitable for intravenous injection may be prepared in aqueous solutions such as saline buffer and other physiologically compatible buffers known in the art. For intravenous injection, the activator is optionally formulated in aqueous solution, preferably a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline buffer.

[0141] Non-enteral injections may include bolus injections or continuous infusions as desired. Injectable formulations may be provided in unit dosage forms, e.g., ampoules, or in multi-dose containers with the addition of preservatives, as desired. In some embodiments, the pharmaceutical compositions described herein are in a form suitable for non-enteral injection as sterile suspensions, solutions, or emulsions in oily or aqueous media, and include formulation agents such as suspensions, stabilizers, and / or dispersants. Non-enteral pharmaceutical formulations include aqueous solutions of the activator in a water-soluble form. Furthermore, the suspension may be prepared as a suitable oily injection suspension as desired.

[0142] In one embodiment, the pharmaceutical composition of the present invention is administered orally. Suitable dosage forms for oral administration may be solid or liquid, and may include, for example, pills, capsules, lozenges, tablets, caplets, gel caplets (gel caps), syrups, aqueous suspensions or solutions, chewable forms, swallowable forms, soluble forms, effervescent forms, granular forms, and oral liquid solutions. In a specific embodiment, the dosage form is a solid dosage form, and more specifically, it includes tablets or capsules or is administered orally by a medium or carrier disclosed herein.

[0143] In one embodiment, the pharmaceutical composition described herein is a unit dosage form suitable for a single dose of a precise dosage. In the unit dosage form, the formulation is divided into unit doses containing an appropriate amount of the activator disclosed herein. In one embodiment, the unit dose is in the form of a package containing separate amounts of the formulation. Non-limiting examples include packaged tablets or capsules and powders in vials or ampoules. In one embodiment, the aqueous suspension composition is packaged in a single-dose, non-resealable container. Alternatively, a multi-dose, resealable container is used, in which case the composition typically contains a preservative. As just one example, formulations for non-enteral injection are provided in unit dosage forms, including but not limited to ampoules, or in multi-dose containers with the addition of a preservative.

[0144] In one embodiment, the pharmaceutical composition is administered intravenously systemically to a subject, particularly a human patient, more specifically, a human patient undergoing surgery.

[0145] In one embodiment, the pharmaceutical composition is administered orally to a subject, in particular a human patient, more specifically a human patient undergoing surgery. [Examples]

[0146] Example 1 Use of neural targeting fluorescence conjugates in ureteral visualization Introduction Phage display screening has been previously used to identify peptides that bind to human neuronal homogenates or isolated human neuroproteins. (See applications '054 and '821'.) Such peptides can be conjugated or tagged to cargo molecules for various applications. When tagged with a fluorescent moiety, for example, such conjugates may be useful for in vivo targeting of neurons during fluorescence-assisted surgery, and when conjugated to an activator, for example, such conjugates may be useful for targeted therapy.

[0147] Examples of peptides were conjugated with fluorescent dyes to produce fluorescent conjugates (also referred to here as conjugates), evaluated in rodent models, and showed detection of sensitive nerves after intravenous administration. These conjugates included FAM-NP41, which contained the fluorescent dye carboxyfluorescein (FAM) conjugated to the C-terminal lysine of acetylated NP41 (Ac-SHSNTQTLAKAPEHTGK; SEQ ID NO: 749) via a lysine amide bond, and FAM-HNP401, which contained FAM conjugated to the C-terminal cysteine ​​of acetylated HNP401 (Ac-SGQVPWEEPYYVVKKSSGGC-CONH2; SEQ ID NO: 748) via a GCC linker. See '054 application; Hingorani et al. 2018, Theranostics 8, 4226-4237; Whitney et al. 2011, Nat. Biotechnol. 29, 352-356.

[0148] Further studies demonstrated fluorescence targeting in resected human (and rodent) nerve tissue after topical administration of FAM-NP41, FAM-HNP401, and other conjugates including N-terminal and C-terminal deletion variants of HNP401 corresponding to SEQ ID NOs. 20, 22-24, and 25-28. Whitney et al. 2011, Nat. Biotechnol. 29, 352-356; Wu et al. 2011, Laryngoscope 121, 805-810; Hussain et al. 2015, PloS One 10, e0119600; Hingorani et al. 2018, Theranostics 8, 4226-4237.

[0149] However, these studies have not clarified the properties of the conjugates, such as their activity, metabolism, and elimination—particularly in the later post-administration period. In rodent models, for example, the serum half-life of NP41 (measured by serum fluorescence) is approximately 10 minutes, indicating rapid elimination of the conjugate from the blood. A similar short half-life (15 minutes) in the blood was observed for FAM-HNP401. In contrast, the neurofluorescence of NP41 has a half-life of approximately 50 minutes and can persist at low levels in tissues for several hours. Similarly, fluorescence of FAM-HNP401 was observed in rat ischial and prostatic nerves several hours after intravenous administration. See, for example, Whitney et al. 2011, Nat. Biotechnol. 29, 352-356; Hingorani et al. 2018, Theranostics 8, 4226-4237.

[0150] The elimination of conjugates bound to nerves (or other tissues) and their subsequent activity are unknown, and it is unknown whether such properties support other applications for conjugates in fluorescence-guided surgery. The studies described here demonstrate that nerve-targeting conjugates, in conjunction with renal excretion, can provide ureteral sensitivity and long-term fluorescence visualization (illumination). The results support the expansion of the repertoire of such conjugates as surgical adjuvants. Nerve highlighting can help prevent nerve injury, making them useful as inductive agents in surgeries such as head and neck surgery. And, as described here, ureteral highlighting makes them useful in other surgeries such as many abdominal and pelvic surgeries where there is a risk of ureteral injury.

[0151] result The dynamics of fluorescence visualization of 5-FAM-QVPWEEPYYVVKKSSGG-NH2 (HNP401-N-2 with GG linker; SEQ ID NO: 747) after intravenous administration were evaluated using a rodent model. The conjugate was administered in a wide range of amounts, and fluorescence was detected using a customized microscope. For example, as shown in Figure 1, the ureter was readily irradiated 2 hours after intravenous administration of approximately 2000 nanomoles (approximately 200 mg / kg) in a mouse model. Further studies exploring amounts in the range of 100 to 2000 nanomoles corresponding to doses of approximately 10 mg / kg to 200 mg / kg demonstrated ureteral illumination after intravenous administration of the fluorescent conjugate, SEQ ID NO: 747, in a mouse model. Similar findings were observed in a rat model. However, such fluorescence visualization did not reflect specific or direct targeting of the ureter. Rather, it reflected the fluorescence of urine flowing from the kidney to the bladder. Furthermore, real-time intraoperative contrast imaging revealed ureteral pulsation, which concentrated the urine and enhanced the fluorescence visualization of the ureters. Further studies demonstrated long-term ureteral illumination after administration of this conjugate. For example, fluorescence visualization of the ureters was observed less than one hour after conjugate administration and similarly up to at least six hours.

[0152] Other studies in rodent models have shown long-term ureteral illumination after administration of other conjugates containing additional peptides and fluorescent moieties. These include molecules consisting of the relevant HNP401 peptide conjugated to FAM and molecules consisting of the relevant HNP401 peptide conjugated to other fluorescent moieties. Furthermore, the studies presented here allow for simultaneous visualization of nerves in conjunction with the ureter.

[0153] Consideration: These observations support the general applicability of the neurotargeting fluorescence conjugate described herein in ureteral illumination. Therefore, the conjugate disclosed herein can be used to help surgeons visualize the ureters during surgical procedures before they are physically encountered and thus potentially damaged. Furthermore, in cases of accidental injury during surgical procedures, i.e., iatrogenic injury, such visualization allows for more rapid recognition of the injury and earlier treatment and intervention.

[0154] Furthermore, administration of the fluorescent conjugates described herein has been observed to provide long-lasting ureteral visualization after administration. While not limited to specific mechanisms, this prolonged visualization is thought to reflect the gradual accumulation, release, metabolism, and excretion of the fluorescent conjugate in neurons or nerves or other tissues, such as the peripheral nervous system, along with subsequent excretion by the urinary system. Therefore, in contrast to systemic administration of fluorescent dyes alone or direct injection of fluorescent dyes, the administration of peptide-fluorescent conjugates can advantageously extend the duration of ureteral illumination with a simple administration method that does not interfere with surgery, providing both early and long-term illumination. Moreover, the administration of such conjugates can provide simultaneous or transient separate detection of nerves or ureters.

[0155] Such fluorescence-assisted imaging is particularly important in surgical procedures with a significant risk of ureteral injury, especially iatrogenic ureteral injury (IUI). Because the ureters extend from the abdomen to the pelvis, this risk can apply to a wide range of surgical procedures. Applicable procedures that may benefit from ureteral illumination may include complex abdominal and pelvic surgeries, such as gynecological, urological, and colorectal surgery, as well as cardiovascular surgery. This also includes surgical procedures performed on organs and systems near the ureters, such as the kidneys, bladder, prostate, uterus, male or female reproductive system, rectum, colon, small or large intestine. Cancer surgery, such as prostate cancer surgery or colorectal cancer surgery, or any procedure where enhanced visualization of the urinary and nerve systems is desired, is also included.

[0156] Example 2 Bioanalysis of fluorescent conjugates in the urine of surgical patients Urine samples were collected 30 minutes before intravenous administration of 500 mg of the parent test drug (fluorescent-conjugated peptide, corresponding to SEQ ID NO: 747), 30 minutes after pre-head and neck surgery infusion, and 22 hours after infusion. The concentration of the intact parent test drug was determined by liquid chromatography with tandem mass spectrometry (LC-MS / MS) using standard biological analytical methods. The presence of metabolites M3-M8 (first identified from in vitro studies) corresponding to shortened versions of the parent peptide conjugated to the fluorescent portion was also evaluated. The results are shown in Table 5. [Table 5] * BQL: Below the limit of quantification (10 ng / ml)

[0157] At 30 minutes, quantifiable levels of the test drug (or fluorescent metabolites M4, M6, and M7) were detected in 4 out of 5 subjects evaluated. At 22 hours, fluorescent metabolites M4 and M6 were detected in both subjects evaluated. As expected, neither the test drug nor the fluorescent metabolites were detected in any of the pre-administration samples.

[0158] These data are consistent with drug administration and ureteral imaging experiments in rodent models, demonstrating that a single dose of the test drug can result in immediate (e.g., 30 minutes) and long-term (e.g., 22 hours) accumulation of the fluorescent molecule in the urine. This, in turn, provides extended ureteral visualization in the early and late stages of surgical procedures and supports simultaneous or separate visualization of nerves and ureters in surgical procedures or other applications.

[0159] Example 3 Illuminating ureters and nerves during surgery The ability of the fluorescent conjugate of the present invention to visualize the ureters (and nerves) can be evaluated using appropriately designed clinical trials in patients undergoing gastrointestinal / genitourinary (GI / GU) surgery, as described below.

[0160] In this hypothetical example, the clinical trial is an open-label, multicenter study evaluating the administration of the investigational drug (fluorescent-conjugate peptide, corresponding to SEQ ID NO: 747) to 80 subjects undergoing GI / GU surgery. Inclusion criteria for this trial include the requirement that subjects undergo a GI / GU surgical procedure that carries a potential risk of nerve (and ureter) damage. For example, such procedures may include colorectal or prostate surgery.

[0161] Participants will receive a single intravenous dose of the test drug (500 mg) 2 to 5 hours prior to surgery. The fluorescence of the test drug will be evaluated by real-time white light reflectance (WLR) and fluorescence (FL) imaging via laparoscopy.

[0162] Multiple endpoints are used to evaluate primary and secondary efficacy objectives.

[0163] Regarding nerve detection, the endpoints include a visualization scoring system to measure the effectiveness of the test drug in contrast enhancement, branching visualization, and improvement of nerve length, using WLR alone or paired (WLR and FL overlay). It also includes the calculation of signal-to-background ratios across multiple regions of nerve (and adjacent non-nerve) tissue to measure the effectiveness of the test drug in improving nerve visualization using WLR versus FL.

[0164] Regarding ureteral detection, the endpoints include a visualization scoring system to measure the effectiveness of the test drug in improving ureteral contrast enhancement using WLR alone versus WLR and FL overlay. It also includes the calculation of signal-to-background ratios across multiple regions of ureteral (and adjacent non-ureteral) tissue to measure the effectiveness of the test drug in improving ureteral visualization using WLR versus FL.

[0165] Significant improvements in the effectiveness of the test drug for nerve and ureter detection are demonstrated across multiple endpoints. Improvements are observed at both early and late stages of the surgical procedure. Therefore, the fluorescence conjugate of the present invention may be useful in fluorescence-guided GI / GU surgical procedures, including enhanced visualization of nerves, ureters, or both. The benefits of such enhanced visualization include a significant improvement in the reliable identification of ureters and nerves in surgical procedures and a significant reduction in the time required for such procedures.

[0166] The above embodiments provide to those skilled in the art a complete disclosure and description of methods for producing and using embodiments of the compositions and methods of the present invention, and are not intended to limit the scope of what the inventors consider to be a disclosure. Modifications of the above embodiments of the invention that would be obvious to those skilled in the art are intended to fall within the scope of the following claims. All patents and publications described herein are indicators of the level of skill of those skilled in the art to which the invention pertains. All references herein are incorporated herein by reference to the same extent as each reference is incorporated by reference in its entirety.

[0167] As will be apparent to those skilled in the art, many modifications and alterations of this application can be carried out without departing from its spirit and scope. The specific embodiments and examples described herein are provided merely as examples, and this application should be limited only by the appended claims, along with the full scope of the equivalents granted by the claims.

[0168] Furthermore, while certain details of the present invention are provided to give a complete understanding of the invention as defined by the appended claims, it will be understood by those skilled in the art that certain embodiments can be carried out without such detail. Moreover, in some cases, well-known methods, techniques or other specific details are omitted to avoid unnecessary ambiguity of the aspects of the invention as defined by the appended claims.

[0169] This application claims priority under U.S. Provisional Application 63 / 137,621, filed January 14, 2021, which is incorporated herein by reference in its entirety. Furthermore, the present invention encompasses the following aspects. 1. A method for visualizing the ureter in a subject, (a) Administer an effective amount of a fluorescent conjugate containing the peptide and the fluorescent portion to the subject; and (b) Detect fluorescence in the fluorescent portion of the ureter after administration. Including; Herein, the fluorescently conjugated peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 16, 18-82, and 84-748. 2. The method described in item 1, in which the subject undergoes a surgical procedure. 3. The method described in item 2, in which the fluorescent conjugate is administered first before the surgical procedure. 4. The method of item 3, wherein the fluorescent conjugate is not administered to the subject any further after the initial dose. 5. Any method described in items 2-4, wherein the surgical procedure includes gynecological, urological, colorectal, or cardiovascular surgical procedures. 6. Any method described in items 2-4, wherein the surgical procedure includes an abdominal or pelvic procedure. 7. Any method described in items 2-4, in which the surgical procedure is performed on the kidney, bladder, prostate, uterus, male or female reproductive system, rectum, colon, small intestine, or large intestine. 8. Any method described in items 2-4, wherein the surgical procedure is an open surgery, laparoscopic surgery, microscopic procedure, or endoscopic procedure. 9. Any method described in items 2-4, wherein the surgical procedure is a cancer surgical procedure, more specifically, a prostate cancer surgical procedure or a colorectal cancer surgical procedure. 10. (a) administer an effective dose of a fluorescent conjugate containing a peptide and a fluorescent moiety to the subject undergoing the surgical procedure; and (b) Visualize the ureter in the subject by detecting fluorescence in the fluorescent portion of the ureter; Herein, the method wherein the fluorescently conjugated peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 16, 18-82, and 84-748. 11. The method of item 10, wherein the fluorescent conjugate is administered first before the surgical procedure. 12. The method of item 11, wherein the fluorescent conjugate is not administered to the subject any further after the initial dose. 13. Any method described in items 10-12, wherein the surgical procedure includes gynecological, urological, colorectal, or cardiovascular surgical procedures. 14. Any method described in paragraphs 10-12, wherein the surgical procedure includes an abdominal or pelvic procedure. 15. Any method described in paragraphs 10-12, in which a surgical procedure is performed on the kidney, bladder, prostate, uterus, male or female reproductive system, rectum, colon, small intestine, or large intestine. 16. Any method described in items 10-12, wherein the surgical procedure is an open surgery, laparoscopic surgery, microscopic procedure, or endoscopic procedure. 17. The surgical procedure is a cancer surgical procedure, more specifically, a prostate cancer surgical procedure or a colorectal cancer surgical procedure, in any of the manner described in items 10-12. 18. A method for reducing injury to the ureter, (a) Identify subjects at risk of ureteral injury before surgical procedures. (b) administer an effective dose of a fluorescent conjugate containing a peptide and a fluorescent moiety to the subject undergoing the surgical procedure; and (c) Visualize the ureter by detecting fluorescence in the fluorescent portion of the ureter. A method comprising, wherein the fluorescently conjugated peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 16, 18-82 and 84-748. 19. The method of item 18, wherein the fluorescent conjugate is administered first before the surgical procedure. 20. The method of paragraph 19, wherein the fluorescent conjugate is not administered to the subject any further after the initial dose. 21. Ureteral injury resulting from ligation, angulation, amputation, laceration, crush, ischemia, or resection, by any of the methods specified in paragraphs 18-20. 22. Any method of paragraphs 18-21, wherein the surgical procedure includes gynecological, urological, colorectal, or cardiovascular surgical procedures. 23. Any method described in paragraphs 18-21, wherein the surgical procedure includes an abdominal or pelvic procedure. 24. Any method described in paragraphs 18-21, in which a surgical procedure is performed on the kidney, bladder, prostate, uterus, male or female reproductive system, rectum, colon, small intestine, or large intestine. 25. Any method described in items 18-21, wherein the surgical procedure is an open surgery, laparoscopic surgery, microscopic procedure, or endoscopic procedure. 26. Any method described in paragraphs 18-21, wherein the surgical procedure is a cancer surgical procedure, more specifically, a prostate cancer surgical procedure or a colorectal cancer surgical procedure. 27. The peptide is a neuronal targeting peptide, using any of the methods described in items 1 to 26. 28. Any method according to items 1 to 27, wherein the fluorescent portion is conjugated to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the peptide. 29. Any method according to items 1 to 28, further comprising detecting fluorescence of the fluorescent portion within a time period of at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours after administration. 30. Any method of items 1 to 29, further comprising detecting fluorescence of the post-administration fluorescent portion more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours after administration; or more than 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours after administration. 31. Any method of items 1 to 30, wherein the detection of fluorescence in a fluorescent portion in the ureter includes the detection of urine flow in the ureter, more specifically, the detection of fluorescence in the peristaltic flow of urine in the ureter. 32. Any method according to items 1 to 31, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. 33. Any method according to items 1 to 31, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 16, 20-28 and 96-102. 34. Any method according to items 1 to 31, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7-14, 20-28 and 96-102. 35. Any method according to items 1 to 31, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 29-82, 84-95 and 103-746. 36. Any method according to items 1 to 31, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 75-82, 84-95, and 103-187. 37. Any method according to items 1 to 31, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 55 to 74 and 188 to 362. 38. Any method according to items 1 to 31, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 33 to 54 and 363 to 446. 39. Any method according to items 1 to 31, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 29-32 and 447-471. 40. Any method according to items 1 to 31, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 472 to 678. 41. Any method according to items 1 to 31, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 679 to 746. 42. Any method according to items 1 to 31, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 25, and SEQ ID NO: 99. 43. The method of item 42, wherein the peptide contains the amino acid sequence of SEQ ID NO: 21. 44. The method of item 42, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof in which 2 to 8 amino acids are deleted from the N-terminus or C-terminus. 45. The method of item 44, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. 46. ​​The method of item 45, wherein the amino acid sequence further comprises a GC, GG, or GGC amino acid sequence at the C-terminus. 47. The method of item 46, wherein the amino acid sequence is selected from the group consisting of SEQ ID NOs: 4, 7, 8, 9, 10, 11, 12, 13, 14, 21, 102, 101, 100, 99, 98, 97, and 96. 48. Any method according to items 1 to 47, wherein the fluorescent portion is selected from the group consisting of fluorescent proteins, fluorescent peptides, fluorescent dyes, and combinations thereof. 49. Any method according to items 1 to 48, wherein the fluorescent portion comprises xanthene; biman; coumarin; aromatic amine; benzofuran; fluorescent cyanine; carbazole; dicyanomethylenepyran; polymethine; oxabenzantran; pyrylium; carbostyryl; perylene; acridone; quinacridone; rubrene; anthracene; coronene; phenanthrencene; pyrene; butadiene; stilbene; porphyrin; phthalocyanine; lanthanide metal chelate complex; rare earth metal chelate complex; or derivatives thereof. 50. Any method according to items 1 to 48, wherein the fluorescent portion comprises carboxyfluorescein, 5-carboxyfluorescein, 6-carboxyfluorescein, 5(6)-carboxyfluorescein, fluorescein isothiocyanate, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate, 5,6-dicarboxyfluorescein, 5-sulfofluorescein, 6-sulfofluorescein, sulfonefluorescein, succinylfluorescein, 5-carboxySNARF-1, 6-carboxySNARF-1, carboxyfluorescein sulfonate, carboxyfluorescein zwitterion, carboxyfluorescein quaternary ammonium, carboxyfluorescein phosphonate, carboxyfluorescein GABA, carboxyfluorescein-cys-Cy5, or fluorescein glutathione. 51. Any method according to items 1 to 48, wherein the fluorescent portion contains carboxyfluorescein (FAM). 52. Any method according to items 1 to 48, wherein the fluorescent portion comprises 5-carboxyrhodamine 110, 6-carboxyrhodamine 110, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxyrhodol derivatives, tetramethyl and tetraethylrhodamine, diphenyldimethyl and diphenyldiethylrhodamine, dinaphthylrhodamine, rhodamine 101 sulfonyl chloride or other rhodamine dyes. 53. Any method according to items 1 to 48, wherein the fluorescent portion comprises Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, or indocyanine green. 54. Any method according to items 1 to 48, wherein the fluorescent portion comprises IR800CW, cyan fluorescent protein (CFP), EGFP, 5-FAM, 6-FAM, FAM, fluorescein, IAEDANS, EDANS, BODIPY FL, TRITC, Cy5, Cy3, YFP, LC Red 640, Alexa Fluor 546, tetramethylrhodamine, dabucil, QSY7, QSY9, QSY21, or BBQ-650 dye. 55. Any method according to items 1 to 31, wherein the fluorescent conjugate contains sequence number 747. 56. Any method according to items 1 to 31, wherein the peptide comprises SEQ ID NO: 20 and the fluorescent portion comprises FAM. 57. Any method according to items 1 to 31, wherein the peptide comprises SEQ ID NO: 21 and the fluorescent portion comprises FAM. 58. The fluorescent conjugate is administered intravenously by any of the methods described in items 1 to 57. 59. The fluorescent conjugate is administered orally by any of the methods described in items 1 to 57. 60. Any method according to items 1 to 59, further comprising the administration of a fluorescent conjugate in a pharmaceutical composition containing a pharmaceutically acceptable additive. 61. Any method from items 1 to 60, further comprising visualization of nerves in an object by fluorescence detection of a fluorescent conjugate. 62. The method of item 61, wherein the visualization of nerves and ureters is detected simultaneously in the subject during surgery. 63. The method of paragraph 61, wherein the visualization of nerves and ureters is detected at different time points in the subject during surgery.

Claims

1. A pharmaceutical composition comprising a fluorescein conjugate peptide for visualizing the ureter in a subject, The fluorescein conjugate peptide contains a peptide having the amino acid sequence of SEQ ID NO: 21 (QVPWEEPYYVVKKSSGG) conjugated to the fluorescein portion at the N-terminus. A pharmaceutical composition in which visualization is achieved by detecting the fluorescence of fluorescein in the peristaltic urinary flow in the ureter after administration of the pharmaceutical composition at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or more than 8 hours after administration of the pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the subject undergoes a surgical procedure.

3. The pharmaceutical composition according to claim 1, for the purpose of reducing injury to the ureter, wherein the subject has been identified as being at risk of ureteral injury prior to a surgical procedure.

4. A pharmaceutical composition according to any one of claims 1 to 3, which is administered first before a surgical procedure.

5. The pharmaceutical composition according to claim 4, wherein no further administration is performed on the subject after the initial administration.

6. Surgical techniques (i) including gynecological, urological, colorectal, or cardiovascular surgical procedures; (ii) including abdominal or pelvic procedures; (iii) Performed in the kidneys, bladder, prostate, uterus, male or female reproductive system, rectum, colon, small or large intestine; (iv) Open surgical procedures, laparoscopic surgical procedures, microscopic procedures or endoscopic procedures; (v) a cancer surgery procedure; and / or (vi) A surgical procedure for prostate cancer or colorectal cancer, A pharmaceutical composition according to any one of claims 2 to 5.

7. A pharmaceutical composition according to any one of claims 3 to 6, wherein the ureteral injury results from ligation, angulation, amputation, laceration, crush, ischemia, or excision.

8. A pharmaceutical composition according to any one of claims 1 to 7, wherein the fluorescence of the fluorescein portion is detected (i) 30 minutes after administration, (ii) 30 minutes and within 1 hour, (iii) within 1 hour, (iv) within 2 hours, (v) within 3 hours, (vi) within 4 hours, (vii) within 5 hours, (viii) within 6 hours, (ix) within 7 hours, or (x) within 8 hours.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the fluorescence of the fluorescein portion is detected at a time of more than 1 hour, more than 2 hours, more than 3 hours, more than 4 hours, more than 5 hours, more than 6 hours, more than 7 hours, or more than 8 hours after administration of the pharmaceutical composition.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the fluorescein portion comprises carboxyfluorescein, 5-carboxyfluorescein, 6-carboxyfluorescein, 5(6)-carboxyfluorescein, fluorescein isothiocyanate, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate, 5,6-dicarboxyfluorescein, 5-sulfofluorescein, 6-sulfofluorescein, sulfonefluorescein, succinylfluorescein, 5-carboxy-SNARF-1, 6-carboxy-SNARF-1, carboxyfluorescein sulfonate, carboxyfluorescein zwitterion, carboxyfluorescein quaternary ammonium, carboxyfluorescein phosphonate, carboxyfluorescein GABA, carboxyfluorescein-cys-Cy5, or fluorescein glutathione.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the fluorescein portion comprises carboxyfluorescein (FAM).

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the fluorescein portion comprises 5-carboxyfluorescein.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the fluorescein conjugate peptide comprises SEQ ID NO: 747 (5-FAM-QVPWEEPYYVVKKSSGG-NH2).

14. The pharmaceutical composition according to any one of claims 1 to 21, wherein the fluorescein conjugate peptide comprises SEQ ID NO: 747 (5-FAM-QVPWEEPYYVVKKSSGG-NH2).

15. A pharmaceutical composition according to any one of claims 1 to 14, which is administered intravenously.

16. A pharmaceutical composition according to any one of claims 1 to 14, which is administered orally.

17. A pharmaceutical composition according to any one of claims 1 to 16, comprising a pharmaceutically acceptable additive.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the nerves in the target are also visualized by fluorescence detection of fluorescein conjugate peptide.

19. The pharmaceutical composition according to claim 18, wherein the visualization of nerves and ureters is simultaneously detected in the subject during surgery.

20. The pharmaceutical composition according to claim 18, wherein the visualization of nerves and ureters is detected at different time points in the subject during surgery.

21. A pharmaceutical composition comprising a fluorescein conjugate peptide for visualizing the ureter in a subject, wherein the fluorescein conjugate peptide comprises a peptide having the amino acid sequence SEQ ID NO: 747 (5-FAM-QVPWEEPYYVVKKSSGG-NH2) conjugated at the N-terminus to a fluorescein portion; and visualization is achieved by detecting the fluorescence of the fluorescein portion in the peristaltic urinary flow of the ureter 30 minutes, 30 minutes and within 1 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or more than 8 hours after administration of the pharmaceutical composition.

22. The pharmaceutical composition according to claim 21, wherein the subject undergoes a surgical procedure.

23. The pharmaceutical composition according to claim 21, for the purpose of reducing injury to the ureter, and for subjects identified as being at risk of ureteral injury prior to a surgical procedure.

24. A pharmaceutical composition according to any one of claims 1 to 23, wherein the subject has iatrogenic ureteral injury.

25. The pharmaceutical composition according to claim 24, wherein the ureteral injury results from ligation, angulation, amputation, laceration, crush, ischemia, or excision.

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