Methods and compositions for visualizing the ureter in surgical procedures - Patents.com

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 sequence listing The sequence listing associated with this application is provided in text form in lieu of a written document 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 142KB, was created on January 13, 2022, and was provided electronically via EFS-Web.

[0002] Field The present invention relates generally to intraoperative imaging methods for surgical procedures, and more specifically to fluorescent visualization of the ureter and nerves. [Background technology]

[0003] background The ureters contain a pair of multi-layered muscular tubes - each about 10 to 12 inches long in an average adult - that transport urine from the kidneys to the bladder. Transport utilizes a peristaltic motion caused by pacemaker-initiated contractions of the muscular layers of the ureter wall. The rhythmic contraction and relaxation of the wall allows urine to flow pulsatilely from the ureter to the bladder.

[0004] As it extends from the abdomen to the pelvis, the ureter is proximal to many structures in the abdominopelvic cavity, such as the gonadal and uterine vessels, the iliac arteries, the inferior mesenteric and sigmoid vessels, as well as the cervix, colon, and rectum. Furthermore, the ureter is a thin-walled tube that is often in a collapsed state and difficult to distinguish from the surrounding tissues. Given its location and physical context, the ureter is at considerable risk of injury in many surgical procedures, including gynecological, urological, colorectal, and cardiovascular. See, e.g., 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. Indeed, ureteral injuries often go unnoticed during surgery, which can lead to delayed diagnosis and treatment that can lead to long-term complications, such as renal failure in some cases. 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 injuries have a significant impact on patient morbidity and associated health care costs. In the United States, approximately 600,000 hysterectomies and 300,000 colon surgeries are performed annually, with ureteral injury rates as high as 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 surgical procedures, the estimated incidence of iatrogenic ureteral injury (IUI) ranges from 0.5% to 1% in cancer surgery, 0.3% to 2.5% in laparoscopic gynecologic surgery, and up to 10% in gynecologic oncology 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 hospital stay of 4 days and costs of over $30,000 per injury.

[0006] Several approaches have been explored to provide a reduction in the risk of ureteral injury during surgery. These include urological surgical procedures involving the insertion of stents or illuminating catheters into the ureter, which may disrupt the workflow of surgery 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 present additional risks and complications, 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. Summary of the Invention [Problem to be solved by the invention]

[0007] There remains a need in the art to identify compositions that have desirable properties, including visualization of the ureter during surgical procedures. The present invention meets these and other needs by providing peptide compositions, particularly fluorescent peptide conjugates, that allow for sensitive and long-term ureter visualization after administration, and that can further support neurovisualization, including simultaneous neuro and ureter visualization. [Means for solving the problem]

[0008] overview Disclosed herein are methods for visualizing the ureter, including methods for fluorescent 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 to the subject an effective amount of a fluorescent conjugate comprising a peptide and a fluorescent moiety, 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 other aspects, the invention relates to methods comprising: (a) administering to a subject undergoing a surgical procedure an effective amount of a fluorescent conjugate comprising a peptide and a fluorescent moiety; and (b) visualizing a ureter in the subject by detecting fluorescence of the fluorescent moiety in the ureter.

[0011] In another aspect, the invention relates to a method of reducing injury to a ureter, the method comprising: (a) identifying a subject at risk of ureteral injury prior to a surgical procedure; (b) administering to the subject undergoing the surgical procedure an effective amount of a fluorescent conjugate comprising a peptide and a fluorescent moiety; and (c) visualizing the ureter by detecting fluorescence of the fluorescent moiety in the ureter. In one embodiment, the ureteral injury results from ligation, angulation, cutting, laceration, crushing, hemorrhage, or resection.

[0012] In some embodiments of the methods, the peptide can be a neurotargeting peptide. In any of the methods, the peptide can consist of or include any of the amino acid sequences disclosed herein, including those set forth in International Patent Application PCT / US2018 / 045054 and International Patent Application PCT / US21 / 61821. In some embodiments, the peptide of the fluorescent conjugate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, 16, 18-82, and 84-748.

[0013] In certain embodiments of any of the methods, the fluorescent moiety can 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 certain embodiments of any of the methods, the fluorescent moiety is selected from the group consisting of a fluorescent protein, a fluorescent peptide, a fluorescent dye, or a combination thereof disclosed herein.

[0014] In certain embodiments, any of the methods may further comprise detecting the fluorescence of the fluorescent moiety 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. In certain embodiments, the methods further comprise detecting the fluorescence of the fluorescent moiety for 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 some embodiments, the fluorescent conjugate in any of the methods is not further administered to the subject after the first administration. In any of the methods, the detection of the fluorescence of the fluorescent moiety in the ureter may include detection of urine flow in the ureter, more specifically detection of urine peristaltic flow 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 certain embodiments, the surgical procedure in any of the methods may include a gynecological, urological, colorectal or cardiovascular surgical procedure; it may involve the abdomen or pelvis; 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 surgical procedure, a laparoscopic surgical procedure, a microscopic procedure or an endoscopic procedure; or it may be a cancer surgical procedure, more specifically a prostate cancer surgical procedure or a colorectal cancer procedure.

[0017] In certain embodiments, the fluorescent conjugate in any of the methods comprises a fluorescent moiety selected from the group consisting of a fluorescent protein, a fluorescent peptide, a fluorescent dye, or a combination thereof described herein.

[0018] In certain embodiments, any of the methods include administration of the fluorescent conjugate in a pharmaceutical composition, which may include a pharma- ceutically acceptable excipient.

[0019] In some embodiments, the fluorescent conjugates can allow for nerve visualization and concomitant (or simultaneous) nerve and ureter visualization in a subject. In some embodiments, the fluorescent conjugates can provide nerve visualization and ureter visualization in a subject at various times during a surgical procedure. [Brief description of the drawings]

[0020] [Figure 1] Brightfield and fluorescent overlay images of the ureter in a live wild-type mouse after intravenous administration of approximately 2000 nmoles of fluorescent conjugate (SEQ ID NO: 747). Images were obtained 2 hours after administration using a customized fluorescent detection microscope. Left panel: white reflectance image. Right panel: fluorescent overlay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description Disclosed herein is a method for visualizing the ureter, more specifically, a method for fluorescent visualization of 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 comprising a neurotargeting peptide, supports sensitive and long-term detection of fluorescence in the ureter during urine discharge from the kidney to the bladder. Advantageously, the use of the fluorescent conjugates herein allows for both early and late ureter detection after administration. These properties highlight the broad utility of such conjugates, which provide short-term and long-term ureter visualization after administration.

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

[0023] In some embodiments, the peptide in the fluorescent conjugate comprises a neural targeting peptide, more specifically, a neural targeting peptide disclosed in International Patent Application No. PCT / US2018 / 045054 or International Patent Application No. PCT / US21 / 61821, both of which are incorporated herein by reference in their entirety. Thus, in some embodiments, the fluorescent conjugates disclosed herein may allow for simultaneous detection of the ureter and nerves.

[0024] In one embodiment, the peptide in the fluorescent conjugate comprises the amino acid sequence QVPWEEPYYVVKKSSGG (HNP401-N-2 with 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 GG linker; SEQ ID NO: 747).

[0025] The present invention can be more fully appreciated with reference to the following description, including examples. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0026] For the sake of brevity, all documents or portions of documents cited herein, including patents, patent applications, articles, books, manuals, and treatises, are expressly incorporated herein by reference in their entirety for all purposes, however, the citation of any such publication shall not be construed as an admission that it is prior art to the present invention.

[0027] The headings and subheadings in the sections of this specification are for organizational purposes only and should not be construed as limiting the subject matter described, which should be read by reference to the specification as a whole. For example, one of skill in the art will recognize that combinations of various aspects from different headings 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 the description herein, the described embodiments and various alternatives thereof can be practiced without the need for specificity in the described examples.

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

[0030] As used herein, the term "about" or "approximately" refers to a range of values ​​including the specified value that one of ordinary skill in the art would consider to be reasonably similar to the specified value. In some embodiments, "about" refers to within a standard deviation using measurements generally accepted in the art. In some embodiments, "about" refers to a range of ±10% of the specified value. In some embodiments, "about" refers to the specified value.

[0031] Whether or not the term "about" is explicitly used, all numerical values ​​given herein refer to the actual given value and approximations of such given value that are reasonably expected based on the prior art in the art, including equivalents or approximations based on experimental and / or measurement conditions of such given value. Thus, for any embodiment of the invention where a numerical value is preceded by "about" or "approximately," the invention includes embodiments in which the exact value is recited. Conversely, for any embodiment of the invention where a numerical value is not preceded by "about" or "approximately," the invention includes embodiments where the value is preceded by "about" or "approximately."

[0032] The terms "comprise" and "include" are used herein in their open, non-limiting sense. Other terms and expressions used herein and variations thereof, unless expressly indicated otherwise, should be construed as open ended, not terminally limiting. As an example of the above, the term "example" is incorporated to provide illustrative examples of the items being discussed, and is not an exhaustive or limiting list thereof. Adjectives such as "conventional," "ordinary," "known," and terms of similar meaning should not be construed to limit the items described to those available at a time or at a point in time, but rather to encompass conventional or ordinary technology that may be available or known at any time now or in the future. Similarly, when this specification describes technology that is obvious or known to one of ordinary skill in the art, such technology includes that which is obvious or known to one of ordinary skill in the art at any time now or in the future.

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

[0034] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs). The term encompasses amino acid chains of any length, including full-length proteins (i.e., neurotargeting molecules) in which the amino acid residues are linked by covalent peptide bonds. As used herein, the term "peptide" refers to a polymer of amino acid residues that typically ranges from 2 to about 50 residues in length. In certain embodiments, peptides range from about 2, 3, 4, 5, 7, 9, 10 or 11 residues to about 50, 45, 40, 45, 30, 25, 20 or 15 residues in length. In certain embodiments, peptides range from about 8, 9, 10, 11 or 12 residues to about 15, 20 or 25 residues in length. When an amino acid sequence is described herein, L-, D- or beta amino acid versions of the sequence are also contemplated, as well as reverse, inverted and reverse-inverted isoforms. Peptides also include amino acid polymers in which one or more amino acid residues are artificial analogs of the corresponding naturally occurring amino acids, and naturally occurring amino acid polymers. In addition, the term also applies to amino acids linked by peptide bonds or other modified bonds (e.g., when peptide bonds are replaced by α-esters, 3-esters, thioamides, phosphonamides, carbamates, hydroxylates, etc. (see, e.g., Spatola 1983, Chem. Biochem. Amino Acids and Proteins 7: 267-357), when amides are replaced by saturated amines (see, e.g., Skiles et al., U.S. Pat. No. 4,496,542; and Kaltenbronn et al., 1990, pp. 969-970 in Proc. 11th American Peptide Symposium, ESCOM Science Publishers, The Netherlands, which are incorporated herein by reference).

[0035] Thus, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids refer to those encoded by genetic code, as well as those that are subsequently 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, carboxyl groups, amino groups, and R groups, such as homoserine, norleucine, and methionine sulfoxide. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0036] Amino acids may be referred to by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUP AC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their commonly accepted single-letter codes.

[0037] Those skilled in the art recognize that individual substitutions, deletions or additions to peptide, polypeptide or protein sequences that change, add or delete a single amino acid or a small percentage of amino acids in the coding sequence are "conservatively modified variants" that result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are also, but do not exclude, polymorphic variants, interspecies homologs and alleles.

[0038] The following eight groups each contain certain amino acids which are conservative substitutions 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, e.g., 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 the amino acid residues in another reference polypeptide sequence, after alignment of the sequences and, if necessary, introduction of gaps as necessary to achieve the maximum percent sequence identity, and without considering any conservative substitutions as sequence identity. 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 the amino acid residues in another reference polypeptide sequence, after alignment of the sequences and, if necessary, introduction of gaps as necessary to achieve the maximum percent sequence homology. Thus, in contrast to sequence identity, sequence homology considers any conservative substitutions (as described above) as part of the sequence alignment. Sequence identity or sequence homology can be determined using well-known methods, including publicly available computer programs, such as the NCBI BLAST 2.0 software defined by Altschul et al. 1997, Nucl. Acids Res. 25, 3389-3402. These programs optimally align sequences using default gap weights to determine the highest level of sequence identity or sequence homology between a sequence and a reference sequence.

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

[0041] The terms "individual", "patient" or "subject" are used interchangeably. As used herein, they refer to any mammal (i.e., any order, family and genus of species within the taxonomic classification Animalia: Chordata: Vertebrata: Mammalia). In some embodiments, the mammal is a cow, horse, sheep, pig, cat, dog, goat, mouse, rat, rabbit, guinea pig, non-human primate or human. In some embodiments, the mammal is a human subject, more specifically a human subject undergoing a surgical procedure. The terms herein include workers who do not require or are not limited to situations characterized by the supervision (e.g., constantly or intermittently) of a medical professional (e.g., a physician, registered nurse, clinical nurse, physician's assistant, handyman or hospice worker).

[0042] As used herein, the terms "administration," "administering," "administering," and the like refer to methods that can be used to enable delivery of an agent or composition to a desired site of biological action. These methods include, but are not limited to, parenteral injection (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravitreal, infusion, or topical). Administration techniques that can be optionally used with the agents and methods described herein are described, for example, in Gennaro: Remington: The Science and Practice of Pharmacy, 21 st Ed. (Lippincott Williams & Wilkins, 2005) and Goodman & Gilman's The Pharmacological Basis of Therapeutics (McGraw-Hill Professional, 2005). In certain embodiments, administration is via systemic intravenous injection into a human patient.

[0043] The term "effective amount" is interchangeable with "therapeutically effective amount" and refers to an amount sufficient to achieve a stated purpose, for example, to achieve an effect in the subject to which it is administered, such as enabling fluorescent visualization during ureteral surgery (the course of surgery, surgical procedure, surgical method, or operation). An example of an "effective amount" is an amount that is sufficient to contribute to the treatment, prevention, or reduction of adverse events caused by a surgical or medical procedure, such as iatrogenic ureteral injury (IUI) during abdominal surgery. The desired effect may, but does not necessarily, occur with one administration. It may also occur after a series of administrations. Thus, an effective amount may be administered in one or more administrations. Determining a therapeutically effective amount of a composition is within the capabilities of one of ordinary skill in the art, especially 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. The dosage in humans may be adjusted by monitoring the effectiveness of the agent or composition disclosed herein and adjusting the dosage upwards or downwards. Adjustment of dosages to achieve maximal efficacy in humans based on the methods described herein and others is well within the capabilities of one of ordinary skill in the art.

[0044] As used herein, the term "pharmacologically acceptable" refers to a substance that does not abrogate the biological activity or properties of the agents 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 pharma- ceutically acceptable substance may be administered to an individual without causing significant undesirable biological effects or interacting significantly in a deleterious manner with any of the components contained in the composition.

[0045] The term "surgery" as used herein refers to any method or procedure that can be used to manipulate, change or cause an effect by physical intervention. Surgical procedures 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 patient. Surgical procedures can include gynecological, urological, colorectal, or cardiovascular surgical procedures. Surgical procedures can also include, but are not limited to, abdominal or pelvic procedures. In some cases, surgical procedures are performed on the kidney, bladder, prostate, uterus, male or female reproductive system, rectum, colon, small intestine, or large intestine. Surgical procedures can be, for example, cancer surgical procedures, including prostate cancer surgical procedures or colorectal procedures. Surgical procedures may also include procedures that carry a risk of ureteral injury, and in some cases may affect nerves as well, since the compositions and methods described herein may support intraoperative neurovisualization, including simultaneous nerve and ureteral visualization in subjects undergoing surgery.

[0046] II. Method The present invention provides a method for visualizing the ureter, particularly during surgery, by using a fluorescent conjugate that comprises a peptide bound to a fluorescent moiety, i.e., a labeled peptide. In some embodiments, the peptide comprises a neurotargeting peptide. In some embodiments, the fluorescent conjugates (and compositions thereof) described herein are useful as surgical adjuncts in such methods to reduce the possibility of ureteral injury and especially iatrogenic ureteral injury (IUI) during surgical procedures.

[0047] As described further herein, it has been observed that neural targeting fluorescent conjugates can be eliminated by renal excretion under conditions that allow significant and long-lasting fluorescence during passage through the ureter.As a result, ureter visualization can be achieved by detecting fluorescent moieties in the flow of urine through the ureter, more specifically, in the peristaltic flow of urine through the ureter.Ureter visualization can persist for extended periods of time, lasting for several hours or more, after administration of certain fluorescent conjugates.

[0048] Thus, in some embodiments, the invention provides a method of visualization of the ureter in a subject using any of the molecules and compositions described herein, hi some embodiments, the subject is undergoing a procedure, such as a diagnostic, medical or surgical procedure.

[0049] In certain embodiments, the method includes: (a) administering to a subject an effective amount of a fluorescent conjugate comprising a peptide and a fluorescent moiety; and (b) detecting fluorescence of the fluorescent moiety in the ureter of the administered subject.

[0050] In other embodiments, the method includes: (a) administering to a subject undergoing a surgical procedure an effective amount of a fluorescent conjugate comprising a peptide and a fluorescent moiety; and (b) visualizing the ureter in the subject by detecting fluorescence of the fluorescent moiety in the ureter.

[0051] In other embodiments, the method includes: (a) identifying a subject at risk for ureteral injury prior to a surgical procedure; (b) administering to the subject undergoing the surgical procedure an effective amount of a fluorescent conjugate comprising a peptide and a fluorescent moiety; and (c) visualizing the ureter by detecting fluorescence of the fluorescent moiety in the ureter. As used herein, a subject at risk for ureteral injury includes a subject in need of a diagnostic, medical, or surgical procedure described herein.

[0052] In some embodiments, the fluorescent conjugate is first administered before the surgical procedure. In some embodiments, the fluorescent conjugate is not administered after the first administration. In some embodiments, the fluorescent conjugate is administered during the surgical procedure. In some embodiments, the fluorescent conjugate is administered before and during the surgical procedure.

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

[0054] In some embodiments of any of these methods, the fluorescent conjugate is administered to a subject described herein, including humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate. In some embodiments, the subject is a human. In some embodiments, the human subject is a pediatric subject (21 years of age or younger), an adult subject (22-65 years of age), or a geriatric subject (65 years of age or older).

[0055] The fluorescent conjugate may be administered by any route, as further described herein. In some embodiments, the fluorescent conjugate is administered intravenously. In some embodiments, the fluorescent conjugate is administered orally. In some embodiments, the fluorescent conjugate administered by any route may be administered as a pharmaceutical composition, as described herein, which may include pharma- ceutically acceptable excipients.

[0056] The fluorescent moiety of the fluorescent conjugate can be detected in the ureter by any suitable method known in the art, for example, the fluorescent moiety can be detected by excitation of the 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 moiety 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 synchronous information from bright field illumination and fluorescent 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 moiety may be used to collect light and create the final image. Live video processing may also be performed to enhance contrast during fluorescence detection and improve the sensitivity of the fluorescent signal.

[0058] In certain embodiments of the method, fluorescence of the fluorescent moiety 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 certain embodiments of the method, fluorescence of the fluorescent moiety is detected in the ureter for 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.

[0060] In certain embodiments of the method, fluorescence of the fluorescent moiety is detected in the ureter greater than 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours or 8 hours after administration.

[0061] In certain embodiments of the method, the fluorescent conjugates allow for concomitant nerve detection and ureter visualization, including intraoperative nerve and ureter visualization. Such nerves may include human nerves, such as motor nerves, sensory nerves, sympathetic and parasympathetic nerves, peripheral nerves, and autonomic nerves, for example, as disclosed in the '054 application.

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

[0063] Additionally, 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 It includes peptides, more specifically, neuro-targeting peptides linked to a fluorescent moiety (or peptides that otherwise include a fluorescent label).

[0065] peptide In some embodiments, the neurotargeting peptide is any peptide, including any deletion or linker variant, disclosed in International Patent Application PCT / US2018 / 045054 (the "'054 Application", published as WO2019028281A2, incorporated herein by reference in its entirety). Thus, such peptides comprise or consist 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 certain embodiments, the peptide comprises or consists of any of SEQ ID NOs: 1-14, 16, 20-28 and 96-102.

[0067] Additionally, the peptides can comprise or consist of any variant of the amino acid sequences described in the '054 application. Such variants can include, but are not limited to, peptides that comprise or consist of an amino acid sequence sharing 75%-99% identity or 75%-99% homology with an amino acid sequence in Table 1 (or as otherwise disclosed in the '054 application).

[0068] In one embodiment, the peptide comprises or consists of an amino acid sequence having at least 75% identity with any of SEQ ID NOs: 1 to 14, 16, 20 to 28, and 96 to 102. In one embodiment, the peptide comprises or consists of an amino acid sequence having at least 80% identity with any of SEQ ID NOs: 1 to 14, 16, 20 to 28, and 96 to 102. In one embodiment, the peptide comprises or consists of an amino acid sequence having at least 85% identity with any of SEQ ID NOs: 1 to 14, 16, 20 to 28, and 96 to 102. In one embodiment, the peptide comprises or consists of an amino acid sequence having at least 90% identity with any of SEQ ID NOs: 1 to 14, 16, 20 to 28, and 96 to 102. In one embodiment, the peptide comprises or consists of an amino acid sequence having at least 95% identity with any of SEQ ID NOs: 1 to 14, 16, 20 to 28, and 96 to 102. In certain embodiments, the peptide comprises or consists of an amino acid sequence having at least 97% identity to 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 certain embodiments, the peptide comprises or consists of an amino acid sequence sharing at least 99% homology with any of SEQ ID NOs: 1-14, 16, 20-28 and 96-102.

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

[0071] In some embodiments, 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 some embodiments, the peptide comprises the amino acid core binding domain of PYY and the N-terminal sequence of QVPWEE (SEQ ID NO: 41). In some embodiments, the peptide comprises the amino acid core binding domain of PYY VV (SEQ ID NO: 83) and the N-terminal sequence of QVPWEE (SEQ ID NO: 41).

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

[0073] In certain embodiments, 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 certain embodiments, 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 a GGC linker; SEQ ID NO:7), Ac-SGQVPWEEPYYVVKKGGC (HNP401-C-2 with a GGC linker; SEQ ID NO:11), QVPWEEPYYVVKKSS (HNP401-N-2; SEQ ID NO:20), QVPWEEPYYVVKKSSGG (HNP401-N-2 with a GG linker; SEQ ID NO:21) and SGQVPWEEPYYVVKK (HNP401-C-2; SEQ ID NO:25) and SGQVPWEEPYYVVKKGG (HNP401-C-2 with a GG linker; SEQ ID NO:99).

[0075] In certain embodiments, the peptide comprises or consists of the amino acid sequence of any of SEQ ID NOs: 1, 4, 7-14, 20-28 and 96-102.

[0076] In certain embodiments, the peptide comprises or consists of the amino acid sequence of any of SEQ ID NOs: 1, 20, and 22-24.

[0077] In certain embodiments, the peptide comprises or consists of the amino acid sequence of any of SEQ ID NOs: 1 and 25-28.

[0078] In certain embodiments, the peptide comprises or consists of the amino acid sequence of any of SEQ ID NOs: 4 and 7-10.

[0079] In certain embodiments, the peptide comprises or consists of the amino acid sequence of any of SEQ ID NOs: 4 and 11-14.

[0080] In certain embodiments, the peptide comprises or consists of the amino acid sequence of any of SEQ ID NOs: 21 and 100-102.

[0081] In certain embodiments, the peptide comprises or consists of the amino acid sequence of any of SEQ ID NOs: 96-99.

[0082] In certain embodiments, the peptides are neural targeting peptides disclosed in PCT Application PCT / US21 / 61821 ("the '821 Application"), which is incorporated herein by reference in its entirety. In certain embodiments, these peptides were derived from a phage display library screen based on selective binding to any of four different neural targeting proteins: myelin protein zero (MPZ), myelin basic protein, myelin proteolipid protein (myelin PLP) and nidogen-2. Such peptides comprise 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 certain embodiments, these peptides were derived from a phage display screen based on selective binding to laminin trimer 421, 521 or both. Such peptides comprise or consist of any of the amino acid sequences listed in Table 3 (SEQ ID NOs: 472-678). In certain embodiments, these peptides were derived from phage display screening based on selective binding to neuronal and neural extracts, but not to muscle extracts. Such peptides comprise 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] Thus, in certain embodiments, the peptide comprises 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 certain embodiments, the peptide comprises 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 certain embodiments, the peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-82, 84-95, and 103-746. In certain embodiments, the peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-74 and 188-362. In certain embodiments, the peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-82, 84-95, and 103-187. In certain embodiments, the peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-54 and 363-446. In certain embodiments, 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 certain embodiments, the peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 472-678. In certain embodiments, the peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 679-746.

[0084] In some embodiments, the peptides comprise or consist of variants of the amino acid sequences set forth in International Patent Application PCT / US21 / 61821 ("the '821 Application"), including, but not limited to, peptides comprising or consisting of amino acid sequences sharing 75%-99% identity or 75%-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 to 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 80% identity to 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 85% identity to 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 90% identity to any of SEQ ID NOs: 18, 19, 29-82, 84-95, and 103-746. In certain embodiments, the peptide comprises or consists of an amino acid sequence having at least 95% identity to 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 certain embodiments, the peptide comprises or consists of an amino acid sequence sharing 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. By way of 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 ("Ac" or "acetyl") at the N-terminus, amidated ("CONH2" or "NH2") at the C-terminus, or both. For example, SEQ ID NO: 747 corresponds to an amidated peptide that terminates with glycinamide.

[0088] Fluorescent part As used herein, the term "fluorescent moiety" refers to any fluorescent molecule, including a fluorescent protein, a fluorescent peptide, a fluorophore (e.g., a fluorescent dye), or any other fluorescent ligand or marker.

[0089] The specific examples of fluorescent moieties described herein are merely illustrative and are not intended to limit the fluorescent moieties that may be used in the peptides disclosed herein.

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

[0091] General classes of fluorophores include, but are not limited to, xanthenes such as rhodamine, rhodol and fluorescein and their derivatives; bimanes; coumarins and their derivatives such as umbelliferone and aminomethylcoumarin; aromatic amines such as dansyl; squaric acid dyes; benzofurans; fluorescent cyanines; carbazoles; dicyanomethylenepyrans; polymethines; oxabenzanthrans; pyryliums; carbostyryls; perylenes; acridones; quinacridones; rubrenes; anthracenes; coronenes; phenanthracenes; pyrenes; butadienes; stilbenes; porphyrins; phthalocyanines; lanthanide metal chelate complexes; rare earth metal chelate complexes; and their derivatives. Fluorescent dyes are disclosed, for example, in U.S. Pat. No. 4,452,720; U.S. Pat. No. 5,227,487; and U.S. Pat. No. 5,543,295.

[0092] Exemplary 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 in, for example, US Pat. No. 6,008,379, US Pat. No. 5,750,409, US Pat. No. 5,066,580 and US Pat. No. 4,439,356.

[0093] In some embodiments, the fluorescent moiety comprises a carboxyfluorescein molecule (FAM), i.e., a fluorescein molecule with a carboxyl group added. Thus, the term "FAM" includes fluorescein molecules with a carboxyl group at the 5-position (5-FAM) or 6-position (6-FAM).

[0094] In some embodiments, 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; dabsyl; QSY7; QSY9; QSY21; and BBQ-650 dye.

[0095] In some embodiments, the fluorescent moiety comprises a rhodamine dye, such as, for example, 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 can be found, for example, in U.S. Patent 6,080,852; U.S. Patent 6,025,505; U.S. Patent 5,936,087; and U.S. Patent 5,750,409.

[0096] In certain embodiments, the fluorescent moiety comprises a cyanine dye, such as, for example, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, and indocyanine green.

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

[0098] In certain embodiments, the excitation and emission spectra of the fluorophores are in the visible region of the spectrum (about 380 nm to about 700 nm).

[0099] In some embodiments, the excitation and emission spectra of the fluorophore are in the near infrared (NIR) range, including the NIR-I spectral region (about 700 nm to about 900 nm) and the NIR-II spectral region (about 1000 nm to about 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 (Molecular Probes AlexaFluor dyes, available from 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 certain embodiments, 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 certain embodiments, the fluorophore is 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)-FAM cadaverine; 5-FAM cadaverine; 5(6)-FAM ethylenediamine; 5-FAM ethylenediamine; 5-FITC (FITC isomer I; fluorescein-5-isothiocyanate); 5-FITC cadaverine; fluorescein-5-maleimide; 5-IAF (5-iodoacetamidofluorescein); 6-JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein); 5-CRl l0 (5-carboxyrhodamine 110); 6-CRl 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 These may include, but are not limited to, C2 maleimide; TR cadaverine; 5-TRITC; G isomer (tetramethylrhodamine-5-isothiocyanate); 6-TRITC; R isomer (tetramethylrhodamine-6-isothiocyanate); dansylcadaverine (5-dimethylaminonaphthalene-l-(N-(5-aminopentyl))sulfonamide); EDANS C2 maleimide; fluorescamine; NBD; and pyrromethene and their derivatives.;

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

[0102] In some embodiments, the fluorescent moiety is a fluorescent peptide or a fluorescent protein. In some embodiments, the fluorescent protein is green fluorescent protein (GFP). In some embodiments, the fluorescent moiety is a fluorescent peptide or a fluorescent protein. In some embodiments, the fluorescent protein is green fluorescent protein (GFP). In some embodiments, the fluorescent moiety is a fluorescent peptide or a fluorescent protein. In some embodiments, the fluorescent moiety is a fluorescent peptide or a fluorescent protein. In some embodiments, the fluorescent protein is green fluorescent protein (GFP). In some embodiments, the fluorescent moiety is a fluorescent peptide or a fluorescent protein. In some embodiments, the fluorescent moiety is a fluorescent peptide or a fluorescent protein. In some embodiments, the fluorescent moiety is a fluorescent peptide or a fluorescent protein. In some embodiments, the fluorescent moiety is a fluorescent peptide or a fluorescent protein. In some embodiments, the fluorescent moiety is a fluorescent peptide or a fluorescent protein. Derivatives or variants of GFP including Orange, Kusabira 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 Citrine.

[0103] In certain embodiments, the fluorescent moiety comprises a fluorescent molecule disclosed in the '054 and '821 applications, which are incorporated herein by reference in their entireties.

[0104] In certain embodiments, the fluorescent moiety 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 be attached to the N-terminus, C-terminus, or internal positions (e.g., internal amino acids) of the peptide. In some embodiments, two, three, four, or more peptides are directly or indirectly attached to fluorescent moieties. In some embodiments, any peptide disclosed herein is attached to two or more fluorescent moieties, which may be the same or different.

[0106] In some embodiments, the fluorescent moieties disclosed herein are directly attached to any of the peptides disclosed herein.

[0107] Linker In some embodiments, the fluorescent moiety is indirectly attached to the peptide, e.g., via a linker. As used herein, a "linker" is any molecule that can be attached (e.g., covalently) to a 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 disulfides, 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 bonds. Linkers also include other linker molecules disclosed or described in the literature, including those described in the '054 and '821 applications incorporated herein.

[0108] In some embodiments, 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. The covalent bond can also be managed by reaction of a functional group of the linker with a functional group of the peptide and the cargo molecule. In some embodiments, the covalent bond comprises 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 condensation reaction of the carboxylic acid functional group at the 5-position of fluorescein with the primary amine at the N-terminus of the peptide, such as in the fluorescent conjugate 5-FAM-QVPWEEPYYVVKKSSGG-NH2 (HNP401-N-2 with GG linker; SEQ ID NO: 747).

[0109] In some embodiments, the linker is flexible. In some embodiments, the linker is rigid. In some embodiments, the linker has a flexible segment and a rigid segment.

[0110] In some embodiments, the linker comprises a linear structure. In some embodiments, the linker comprises a non-linear structure. In some embodiments, the linker comprises a branched structure. In some embodiments, the linker comprises a cyclic structure.

[0111] In some embodiments, the linker is alkyl. In some embodiments, the linker is heteroalkyl.

[0112] In some embodiments, the linker is an alkylene. In some embodiments, the linker is an alkenylene. In some embodiments, the linker is an alkynylene. In some embodiments, the linker is a heteroalkylene.

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

[0114] An "alkyl" moiety may have 1 to 10 carbon atoms (numerical ranges such as "1 to 10", as used herein, refer to each integer in the range; for example, "1 to 10 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, but this definition also encompasses the case of the term "alkyl" where no numerical range is specified). An alkyl group may also be "lower alkyl", having 1 to 6 carbon atoms. The alkyl group of the compounds described herein may be designated as "C1-C4 alkyl" or a similar designation. By way of example only, "C1-C4 alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Exemplary alkyl groups include, but are not limited in any manner to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, and the like.

[0115] In some embodiments, the linker comprises a ring structure (e.g., aryl). As used herein, the term "ring" refers to any covalently closed ring structure. Rings include, for example, carbocycles (e.g., aryl and cycloalkyl), heterocycles (e.g., heteroaryl and non-aromatic heterocycles), aromatics (e.g., aryl and heteroaryl), and non-aromatic (e.g., cycloalkyl and non-aromatic heterocycles). Rings may be optionally substituted. Rings 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. The aryl ring can be formed from 5, 6, 7, 8, 9 or more carbon atoms. The aryl group can be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl, naphthalenyl, phenanthrenyl, anthracenyl, fluorenyl, and indenyl. Depending on the structure, the 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 atoms forming the ring (i.e., the skeletal atoms) is a carbon atom. Cycloalkyls can be saturated or partially unsaturated. Cycloalkyl groups include groups having 3 to 10 ring atoms. Cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0118] In certain embodiments, the ring is a cycloalkane. In certain embodiments, the ring is a cycloalkene.

[0119] In some embodiments, the ring is an aromatic ring. The term "aromatic" refers to a planar ring with a delocalized π-electron system containing 4n+2 π-electrons (where n is an integer). An aromatic ring can be formed from 5, 6, 7, 8, 9 or more atoms. An aromatic can be optionally substituted. 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-ring polycyclic (i.e., rings shared by adjacent pairs of carbon atoms) groups.

[0120] In certain embodiments, the ring is a heterocycle. The term "heterocycle" refers to heteroaromatic and heteroalicyclic groups containing 1 to 4 heteroatoms each selected from O, S and N, with the proviso 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. Non-aromatic 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-fused ring systems. An example of a 3-membered heterocyclic group is aziridinyl. 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 are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyran ... These are 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 quinolizinyl.Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl and furopyridinyl. The groups may be C- or N-bonded if possible. For example, the group derived from pyrrole may be pyrrol-1-yl (N-bonded) or pyrrol-3-yl (C-bonded). Additionally, the imidazole derived groups can be imidazol-1-yl or imidazol-3-yl (both N-linked) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-linked). Heterocyclic groups include benzo-fused ring systems and ring systems substituted with one or two oxo (=0) moieties, such as pyrrolidin-2-one. Depending on the structure, heterocyclic groups can be monoradicals or diradicals (i.e., heterocyclene groups).

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

[0122] The term "carbocyclic" or "carbocycle" refers to a ring in which each of the atoms forming the ring is a carbon atom. Carbocycle includes aryl and cycloalkyl. The term thus distinguishes between carbocycle and heterocycle ("heterocyclic") in which the ring backbone contains at least one atom different from carbon (i.e., a heteroatom). Heterocycle includes heteroaryl and heterocycloalkyl. Carbocycle and heterocycle may be optionally substituted.

[0123] In some embodiments, the linker is substituted. The term "optionally substituted" or "substituted" refers to a group that is i -C e Alkyl, C3-C g It is meant that they may be substituted with one or more further groups individually and independently selected from cycloalkyl, aryl, heteroaryl, C2-C6 heteroalicyclic, hydroxy, C1-C6 alkoxy, aryloxy, C1-C6 alkylthio, arylthio, C1-C6 alkylsulfoxide, arylsulfoxide, C1-C6 alkylsulfone, arylsulfone, cyano, halo, C2-C8 acyl, C2-C8 acyloxy, nitro, C1-C6 haloalkyl, C1-C6 fluoroalkyl, and amino, including C1-C6 alkylamino, and protected derivatives thereof. As an example, the optional substituents may be LSRS, where each L is independently selected from a bond, -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). Protecting groups that may form protected derivatives of the above substituents are known to those of skill in the art.

[0124] In some embodiments, 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 the desired conjugate. Alternatively, derivatization is performed to provide the functional group. Thus, for example, methods are also known for creating free sulfhydryl groups on peptides (see U.S. Pat. No. 4,659,839). The linker may alternatively comprise a heterobifunctional crosslinker that comprises two or more different reactive groups that form a heterocyclic ring that can interact with the targeting molecule. For example, a heterobifunctional crosslinker such as cysteine ​​may comprise an amine reactive group, and a thiol reactive group 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 cross-linkers 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 be chemically stable to the extracellular environment, e.g., chemically stable in the bloodstream, and / or may contain bonds that are not stable. The linker may contain bonds that are designed to be specifically or non-specifically cleaved and / or immolated or otherwise destroyed within the cell. The cleavable linker may be susceptible to enzymes at specific sites, such as extracellular proteases.

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

[0127] The non-cleavable linker is generally insensitive to proteases and to intracellular processes. The non-cleavable linker may comprise a maleimide group. The non-cleavable linker may comprise a succinimide group. The non-cleavable linker may be a maleimido-alkyl-C(O)-linker. The non-cleavable linker may be a maleimidocaproyl linker. The maleimidocaproyl linker may be N-maleimidomethylcyclohexane-1-carboxylate. The maleimidocaproyl linker may comprise a succinimide group. The maleimidocaproyl linker may comprise a pentafluorophenyl group.

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

[0129] In some embodiments, the fluorescent conjugate of the present invention can be conjugated to a high molecular weight molecule that increases the multivalency and avidity of binding as desired. In some embodiments, the high molecular weight molecule is a water-soluble polymer. Examples of suitable water-soluble polymers include, but are not limited to, peptides, sugars, poly(vinyl), poly(ether), poly(amine), poly(carboxylic acid), etc. In some embodiments, the water-soluble polymer is dextran, polyethylene glycol (PEG), polyoxyalkylene, polysialic acid, starch or hydroxyethyl starch. Peptides are conjugated to water-soluble polymers using any suitable method (see Hermanson G., Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008).

[0130] In some embodiments, the fluorescent conjugates of the present invention may be modified to increase solubility. Peptide modifications that increase solubility include the addition of hydrophilic amino acids, PEG moieties, 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 1-10 (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 positions, 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 includes 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 a GG linker; SEQ ID NO: 747), which may be used in any of the methods described herein.

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

[0133] IV. Pharmaceutical Compositions In some embodiments, 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, including additives and auxiliary agents that facilitate processing of the active agent into a pharmaceutical preparation. The appropriate formulation depends on the selected route of administration. Such compositions can be prepared, for example, as described in Gennaro: Remington: The Science and Practice of Pharmacy, 21 st 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) using methods and additives known in the art.

[0134] In certain embodiments of the pharmaceutical composition, the peptide in the fluorescent conjugate may consist of or comprise any amino acid sequence, including all variants and combinations described herein. In certain embodiments, the peptide consists of or comprises any amino acid sequence listed in Table 1 described herein, or any variant thereof. In certain embodiments, the peptide comprises or comprises any of SEQ ID NOs: 1-14, 16, 20-28, and 96-102. In certain embodiments, the peptide comprises or comprises any amino acid sequence listed in Tables 2-4, or any variant thereof. In certain embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NOs: 75-82, 84-95, and 103-187. In certain embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NOs: 55-74, and 188-362. In certain embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NOs: 33-54, and 363-446. In certain embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NOs: 18, 19, and 29-32, and 447-471. In certain embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NOs: 472 to 678. In certain embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NOs: 679 to 746. In certain embodiments, 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 comprises SEQ ID NO:747.

[0136] In some embodiments, the pharmaceutical compositions disclosed herein further comprise a pharma- ceutically acceptable diluent, excipient, carrier or solvent.Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene-glycol, glycerol, cremophor, etc.), suitable mixtures thereof, vegetable oils (e.g. olive oil) and injectable organic esters such as ethyl oleate.Other commonly used surfactants such as Tween, Span and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma-ceutically acceptable solid, liquid or other dosage forms can also be used for formulation purposes.Bioavailability enhancers can include penetration or permeation enhancers. See, e.g., 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, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[0137] In some embodiments, the pharmaceutical composition contains other medicinal or pharmaceutical agents, carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution enhancers, salts for regulating the osmotic pressure and / or buffers. In addition, the pharmaceutical composition may also contain other therapeutically valuable substances.

[0138] In some embodiments, the peptide conjugates disclosed herein are delivered to a subject by a drug delivery vehicle or carrier.In some embodiments, the delivery vehicle is made of natural or synthetic materials or both.In some embodiments, the delivery vehicle is a nanoparticle, a microparticle, a polymeric micelle, a nanocapsule, a dendrimer, a large PEG, a nanogel, a liposome, a fullerene, a nanostructured lipid carrier, a nanoshell, a quantum dot, a protein-based nanocarrier (e.g., albumin, elastin, gliadin, legumin, zein, soy protein, milk protein, whey-based nanocarrier), an organic nanocarrier (e.g., gelatin, dextran, guar gum, chitosan, collagen), a polysaccharide-based carrier (e.g., dextran, chitosan, pectin), a lipid emulsion, or a combination thereof.

[0139] In some embodiments, the pharmaceutical compositions disclosed herein are administered to a subject by any suitable route of administration, including but not limited to topical, oral, rectal, vaginal, intranasal, inhalation, or parenteral (intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, or intravitreal injection) administration. In some embodiments, the pharmaceutical compositions disclosed herein are administered to a subject locally or systemically. In some embodiments, the pharmaceutical compositions disclosed herein are administered intravenously.

[0140] Preparations 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. Preparations suitable for subcutaneous injection also include optional additives such as preserving, wetting, emulsifying and dispersing agents as known in the art. In some embodiments, preparations suitable for intravenous injection can be prepared in aqueous solution, such as saline buffer and other physiologically compatible buffers known in the art. For intravenous injection, the active agent is optionally formulated in aqueous solution, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer.

[0141] Parenteral injections include bolus injections or continuous infusions as desired. Injectable preparations are optionally provided in unit dosage form, for example, ampoules, or in multidose containers with the addition of preservatives. In some embodiments, the pharmaceutical compositions described herein are in a form suitable for parenteral injection as a sterile suspension, solution, or emulsion in an oily or aqueous medium, and include formulation agents such as suspending, stabilizing, and / or dispersing agents. Pharmaceutical preparations for parenteral administration include aqueous solutions of the active agent in water-soluble form. Furthermore, suspensions are optionally prepared as suitable oily injection suspensions.

[0142] In some embodiments, the pharmaceutical composition of the present invention is administered orally.The dosage form suitable for oral administration can be solid or liquid, and can include, for example, pill, capsule, troche, tablet, caplet, gel caplet (gel cap), syrup, aqueous suspension or solution, chewable form, swallowable form, dissolvable form, effervescent, granular form and oral liquid solution.In a specific embodiment, the dosage form is a solid dosage form, more specifically, includes tablet or capsule or is administered orally by vehicle or carrier disclosed herein.

[0143] In some embodiments, the pharmaceutical compositions described herein are in unit dosage form suitable for single administration of precise dosage amounts. In unit dosage form, the formulation is divided into unit doses containing appropriate amounts of the active agent disclosed herein. In some embodiments, the unit dosage is in the form of a package containing discrete amounts of the formulation. Non-limiting examples are packaged tablets or capsules and powders in vials or ampoules. In some embodiments, aqueous suspension compositions are packaged in single-dose non-reclosable containers. Alternatively, multi-dose reclosable containers are used, in which case it is typical to include a preservative in the composition. By way of example only, parenteral injection formulations are provided in unit dosage form, including but not limited to ampoules, or in multi-dose containers with the addition of a preservative.

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

[0145] In certain embodiments, the pharmaceutical composition is orally administered to a subject, particularly a human patient, more particularly a human patient undergoing surgery. EXAMPLES

[0146] Example 1 Use of neural targeting fluorescent conjugates for ureter visualization Introduction Phage display screening has previously been used to identify peptides that bind to human neural homogenates or isolated human neural proteins. See the '054 and '821 applications. Such peptides can be conjugated or tagged to cargo molecules for a variety of applications. When tagged with a fluorescent moiety, for example, such conjugates can be useful for in vivo targeting of nerves during fluorescence assisted surgery, and when conjugated to an active agent, for example, such conjugates can be useful for targeted therapy.

[0147] Exemplary peptides were conjugated to fluorescent dyes to produce fluorescent conjugates (also referred to herein as conjugates) and evaluated in rodent models to demonstrate sensitive neurodetection after intravenous administration. These conjugates included FAM-NP41, which contains the fluorescent dye, carboxyfluorescein (FAM), linked to the C-terminal lysine of acetylated NP41 (Ac-SHSNTQTLAKAPEHTGK; SEQ ID NO: 749) via a lysine amide bond, and FAM-HNP401, which contains FAM conjugated to the C-terminal cysteine ​​of acetylated HNP401 (Ac-SGQVPWEEPYYVVKKSSGGC-CONH2; SEQ ID NO: 748) with 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 have demonstrated fluorescent targeting of human (and rodent) resected neural tissues following local administration of other conjugates, including FAM-NP41, FAM-HNP401 and N- 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 do not reveal the properties of the conjugates, such as activity, metabolism and elimination - especially at later times after administration. In rodent models, for example, the serum half-life of NP41 (as measured by serum fluorescence) is about 10 minutes, indicating rapid clearance of the conjugate from the blood. A similar short half-life in blood (15 minutes) was observed with FAM-HNP401. In contrast, the neurofluorescence of NP41 has a half-life of about 50 minutes and can persist at low levels in tissues for several hours. Similarly, fluorescence of FAM-HNP401 was observed in rat sciatic and prostatic nerves for several hours after intravenous administration. See, e.g., Whitney et al. 2011, Nat. Biotechnol. 29, 352-356; Hingorani et al. 2018, Theranostics 8, 4226-4237.

[0150] The clearance and subsequent activity of conjugates bound to nerves (or other tissues) is unknown, and it is unknown whether such properties may support other uses for the conjugates in fluorescence-guided surgery. The studies described herein show that nerve-targeting conjugates can provide sensitive and long-term fluorescent visualization (illumination) of the ureter in conjunction with renal excretion. The results support the expansion of the repertoire of such conjugates as surgical adjuncts. Highlighting of the nerves makes them useful as guidance agents in procedures such as head and neck surgical procedures, where they may help prevent nerve injury. And, as described herein, highlighting of the ureter makes them useful in other procedures such as many abdominal and pelvic surgical procedures, where there is a risk of ureteral injury.

[0151] result A rodent model was used to evaluate the kinetics of fluorescent visualization of 5-FAM-QVPWEEPYYVVKKSSGG-NH2 (HNP401-N-2 with GG linker; SEQ ID NO: 747) after intravenous administration. The conjugate was administered in a wide range of doses, and fluorescence was detected using a customized microscope. For example, as shown in Figure 1, the ureter is readily illuminated 2 hours after intravenous administration of about 2000 nanomolar (about 200 mg / kg) in a mouse model. Further studies exploring doses ranging from 100 nanomolar to 2000 nanomolar, corresponding to doses of about 10 mg / kg to about 200 mg / kg, demonstrated ureter 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 fluorescent visualization did not reflect specific or direct targeting of the ureter. Rather, it reflected the fluorescence of urine flowing down the ureter from the kidney to the bladder. Moreover, real-time intraoperative imaging shows ureteral pulsation, which concentrates the urine and enhances fluorescent visualization of the ureter. Further studies have demonstrated prolonged ureteral illumination following administration of this conjugate. For example, fluorescent visualization of the ureter was observed in less than 1 hour and as well for at least up to 6 hours following administration of the conjugate.

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

[0153] Consideration: These observations support the general applicability of the neural targeting fluorescent conjugates described herein in ureter illumination. Thus, the conjugates disclosed herein can be used to help surgeons visualize ureters during surgical procedures before they physically encounter them and thus potentially damage them. Furthermore, in the case of inadvertent damage during a surgical procedure, i.e., iatrogenic injury, such visualization allows for more rapid recognition of the injury and early treatment and intervention.

[0154] Furthermore, it has been observed that administration of the fluorescent conjugates described herein can provide ureter visualization for extended periods of time after administration. Without being limited to a specific mechanism, this extended visualization is believed to reflect gradual accumulation of the fluorescent conjugate in neurons or nerves or other tissues, such as the peripheral nervous system, and gradual release, metabolism, and excretion by the urinary system. Thus, in contrast to systemic administration of fluorescent dyes alone or direct injection of fluorescent dyes, administration of peptide-fluorescent conjugates can advantageously extend the time of ureter illumination and provide both early and long-term illumination with a simple administration that does not interfere with surgery. Furthermore, administration of such conjugates can provide simultaneous or transient nerve or ureter detection separately.

[0155] Such fluorescent assisted assistance is particularly important during surgical procedures where there is a substantial risk of ureteral injury during the surgical procedure, especially iatrogenic ureteral injury (IUI). This risk may apply to a wide range of surgical procedures, as the ureter extends from the abdomen to the pelvis. Applicable procedures that may benefit from ureteral illumination may include complex abdominal and pelvic surgeries, such as gynecological, urological and colorectal surgical procedures, as well as cardiovascular surgical procedures. Also included are surgical procedures performed on organs and systems near the ureter, such as the kidney, bladder, prostate, uterus, male or female reproductive system, rectum, colon, small intestine or large intestine. Also included are cancer surgical procedures, such as prostate cancer surgical procedures or colorectal cancer procedures or procedures where enhanced urinary and neurological visualization is desired.

[0156] Example 2 Bioanalysis of fluorescent conjugates in urine of surgical patients Urine samples were collected 30 min before intravenous administration of 500 mg parent test drug (corresponding to fluorescent-conjugated peptide, SEQ ID NO: 747), 30 min after pre-head and neck surgery injection, and 22 h after. Concentrations of intact parent test drug were determined by liquid chromatography with tandem mass spectrometry (LC-MS / MS) using regular bioanalytical methods. The presence of metabolites M3-M8 (originally identified from in vitro studies), which correspond to truncated versions of the parent peptide conjugated to fluorescent moieties, was also assessed. Results are shown in Table 5. [Table 5] *BQL: below limit of quantification (10ng / ml)

[0157] At 30 minutes, quantifiable levels of test drug (or fluorescent metabolites M4, M6, and M7) were detected in 4 of 5 subjects evaluated. At 22 hours, fluorescent metabolites M4 and M6 were detected in both subjects evaluated. As expected, test drug and fluorescent metabolites were not detected in any of the pre-dose samples.

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

[0159] Example 3 Intraoperative ureteral and nerve illumination The ability of the fluorescent conjugates of the invention to image the ureter (and nerves) can be evaluated using a properly designed clinical trial, as described below, in patients undergoing gastrointestinal / genitourinary (GI / GU) surgery.

[0160] In this prospective example, the clinical trial is an open-label, multi-center study that evaluates the administration of a test drug (corresponding to a fluorescent-conjugated peptide, SEQ ID NO: 747) in 80 subjects undergoing GI / GU surgery. The inclusion criteria for enrollment in this study include that subjects must undergo a surgical procedure in the GI / GU region that may be at risk of nerve (and ureter) damage. For example, such procedures may include colorectal or prostate surgery.

[0161] Enrolled subjects will receive a single dose of study drug (500 mg) via intravenous infusion 2-5 hours before surgery. Study drug fluorescence will be assessed by real-time white light reflectance (WLR) and fluorescence (FL) imaging via laparoscopy.

[0162] Multiple endpoints will be used to assess the primary and secondary efficacy objectives.

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

[0164] For ureter detection, endpoints include a visualization scoring system to measure the effectiveness of test agents in improving ureteral contrast enhancement using WLR alone versus WLR and FL overlay, as well as calculation of signal-to-background ratios in multiple regions of ureteral (and adjacent non-ureteral) tissue to measure the effectiveness of test agents in improving ureteral visualization using WLR versus FL.

[0165] Significant improvements in the efficacy of nerve and ureter detection with the test drug are shown at multiple endpoints. Improvements are observed at early and late time points of the surgical procedure. Thus, the fluorescent conjugates of the present invention may be useful in fluorescence-guided GI / GU surgical procedures that include enhanced visualization of nerves, ureters, or both. The benefits of such enhanced visualization include a significant improvement in the surgeon's ability to positively identify ureters and nerves in the surgical procedure and a significant reduction in the time of such surgical procedures.

[0166] The above examples provide those skilled in the art with as complete a disclosure and description of how to make and use the 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 their disclosure. Modifications of the above modes of carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications mentioned herein are indicative of the level of skill of those skilled in the art to which the invention pertains. All references cited herein are incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety.

[0167] As will be apparent to those skilled in the art, many modifications and variations of this application can be made without departing from its spirit and scope. The specific embodiments and examples described herein are offered by way of example only, and this application is to be limited only by the scope of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0168] Furthermore, while certain details of the invention are provided to provide a thorough understanding of the invention as defined by the appended claims, it will be understood by those skilled in the art that certain embodiments may be practiced without such details. Further, in some instances, details of well-known methods, procedures or other specific details have not been described to avoid unnecessarily obscuring aspects of the invention as defined by the appended claims.

[0169] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 137,621, filed January 14, 2021, which is incorporated herein by reference in its entirety.

Claims

1. 1. A pharmaceutical composition for visualizing the ureter in a subject, comprising a fluorescent conjugate comprising a peptide and a fluorescent moiety, visualization by detecting fluorescence of a fluorescent moiety in the ureter following administration of the pharmaceutical composition; wherein the fluorescent conjugate peptide comprises the amino acid sequence of SEQ ID NO: 21 (QVPWEEPYYVVKKSSGG),

2. The pharmaceutical composition of claim 1, wherein the subject is undergoing a surgical procedure.

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

4. A pharmaceutical composition of any of claims 1 to 3 which is first administered prior to a surgical procedure.

5. The pharmaceutical composition of claim 3, which is not further administered to the subject after the initial administration.

6. The surgical procedure (i) Includes gynecological, urological, colorectal or cardiovascular surgical procedures; (ii) involves abdominal or pelvic procedures; (iii) performed on the kidney, bladder, prostate, uterus, male or female reproductive system, rectum, colon, small intestine, or large intestine; (iv) It is an open surgical procedure, a laparoscopic surgical procedure, a microscopic procedure, or an endoscopic procedure; (v) a cancer surgical procedure; and / or (vi) prostate cancer surgery or colorectal cancer surgery, The pharmaceutical composition according to any one of claims 2 to 5.

7. The pharmaceutical composition according to any one of claims 3 to 6, wherein the ureteral injury is caused by ligation, angulation, cutting, laceration, crushing, ischemia or resection.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the peptide is a neurotargeting peptide.

9. 9. The pharmaceutical composition of any of claims 1 to 8, wherein the fluorescent moiety is conjugated to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus of the peptide.

10. Any of the pharmaceutical compositions of claims 1 to 9, wherein the fluorescence of the fluorescent moiety is detected within (i) 30 minutes, (ii) 30 minutes but 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 after administration of the pharmaceutical composition.

11. Any of the pharmaceutical compositions of claims 1 to 10, wherein fluorescence is detected 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.

12. 12. The pharmaceutical composition of any of claims 1 to 11, wherein detecting the fluorescence of the fluorescent moiety in the ureter comprises detecting the fluorescent moiety in the urine flow in the ureter.

13. The pharmaceutical composition of claim 12, wherein detecting the fluorescence of the fluorescent moiety in the ureter comprises detecting the fluorescent moiety in the peristaltic flow of urine in the ureter.

14. The pharmaceutical composition of any of claims 1 to 13, wherein the fluorescent moiety is selected from the group consisting of fluorescent proteins, fluorescent peptides, fluorescent dyes, and combinations thereof.

15. 15. The pharmaceutical composition of any of claims 1-14, wherein the fluorescent moiety comprises xanthene; bimane; coumarin; aromatic amine; benzofuran; fluorescent cyanine; carbazole; dicyanomethylenepyran; polymethine; oxabenzanthrane; pyrylium; carbostyril; perylene; acridone; quinacridone; rubrene; anthracene; coronene; phenanthracene; pyrene; butadiene; stilbene; porphyrin; phthalocyanine; lanthanide metal chelate complex; rare earth metal chelate complex; or a derivative thereof.

16. 16. The pharmaceutical composition of any of claims 1-15, wherein the fluorescent moiety 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.

17. 17. The pharmaceutical composition of any of claims 1 to 16, wherein the fluorescent moiety comprises carboxyfluorescein (FAM).

18. 18. The pharmaceutical composition of any of claims 1 to 17, wherein the fluorescent moiety 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.

19. 19. The pharmaceutical composition of any of claims 1 to 18, wherein the fluorescent moiety comprises Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, or indocyanine green.

20. 20. The pharmaceutical composition of any of claims 1-19, wherein the fluorescent moiety 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, dabsyl, QSY7, QSY9, QSY21, or BBQ-650 dye.

21. The pharmaceutical composition of any of claims 1 to 20, wherein the fluorescent conjugate comprises SEQ ID NO: 747 (5-FAM-QVPWEEPYYVVKKSSGG-NH2).

22. 22. The pharmaceutical composition of any of claims 1 to 21, wherein the peptide comprises SEQ ID NO:21 (QVPWEEPYYVVKKSSGG) and the fluorescent moiety comprises FAM.

23. A pharmaceutical composition of any of claims 1 to 22, administered intravenously.

24. A pharmaceutical composition of any of claims 1 to 22, which is administered orally.

25. The pharmaceutical composition according to any one of claims 1 to 24, further comprising a pharma-ceutically acceptable excipient.

26. The pharmaceutical composition of any one of claims 1 to 25, further comprising a fluorescent conjugate for detecting nerves in a subject.

27. 27. The pharmaceutical composition of claim 26, wherein visualization of the nerves and ureters is detected simultaneously in a subject during surgery.

28. 27. The pharmaceutical composition of claim 26, wherein visualization of the nerves and ureters is detected in a subject at different time points during surgery.

29. The pharmaceutical composition of any of claims 1 to 28, wherein the fluorescence of the fluorescent moiety is detectable more than 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours or 8 hours after administration.

30. A pharmaceutical composition comprising a fluorescent conjugated peptide for visualizing the ureter in a subject, wherein the fluorescent conjugated peptide comprises the amino acid sequence of SEQ ID NO: 21 (QVPWEEPYYVVKKSSGG) conjugated to a fluorescent moiety at its N-terminus; and wherein the visualization is by detection of fluorescence of the oral portion of the peristaltic 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.

31. The pharmaceutical composition of claim 30, wherein the subject is undergoing a surgical procedure.

32. The pharmaceutical composition of claim 30, for reducing injury to the ureter, wherein the subject has been identified as being at risk for ureteral injury prior to the surgical procedure.

33. A pharmaceutical composition according to any one of claims 1 to 32, wherein the subject has iatrogenic ureteral injury.

34. The pharmaceutical composition of claim 32, wherein the ureteral injury results from ligation, angulation, amputation, laceration, crushing, ischemia or resection.

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