Compositions and methods for imaging and treating endometriosis

Abstract

Provided herein are peptides that bind to beta-catenin and compositions comprising the peptides. Agents utilizing peptides that bind directly to beta-catenin can be used for imaging, diagnosis, and therapeutic purposes of diseases driven by beta-catenin dysfunction. Also provided herein are methods of using the peptides and compositions in the imaging, diagnosis, and treatment of tissue infiltrating conditions, including endometriosis.

Description

Cross Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 504,185, filed May 24, 2023, which is hereby incorporated by reference in its entirety. Government Interest Statement

[0002] This invention was made with U.S. Government support under Grant No. HD100289 awarded by the National Institute of Health. The U.S. Government has certain rights in the invention. SEQUENCE LISTING

[0003] This application contains a Sequence Listing submitted in ST.26 format, entitled “170502-2010 Sequence Listing,” created on May 24, 2024, having 566,033 bytes. The content of the Sequence Listing is incorporated herein in its entirety. BACKGROUND

[0004] Endometriosis (EMS) affects approximately 10% of women and adolescents: over 7.4 million women in the United States, and over 176 million women globally. EMS is the number one cause of disability and infertility in women of reproductive age; 40% of infertile women have EMS. The average age at diagnosis is 28 years. Debilitating symptoms include menstrual pain, chronic pain, painful intercourse, and infertility. In addition to human suffering, the annual productivity loss and direct medical costs associated with symptoms in the United States exceed $90 billion. EMS occurs via retrograde menstruation, in which surviving endometrial tissue flows back through the fallopian tubes and into the peritoneal cavity. There, it attaches to multiple foreign sites (e.g., fallopian tubes, ovarian fossae, peritoneal wall, ligaments, and bowel) and responds to hormones (Sampson, J. A., Am J Pathol 3, 93-110 143, 1927). The gold standard for endometriosis diagnosis is laparoscopic visual inspection, preferably with histological confirmation. Since there is no good non-invasive test for endometriosis, diagnosis of this disease is often significantly delayed. Thus, there is a need for non-invasive methods for detecting endometriosis.

[0005] Currently, the only way to diagnose endometriosis is through laparoscopic surgery, with an average delay of 4-12 years from symptom onset to diagnosis. Efforts using scanning techniques are at best experimental. Ultrasound is limited by the field of view and depends on the operator's skill, while experimental MRI for endometriosis exhibits significant variability in protocol (T1-T2) and utilizes gadolinium, which carries toxic risks (especially for women of reproductive age). Due to the high inconsistency of results and low sensitivity in detecting certain common subtypes of endometriosis, these methods are not qualified as a substitute for surgery. Overview

[0006] To begin bridging this significant unmet need in the diagnosis of endometriosis, this article discloses targeted, non-invasive diagnostic agents for endometriosis. Non-invasive diagnostics with broad market access can provide early detection, including for superficial endometriosis, which may be prevalent and cause painful symptoms. For patients, this could eliminate years of suffering, anxiety, and confusion currently experienced before a correct diagnosis, and transform healthcare costs. Early intervention can also lead to better outcomes for women with endometriosis, including fertility preservation.

[0007] Developing specific, targeted diagnostic methods for endometriosis lesions promises to enhance both the reliability and reproducibility of results, minimizing reliance on variables such as equipment and operator expertise. The disclosed targeted diagnostic strategy overcomes current limitations in laparoscopic assessment and contrast agents, providing a more reliable and reproducible means of diagnosing endometriosis.

[0008] The disclosed agent can be used for whole-body scans with readily available conventional MRI machines, including lower Tesla models. This includes compatibility with mobile and open MRI systems, enhanced accessibility and comfort, especially for those with claustrophobia.

[0009] The lack of precision caused by contrast agents such as gadolinium necessitates the use of more powerful Tesla machines or PET / MRI scans. These machines are limited to select imaging centers and can only view a limited number of subtypes of endometriosis. Our technology's potential as a targeted probe lies in its ability to significantly improve lesion detection without requiring the highest resolution equipment. Most available MRI machines are conventional models with lower magnetic field strengths, which are cheaper and ineffective against gadolinium contrast agents. The disclosed agents and methods have the potential to make endometriosis diagnosis more accessible to all populations, including underserved areas, particularly in environments where only low-intensity MRI machines are available. Furthermore, the disclosed solution can be rapidly brought to market.

[0010] The disclosed diagnostic agents can be delivered to all women suspected of having endometriosis or at high risk of this condition, regardless of age, race, or economic status.

[0011] The imaging agent can be used with whole body MRI scans to diagnose all subtypes and stages of endometriosis lesions on any organ system. The goal is to provide patients and clinical providers with a deeper understanding of disease progression and prognosis to enable data-driven care. For example, endometriosis on the bladder can present itself as a painful UTI, while endometriosis on the appendix can present itself as a painful appendicitis. Critically, this MRI approach can also enable detection and characterization of malignancies, providing patients with critical information so that they can receive appropriate care.

[0012] The disclosed targeted imaging agent involves a peptide conjugated to an imaging moiety, such as an iron-based moiety. Since iron / iron oxide has been proven safe, this solution not only shows promise over gadolinium, but also avoids the severe side effects of gadolinium.

[0013] A key feature of effective diagnosis is its retention in the targeted lesion while rapid clearance from the bloodstream, enabling an imaging opportunity window from blood clearance of the imaging agent to breakdown of the imaging agent in the lesion. From our studies, our pharmacokinetic (PK) data in both the lesion and the bloodstream suggest an imaging time range of at least one day. This duration is beneficial because it provides flexibility in scheduling, allowing scans to be performed even in the presence of appointment delays. Other experimental techniques based on contrast technology have shorter imaging windows and can be impractical or difficult for the clinic.

[0014] Currently, the tracking of success for clinical trials for endometriosis therapy is limited to pain, which is highly subjective and non-specific. A non-surgical diagnostic method would be revolutionary and allow for data-driven assessment of the impact of treatment on the extent of disease.

[0015] The disclosed agents will not only serve as efficacy biomarkers, but can also help assess the appropriate treatment duration needed for disease elimination. This approach can revolutionize the development of endometriosis therapies by eliminating the need for a second laparoscopy. Also disclosed are beta-catenin binding agents that can be used to treat endometriosis. In sum, these diagnostic and therapeutic agents can be used to eliminate endometriosis as a long-term condition.

[0016] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 In vivo and ex vivo imaging of lesions is consistent. This is for illustrative purposes, and has also been performed with other compounds, including compound 495.

[0018] Figure 2 Biodistribution of compounds 491 and 492 is shown via the Cy7 labeled version of the peptide (compound 492). Compound 491 has high specificity for the lesion tissue, with little to no detectable amount in other organs, including heart, lung, uterus, ovary, GI organs, and bone. This is for illustrative purposes, and has also been performed with other compounds, including compounds 493-495.

[0019] Figure 3 Vaginal administration of compound 494 is shown, which supports vaginal administration as a viable option for distribution of the agent to the lesion. This has been performed with other labeled peptides, as well as other routes of administration, including but not limited to SQ, IV, and PO dosing.

[0020] Figure 4 Peptides are shown to permeate through vaginal tissue, and vaginal administration of the peptide takes approximately 3 hours to cross human vaginal tissue. This is consistent with imaging via animal models using labeled agents (compound 491 is shown here for illustrative purposes).

[0021] Figure 5 Labeled agents / peptides can be used to determine the elimination route, for the purposes of this figure, the route is fecal for compound 492.

[0022] Figure 6 A spectral imaging time course of a C57B1 / 6 albino mouse treated with compound 494 is shown. This shows that the label can be used in live mice to identify endometriosis lesions after blood clearance (~24h, SQ delivery is shown here), and multiple time points can be scanned to show retention time of the agent in the endometriosis lesion, in this case 24 hours to at least 48 hours post administration. This is for illustrative purposes, and has also been performed with other compounds, including compounds 492-495, and with different routes of administration with different serum pharmacokinetic profiles.

[0023] Figure 7 Tracking of lesion regression within the same mouse after 2 SQ doses administered weeks apart is shown, showing that daily dosing is not necessary for efficacy. This is for illustrative purposes, and has also been performed with other compounds, including compounds 492-495 (492 is shown here).

[0024] Figure 8 Longer treatment times are shown to improve efficacy, especially at lower doses.

[0025] Figure 9 Pharmacokinetic drug exposure analyzed in serum is shown. Pharmacodynamic effects last longer than pharmacokinetic drug exposure analyzed in serum. Figure 6 and Figure 7 Pharmacodynamic effects last longer than pharmacokinetic drug exposure analyzed in serum.

[0026] Figure 10 Cell proliferation assays are shown that confirm that one dose of Compound 491 has a longer pharmacodynamic effect within the lesion than serum pharmacokinetics (showing 5 days post-treatment). It also shows that more frequent dosing is helpful but not necessary for efficacy.

[0027] Figure 11 Compound 492 is shown to bind directly to the target β-catenin within the cell, Compound 492 is cell permeable, and Compound 492 binds to the target within the cytoplasm of the cell, not the nucleus, and is able to image without the cell localizing the peptide quenching signal or imaging agent.

[0028] Figure 12A and Figure 12B Compound 492 is shown to bind to β-catenin at 1 hour of incubation, Compound 492 is in the cytoplasm, and the signal is stable for at least 3 days.

[0029] Figure 13 Downstream effects of the binding described in Figure 12 are shown. Figure 13 Endogenous cytoplasmic β-catenin protein begins to decrease after Compound 492 β-catenin binding occurs in the cytoplasm is shown. The same trend is observed in the nuclear fraction, even though the peptide does not directly interact with the nucleus; rather it sequesters excess cytoplasmic β-catenin and prevents its nuclear translocation and redirection to the membrane.

[0030] Figures 14A to 14C Protein of endogenous β-catenin remains stable in the non-nuclear / non-cytoplasmic (membrane) fraction, and binding of the peptide to cytoplasmic β-catenin does not inhibit β-catenin membrane binding is shown. Figures 14A-14C β-catenin amounts after incubation with 0.5 μΜ Compound 492 in the cytoplasmic fraction ( Figure 14A ), the membrane fraction ( Figure 14B ), or the nuclear fraction ( Figure 14C ) are shown.

[0031] Figure 15Imaging peptides were shown to not be detected in healthy uterine tissue of endometriosis animals, but were found in lesions, confirming specificity to disease. The figure also shows cellular localization of the peptides, after reaching equilibrium, most of the peptides are on the membrane, which hints at Wnt4 activity of the peptides.

[0032] Figure 16 Subcellular localization observed in diseased lesions in Figure 15 confirmed mechanism of action and Figure 15 observations in.

[0033] Figure 17 Reduction in cell proliferation in epithelial cells of endometriosis lesions after administration of compound 491 to animals was shown, confirming mechanism of action and on target activity in.

[0034] Figure 18 Increase in apoptosis in epithelial cells of endometriosis lesions after administration of compound 491 to animals was shown, confirming mechanism of action and on target activity in.

[0035] Figure 19 Activated macrophages were recruited to the lesion site in animals after administration of compound 491 was shown, confirming mechanism of action and on target activity in.

[0036] Figure 20 Lesions extracted from animals treated with compound 491 showed reduction in RNA of beta-catenin downstream target genes was shown, confirming mechanism of action and on target activity in.

[0037] Figure 21 Lesions extracted from animals treated with compound 491 showed reduction in protein of beta-catenin target genes was shown, confirming mechanism of action and on target activity in.

[0038] Figure 22 Imaging of endometriosis lesions in animals was shown, which showed successful targeting and binding of the labeled peptide to the beta-catenin downstream target protein MMP2.

[0039] Figure 23 Beta-catenin peptides (here compound 491 for illustrative purposes) do not alter uterine or ovarian histology was shown. Top: In uterine cross-sections, normal proliferation, glands, stroma, and normal myometrium were observed as expected. Magnification = 100x. Bottom: Ovaries were normal and showed all stages of folliculogenesis from primordial follicles to corpora lutea. Magnification = 400x. Sections were stained with H&E.

[0040] Figure 24A and Figure 24BZeta potential analysis before and after conjugation of the functionalized iron oxide with compound 496 confirmed the successful synthesis of the MRI agent. DETAILED DESCRIPTION

[0041] Unless otherwise defined, all technical and scientific terms and other technical and scientific terms or terminology used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some instances, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to mean that the terms are used inconsistently or inconsistently throughout the art.

[0042] Throughout this application, various embodiments can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as limiting the scope of the disclosure to a range. Therefore, the description of a range should be considered to have specifically disclosed the sub-ranges as well as individual numbers within that range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. DEFINITIONS

[0043] As used in the specification and claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a sample" includes more than one sample, including mixtures thereof.

[0044] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably in the present disclosure to refer to the act of measuring a quantity. These terms can include quantitative, qualitative or both quantitative and qualitative determinations. Optionally, the assessment is relative or absolute. "Detecting the presence of" includes determining the amount of something present, as well as determining whether it is present or not.

[0045] The terms "subject," "individual," or "patient" are often used interchangeably herein. A "subject" can be a biological entity that comprises expressed genetic material. The biological entity can be a plant, an animal, or a microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject can be a tissue, a cell, and their progeny of a biological entity that is obtained in vivo or cultured in vitro. A subject can be a mammal. The mammal can be a human. A subject can be diagnosed or suspected to be at high risk for a disease. The disease can be endometriosis. In some cases, a subject is not necessarily diagnosed or suspected to be at high risk for a disease.

[0046] The term "in vivo" is used to describe an event that occurs in the body of a subject.

[0047] The term "ex vivo" is used to describe an event that occurs outside of the body of a subject. An "ex vivo" assay is not performed on a subject. Rather, it is performed on a sample that is isolated from a subject. An example of an "ex vivo" assay performed on a sample is an "in vitro" assay.

[0048] The term "in vitro" is used to describe an event that occurs in a container used to house laboratory reagents, such that it is separated from the living organism from which the material was obtained. An in vitro assay can encompass a cell-based assay in which living or dead cells are employed. An in vitro assay can also encompass a cell-free assay in which no intact cells are used.

[0049] As used herein, the term "about" a number refers to the number plus or minus 10% of the number. The term "about" a range refers to the range minus 10% of the lowest value and plus 10% of the highest value.

[0050] As used herein, the term "treatment" or "treating" refers to a pharmaceutical or other intervention regimen used in an attempt to obtain a beneficial or desired result in a recipient. The beneficial or desired result can include, but is not limited to, therapeutic benefit and / or prophylactic benefit. A therapeutic benefit can refer to the eradication or amelioration of symptoms of the underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that the improved is observed, even though the subject can still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or a subject reporting one or more of the physiological symptoms of a disease, can undergo treatment even though a diagnosis of this disease can not have been made.

[0051] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0052] As used herein, "peptide mimetic" means a mimic of a peptide that includes some alteration of normal peptide chemistry. Peptide mimetics generally enhance certain properties of the original peptide, such as increased stability, increased efficacy, enhanced delivery, increased half-life, etc. Methods for preparing peptide mimetics based on known polypeptide sequences are described, for example, in U.S. Pat. Nos. 5,631,280; 5,612,895; and 5,579,250. The use of peptide mimetics can involve the incorporation of non-amino acid residues with non-amide linkages at given positions. Some non-limiting examples of non-natural amino acids that can be suitable amino acid mimetics include beta-alanine, L-alpha-aminobutyric acid, L-gamma-aminobutyric acid, L-alpha- aminoisobutyric acid, L-epsilon-amino hexanoic acid, 7-amino heptanoic acid, L- aspartic acid, L-glutamic acid, N-epsilon-Boc-N-alpha-CBZ-L-lysine, N-epsilon-Boc-N-alpha-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-alpha-Boc-N-delta CBZ-L-ornithine, N-delta-Boc-N-alpha-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.

[0053] Abbreviation definitions for many of the terms used in the sequences herein are set forth in Table 1.

[0054]

[0055] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Methods

[0056] Beta-catenin binding peptides that can be used to treat endometriosis are disclosed in WO 2020 / 106995, which is incorporated by reference in its entirety to teach these peptides and methods of making and using them. As disclosed herein, these peptides can also be used for diagnosis due to their specificity for endometriosis.

[0057] In healthy cells, β-catenin binds to E-cadherin at the cell membrane; high concentrations of β-catenin are found at the membrane, and low concentrations are found in the cytoplasm and nucleus. In contrast, in the pathogenesis of endometriosis, β-catenin dissociates from the membrane through receptor misregulation or somatic mutation and accumulates in the cytoplasm. Cytosolic β-catenin is thought to bind to the estrogen receptor (ESR1) and translocate to the nucleus. Nuclear transcription factor activity of β-catenin upregulates matrix metalloproteinases (MMPs), chloride ion 3 channels (ClC-3), and other proteins that can initiate transformation, invasion, migration, and fibrogenesis of endometriosis cells, leading to lesions. Epithelial-to-mesenchymal transition (EMT) involves TGF-β and Wnt81, and this transition, by disrupting the cytoskeleton and cell-cell organization of epithelial cells, here produces highly mobile mesenchymal cells that are critical for the establishment and proliferation of endometriosis lesions. Furthermore, human endometriosis tissue exhibits high expression levels of β-catenin-activated ClC-3, matrix metalloproteinase-9 (MMP9), and SOX proteins. MMP9 supports angiogenesis, thus enabling endometrial cells to grow outside the uterus. Notably, high ClC-3 expression levels correlate with endometriosis symptoms, including infertility, dysmenorrhea, pelvic pain, dyspareunia, and lesion diameter.

[0058] Downstream events of the pathogenesis of endometriosis all occur through β-catenin (e.g., mutated Wnt4 [germline], mutated β-catenin [somatic], and excess estrogen (E2) production [hormone]). GWAS studies revealed a strong association between WNT4 mutations and endometriosis. This is expected given the increased likelihood of the condition in individuals with a family history of endometriosis. Furthermore, WNT4 gene expression in eutopic endometrium was significantly higher in diseased eutopic endometrium. Importantly, wild-type WNT4 protein binds to and transports cytosolic β-catenin to the membrane, thus preventing β-catenin nuclear translocation and misregulation. Mutated or downregulated Wnt4 protein allows cytosolic accumulation, nuclear translocation, and subsequent transcription factor activity of β-catenin to activate IL6, MMP2, MMP7, MMP9, ClC-3, and other β-catenin-activated proteins. Our unique peptide candidates specifically target cytosolic β-catenin and inhibit nuclear translocation of excess cytosolic β-catenin by redirecting it to the membrane, similar to Wnt4 activity.

[0059] Accordingly, cytoplasmic β-catenin specific markers are an attractive approach to target endometriosis. β-catenin is considered an undruggable target due to its protein-protein interactions; however, using a combination of in vitro, in vivo, and in silico tools, suitable cell-permeable cyclic peptide candidates with acceptable ADME profiles were identified for pharmaceutical use. β-catenin cytoplasmic accumulation is observed across all stages of endometriosis, including lesions of atypical endometriosis, typical endometriosis, and cancer caused by endometriosis, making it a prime target for diagnosis and treatment.

[0060] The spectrum of endometriosis can range from small, solitary lesions to large areas of invasive endometrial tissue affecting pelvic organs, including ovaries, fallopian tubes, intestines, and bladder. The American Society for Reproductive Medicine classification system divides endometriosis into four stages based on clinical findings of lesion characteristics (size, location, and superficial lesions or deep infiltrating endometriosis (DIE)) and the extent of pelvic adhesions at the time of surgery. Genetic testing of lesions has confirmed that all classifications and organ locations carry somatic β-catenin mutations or mutations in genes that regulate cytoplasmic β-catenin. Furthermore, endometriosis mesenchymal stem cells promote fibrogenesis in ovarian endometriomas through the Wnt / β-catenin pathway by producing TGF-β1 and Wntl through paracrine. Multiple types of endometriosis lesions (e.g., atypical, DIE, rectovaginal, and ovarian endometrioma subtypes) can carry oncogenic mutations in genes known to upregulate β-catenin, including ARID1A, PIK3CA, KRAS, and PPP2R1A. Altogether, this suggests that cytoplasmic β-catenin-targeted diagnostic agents can address all stages and subtypes of endometriosis.

[0061] Importantly, MRI findings have been successful in identifying malignant tumors. In combination with targeted diagnostic agents, this approach can distinguish cancerous lesions, including those that are cancerous lesions of endometriosis. Early detection of malignant transformation is critical for proper management. Endometriosis-associated ovarian cancer (EAOC) has been successfully detected in MRI findings by: (1) identification of enhanced wall nodules, (2) growth in tumor size—particularly tumors of 9 cm or larger have been used as an independent predictor of ovarian cancer, and (3) absence of T2 shadowing suggests is caused by malignant tumor secretion diluting the hemorrhagic fluid.

[0062] The disclosed agents and methods utilize an innovative approach by using pH-sensitive peptides as agents for targeted endometriosis diagnosis. The specificity for endometriosis tissue is attributed to a novel mechanism driven by the pKa of the weakly acidic peptide, which allows selective permeability in diseased tissue. Furthermore, to our knowledge, our group is the first (and only) to identify a 3-point pH difference between endometriosis and healthy cells.

[0063] Our initial toxicity findings show no observable toxicity associated with the peptides in our library. We attribute this to our unique approach of specifically targeting cytoplasmic β-catenin within endometriosis tissue via Wnt4-like activity. Maintaining membrane adhesion of β-catenin promotes cell health: disruption of β-catenin membrane binding can lead to proto-oncogenic effects, while Wnt4 activity is critical for uterine decidualization, GI tract maintenance, and prevention of anemia. Additionally, our approach is non-hormonal.

[0064] The disclosed peptides have shown to be both effective and exhibit a high safety profile, even at 2000-fold over the effective exposure level. Additionally, the peptide degradation products have little to no toxicity risk (unlike small molecules). Thus, MRI-compatible iron-conjugated peptides are expected to be exceptionally safe, as the iron moiety does not have the toxicity typically associated with gadolinium.

[0065] The use of targeted molecular agents for diagnosing endometriosis significantly enhances precision by specifically binding to diseased cells, superior to traditional methods that rely on nonspecific contrast agent uptake. The disclosed method involves highly specific peptides designed for precision localization accuracy in detecting endometriosis lesions down to sub-millimeter levels. This method is expected to improve reproducibility, reliability, sensitivity, and specificity across all lesion subtypes and organs using standard MRI equipment, thereby enhancing accessibility and reducing scan costs.

[0066] β-catenin binding peptides Disclosed herein are β-catenin binding agents that can be useful in a variety of imaging, diagnostic, and / or therapeutic settings, including, for example, detecting, treating, inhibiting, preventing, monitoring, staging, or reducing endometriosis (EMS) and its associated symptoms.

[0067] In some embodiments, these agents can be useful in imaging, diagnostic, and / or therapeutic settings for other diseases associated with a dysregulated Wnt / β-catenin pathway, including colorectal cancer, hepatocellular carcinoma, endometrial cancer, ovarian cancer, medulloblastoma, glioblastoma, melanoma, and Alzheimer’s disease.

[0068] In some embodiments, the agents can be useful in diagnosing, imaging, and treating other gynecological conditions, including endometriosis, uterine fibroids, adenomyosis, ovarian cysts, polycystic ovary syndrome (PCOS), endosalpingiosis, and cervical, uterine, and vaginal polyps and / or symptoms thereof.

[0069] In some embodiments, the beta-catenin binding agent comprises a peptide. Beta-catenin binding peptides are described in WO 2020 / 106995, which is incorporated by reference in its entirety to teach the peptides and methods of making and uses thereof.

[0070] As disclosed herein, the disclosed beta-catenin binding peptides can be used to selectively target agents to endometriosis lesions. In some embodiments, the peptides target diseased tissue that has a pathogenesis associated with beta-catenin dysregulation. In some embodiments, the agents and peptides are capable of identifying diseased tissue. In some embodiments, the agents and peptides have a higher selectivity for diseased tissue than healthy tissue. In some embodiments, the peptides are not detected in healthy tissue, such as uterine tissue. Without wishing to be bound by theory, the peptides, having a cell-permeable conformation, can be selective for beta-catenin and are intended to be available to the cytoplasm (cytoplasmic beta-catenin) in diseased tissue where there is an excess of the target.

[0071] The disclosed peptides can also be capable of directly visualizing and / or addressing the EMS etiology, reversing disease progression, and preventing or delaying EMS recurrence. In some cases, the beta-catenin binding peptides specifically bind to EMS lesions. In some cases, it can cause existing EMS lesions to shrink. In some cases, the beta-catenin binding peptides can inhibit dysregulation of the Wnt pathway, which is thought to be one of the underlying causes of EMS. The beta-catenin binding peptides can sometimes limit the EMS pathogenesis by preventing endometrial cell invasion. The beta-catenin binding peptides can sometimes limit the EMS pathogenesis by inducing apoptosis. The beta-catenin binding peptides can sometimes induce macrophages that help clear lesions. Alternatively or additionally, the beta-catenin binding peptides can inhibit epithelial to mesenchymal transition (EMT). This inhibition can limit the proliferation of endometriosis lesions. Alternatively or additionally, the beta-catenin binding peptides can sometimes reduce the paracrine production of TGF-betal and Wntl, which in turn can reduce fibrogenesis in ovarian endometriomas. In some cases, the beta-catenin binding peptides reduce disease burden and inflammation caused by the disease. In some cases, downregulating cytoplasmic beta-catenin can reduce the expression of the precursor cytokine IL-6 by inhibiting the complex of beta-catenin with TCF / LEF transcription factors, and thereby reduce the expression of target genes, including but not limited to IL-6.

[0072] In some cases, the beta-catenin binding peptides bind to cytoplasmic beta-catenin and shuttle back to the membrane with Wnt4-like activity. In some cases, the Wnt4-like activity / rescue mechanism can help maintain bone and colon regeneration while downregulating integrity. In some cases, the peptide agents that display Wnt4 activity can help recurrent implantation failures. In some cases, the Wnt4 rescue mechanism can cause downstream cytoplasmic beta-catenin specific canonical Wnt antagonism by redirecting the nuclear localization of beta-catenin to the membrane, which can inhibit or modulate aberrant transcription factor activity while maintaining critical cell-cell connections. These are attractive approaches for imaging, diagnosing, and treating diseases driven by dysregulated beta-catenin, including but not limited to endometriosis, adenomyosis, fibromas, colon cancer, skin cancer, endometrial cancer, and ovarian cancer.

[0073] In addition, the beta-catenin binding peptides can sometimes reduce the expression of downstream proteins, including but not limited to matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 7 (MMP7), SOX (SRY-related high-mobility group box transcription factor), interleukin-6 (IL6), fibronectin 1 (Fn1), or chloride C3 channel (ClC-3). In some embodiments, it can cause reduced angiogenesis. Inhibiting or reducing angiogenesis can inhibit the ability of endometrial cells to grow outside the uterus.

[0074] The peptides described herein can be optimized for affinity for their intended target, absorption or cellular uptake, stability, protease resistance, and other factors. Various methods can be used to alter the affinity of a peptide for its intended target. For example, affinity can be improved by amino acid substitution. Additionally, modifying the size of the peptide, modifying the location of cyclization, increasing hydrophobicity, and modifying or incorporating artificial amino acids into the structure of the peptide can affect the affinity of the peptide. Exemplary modifications include incorporation of N-methyl and alpha-methyl amino acids.

[0075] Among the embodiments provided are peptides, peptidomimetics, and / or beta-catenin binding agents comprising the same or similar structure and properties as the molecules described herein. Such peptidomimetic agents include small protein-like chains designed to mimic peptides. These include peptidomimetic agents comprising peptide modifications, or analogous systems that mimic peptides by design, such as peptoids and beta-peptides.

[0076] Accordingly, disclosed herein are peptides comprising a structure having the formula X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X16 peptide and peptidomimetic agents having an amino acid sequence of Formula R-X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 is C, S, A, Abu, C(me), or S(Bzl); X 11 is F, H, A, K, E, Chg, Cng, or Orn; X 12 is W, Y, A, or F; and X 13 is G, GABA, or absent; X 14 is V, I, L, norvaline, or absent; X 15 is W, Y, F, or absent; and X 16 is R, G, or absent.

[0077] Provided herein are peptide and peptidomimetic agents comprising an amino acid sequence of Formula R-X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 is G, GABA, or absent; X 10 is H or methyl-H; X 11 is V, a-methyl-V, Chg, L, I, or norvaline; X 12 is Q, Aib, S, R, or N; X 13 is R, K, citrulline, Orn, D, or E; X 14 is V, I, L, or norvaline; X15 is W, Y, or F; and X 16 is R, G, or absent.

[0078] In some embodiments, the peptide or peptidomimetic has the amino acid sequence Ac-CW-{NMeA}-Q-K*ILDD*-H-{Chg}-QRVWG-NH2 (SEQ ID NO: 496) or (Ahx)-NH-W-NMeA-QK*ILDD*H-Chg-QRVWG-NH2 (SEQ ID NO: 497).

[0079] Accordingly, disclosed herein are peptides and peptidomimetic agents comprising the amino acid sequence of any one of SEQ ID NO: 1-SEQ ID NO: 499, or a peptide and peptidomimetic agent comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 499.

[0080] In some embodiments, the imaging moiety is an iron oxide nanoparticle. Accordingly, in some embodiments, the imaging agent has the following formula:

[0081] or .

[0082] In some embodiments, the disclosed peptides or peptidomimetics can be covalently conjugated to superparamagnetic iron oxide nanoparticles (SPIONs) (e.g., PEGylated SPIONs) via a linker using the method described in Liu et al. Nanoscale 2012 4:2306, which is incorporated by reference in its entirety for the teachings of this method.

[0083] In some embodiments, the imaging moiety is iron chelated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Accordingly, in some embodiments, the imaging agent has the formula {DOTA-[iron]}-(Ahx)-NH-W-NMeA-QK*ILDD*H-Chg-QRVWG-NH2 (Compound 499, SEQ ID NO: 499).

[0084] In some embodiments, the peptide has a pKa value of about 4.5 to about 7.2, including a pKa value of about 4.5 to about 6, about 5.5 to about 7.0, about 5 to about 7.2, and about 4.5 to about 7.0.

[0085] In some embodiments, the peptide directly binds to β-catenin. In some embodiments, the binding occurs in the cytoplasm. In some embodiments, the peptide is an inhibitor of β-catenin translocation to the nucleus. In some embodiments, the peptide allows β-catenin to bind to the membrane. In some embodiments, the peptide acts like a Wnt4 rescue. In some embodiments, the peptide does not reduce membrane binding of β-catenin. In some embodiments, the peptide prevents β-catenin from acting as a transcription factor for oncogenes, MMP2, MMP7, SOX, IL6, Fn1, or chloride C3 channels (ClC-3). In some embodiments, the peptide prevents transformation, invasion, migration, fibrogenesis, or any combination thereof, of endometriosis (EMS) cells. In some embodiments, the peptide activates apoptosis and macrophages in diseased tissue. In some embodiments, no systemic necrosis occurs. In some embodiments, the peptide does not affect the HPX axis (i.e., the hormonal periodicity). In some embodiments, the peptide acts like a Wnt4 agonist. In some embodiments, the peptide does not reduce β-catenin E-cadherin binding. In some embodiments, the peptide prevents oncogenic transcription factor activity. In some embodiments, the peptide has an EC50 of less than or less than about 50 μΜ, about 30 μΜ, about 10 μΜ, about 5 μΜ, about 1 μΜ, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 50 nM, about 30 nM, about 10 nM, about 5 nM, about 3 nM, about 1 nM, about 800 pM, about 600 pM, about 400 pM, about 200 pM, about 100 pM, about 50 pM, about 30 pM, about 20 pM, about 10 pM, or about 5 pM. 50downregulate Wnt pathway activity. In some embodiments, the peptide is non-naturally occurring. In some embodiments, the peptide is a cyclic peptide. In some embodiments, the peptide is a bicyclic peptide. In some embodiments, the peptide is cyclized with a Cys-Cys disulfide bond. In some embodiments, the peptide is cyclized with an amide bond. In some embodiments, the amide bond is a head-to-tail amide bond between the N-terminus and the C-terminus. In some embodiments, the amide bond is a head-to-side chain amide bond between the N-terminus and an internal COOH. In some embodiments, the amide bond is a side chain-to-tail amide bond between an internal NH2 and the C-terminus. In some embodiments, the amide bond is a side chain-to-side chain amide bond between an internal NH2 and an internal COOH. In some embodiments, the peptide is cyclized using a hydrocarbon staple. In some embodiments, the peptide is cyclized using click chemistry. In some embodiments, the peptide is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, or at least 81 amino acid residues.In some embodiments, the peptide is less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 16, less than 17, less than 18, less than 19, less than 20, less than 21, less than 22, less than 23, less than 24, less than 25, less than 26, less than 27, less than 28, less than 29, less than 30, less than 31, less than 32, less than 33, less than 34, less than 35, less than 36, less than 37, less than 38, less than 39, less than 40, less than 41, less than 42, less than 43, less than 44, less than 45, less than 46, less than 47, less than 48, less than 49, less than 50, less than 51, less than 52, less than 53, less than 54, less than 55, less than 56, less than 57, less than 58, less than 59, less than 60, less than 61, less than 62, less than 63, less than 64, less than 65, less than 66, less than 67, less than 68, less than 69, less than 70, less than 71, less than 72, less than 73, less than 74, less than 75, less than 76, less than 77, less than 78, less than 79, less than 80, or less than 81 amino acid residues. In some embodiments, the peptide comprises one or more non-natural amino acids. In some embodiments, the one or more non-natural amino acids are N-methyl amino acids.

[0086] Exemplary peptide sequences include Compounds 1-499 (SEQ ID NO: 1- SEQ ID NO: 499), as listed in Table 2.

[0087]

[0088] Proteins downstream of β-catenin can also be targeted for endometriosis and other gynecological diseases for both therapy and imaging. A marker peptide targeting MMP2 shows that lesions can be visualized (see, e.g., Figure 13). These agents can also be used for other diseases affected by dysregulation of beta-catenin.

[0089] Peptide synthesis The peptides described herein can be produced by synthetic methods. For example, solid phase peptide synthesis (SPPS) allows rapid assembly of a peptide chain through sequential reactions of amino acid derivatives on an insoluble porous support. The solid support typically comprises small polymeric resin beads functionalized with a reactive group such as an amine or hydroxyl group that is linked to the nascent peptide chain. As the peptide remains covalently attached to the support throughout the synthesis, excess reagents and byproducts can be removed by washing and filtration. Each amino acid coupled to the N-terminus of the peptide chain can be protected at its N-terminus and side chain with appropriate protecting groups such as Boc (acid-labile) or Fmoc (base-labile) depending on the side chain and the protection strategy used (see below).

[0090] The general SPPS procedure is typically one of alternating cycles of N-terminal deprotection and coupling reactions. The resin can be washed between each step. First, an amino acid is typically coupled to the resin. Subsequently, the amine will typically be deprotected and then coupled to the free acid of a second amino acid. This cycle can be repeated until the desired sequence is synthesized. The SPPS cycle can also include a capping step that can block the end of unreacted amino acids from reacting. At the end of the synthesis, the crude peptide can be cleaved from the solid support. This step can typically include the use of a strong acid such as trifluoroacetic acid or a nucleophile to simultaneously remove all protecting groups. The crude peptide can be precipitated from a non-polar solvent such as diethyl ether to remove organic-soluble byproducts. The crude peptide can be purified using reverse phase HPLC. Byproducts can be removed using a continuous chromatography method such as MCSGP to maximize yield without sacrificing purity levels.

[0091] The peptides described herein can be cyclic peptides. In certain cases, cyclic peptides can offer several advantages. For example, cyclic peptides are generally metabolized more slowly due to their higher resistance to proteases than non-cyclic counterparts; on the other hand, they have a longer acting depot effect than their linear counterparts. They can be used to mimic the structure of biologically active peptides (e.g., peptide hormones) and are able to bind drug targets in vivo.

[0092] The peptides described herein can have some flexibility in the loop. Flexible loops allow for different conformations and allow for flexibility in binding to targets that undergo different conformational structures. The peptides are able to penetrate cells and can exert their action on intracellular protein targets (cell permeable peptides). The large surface area of cell permeable peptides gives them an advantage over small molecules in their ability to disrupt specific signaling pathways by inhibiting targeted protein-protein interactions. This occurs when the peptide binds to β-catenin, which allows binding to occur when β-catenin changes its structure from cytoplasmic (stable) to an unstable structure that allows membrane binding. The ability of the flexible peptide to bind to cytoplasmic β-catenin allows the peptide to show activity and efficacy, while the ability of the same β-catenin-bound peptide to enter the cell membrane allows safety during use. Thus, flexible peptides are advantageous for diagnostic and imaging uses, can act as Wnt4 agonists, as they are flexible and allow for safety and efficacy. The peptides have conformational structures that can mimic the molecular structures typically found at protein-protein interaction interfaces.

[0093] The peptides described herein can be cyclized by using disulfide bridges. Peptide disulfide bonds can link two thiol (SH) groups from cysteine or cysteine analog side chains. Unwanted ligation can be prevented by using appropriate protecting group chemistry to effect specific intra- or intermolecular oxidation. Generally, disulfide bridges can be formed as follows: intermolecularly (two peptide molecules linked via a disulfide bridge), resulting in: homodimers (two identical peptides) or heterodimers (two different peptides), or intramolecularly (cyclization within one peptide molecule).

[0094] Cyclic peptides can also be synthesized by linking the amino (N) terminus with the carboxyl (C) terminus of a peptide via an amide bond. The amino side chains of Lys and Orn and the carboxyl side chains of Asp and Glu can also be used to construct cyclic peptides via amide bonds. Depending on the functional groups of the peptide, cyclic peptide synthesis generally uses one of four different methods: head-to-tail amide bond between the N terminus and the C terminus; head-to-side chain amide bond between the N terminus and an internal COOH (e.g., the β-COOH-group of Asp or the γ-COOH group of Glu); side chain-to-tail amide bond between an internal NH2 (e.g., the ε-NH2-group of Lys) and the C terminus; and side chain-to-side chain amide bond between an internal NH2 and an internal COOH (e.g., the ε-NH2-group of Lys and the β-COOH-group of Asp or the γ-COOH-group of Glu).

[0095] The peptides described herein can be cyclized using stapled peptide synthesis. Peptide stapling is typically achieved by incorporation of a, a disubstituted unnatural amino acid bearing terminal olefin pendants of varying length. Subsequent olefin metathesis generates carbon-carbon bond pendants between the amino acid side chains to cyclize the peptide. Alternatively, stapled peptides can be produced using Fmoc solid phase synthesis chemistry generally known in the art.

[0096] The peptides described herein can also be cyclized by using click chemistry. Clickable functional groups can be incorporated into the peptides at the time of synthesis using different combinations of protected amino acids modified with alkyne groups, followed by a click reaction with azido acid. The resulting peptides are cleaved from the resin to give triazole-containing peptides. These functional groups can also be introduced via post-synthetic modification to produce structurally constrained peptides.

[0097] The peptides described herein can have modifications to increase stability. It has been generally shown that N-methyl modifications can increase the function and stability of peptide ligands (Fiacco et al., Chembiochem 2008, 9(14):2200; Fiacco et al., Chembiochem 2016, 17(17): 1643, each of which disclosures are incorporated herein by reference for such disclosure). In some embodiments described herein, the peptides are modified with one or more N-methyl analogs of natural amino acids. Incorporation of one or more N-methyl amino acids can increase proteolytic resistance by 10-fold to 10,000-fold compared to that of a peptide composed of natural residues. In some embodiments, the proteolytic resistance of a peptide comprising one or more N-methyl amino acids is at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000-fold, at least 9000-fold, at least 10,000-fold compared to that of a peptide composed of natural residues.

[0098] Imaging agents and methods Disclosed herein are imaging agents involving one or more of the disclosed beta-catenin binding peptides conjugated to an imaging moiety. Also disclosed are methods of detecting diseased tissue in a subject, the method comprising administering to the subject a disclosed imaging agent and imaging the subject or object to determine the presence of the imaging moiety in the subject.

[0099] In some embodiments, the diseased tissue is an endometriosis lesion. In some embodiments, the diseased tissue is a fibroid. In some embodiments, the diseased tissue is a colon cancer tumor, an ovarian cancer tumor, an endometrial cancer tumor, and a skin cancer tumor.

[0100] Described herein are methods in all living cells and species. In some embodiments, the subject is a human. In some embodiments, the subject is an animal. In some embodiments, the subject is a cultured cell. In some embodiments, the subject is an ex vivo diseased tissue taken from an animal. In some embodiments, the subject is an ex vivo diseased tissue taken from a human.

[0101] In some embodiments, the imaging moiety is intrinsically cell-permeable. In other embodiments, the imaging moiety is not inherently cell-permeable, but becomes permeable when conjugated to a cell-permeable peptide, effectively acting as a cargo for the cell-permeable peptide.

[0102] The disclosed peptides and peptidomimetics can be labeled with an imaging moiety for use as an in vivo imaging probe. In some embodiments, the label is in the N-terminus. In some embodiments, the label is in the C-terminus. In some embodiments, the label is attached to an amino acid side chain. In some embodiments, the label replaces an atom or molecule within the amino acid sequence (i.e., radiolabeling). In some embodiments, the labeling is followed by a further modification. In some embodiments, the modification is a lipid, acetylation, and / or amidation.

[0103] An advantage of using the imaging agents described herein is that the peptides bind to the target in the diseased tissue and thereby aid in the contrast of the signal in the diseased tissue to the background noise of healthy tissue. This means that the agents can help visualize very small lesions (<1 mm, as described in the example in mice) and / or lesions deep in the body without the detection limits of the scanning technology of the imaging agents being much higher or being non-specific. This is crucial for detecting metastatic diseases such as endometriosis or cancer where the size and spread of the diseased tissue in the patient is unknown. Ensuring that all lesions or tumors are identified is crucial for determining the pathology, staging, and recurrence.

[0104] These imaging agents can revolutionize medical imaging, disease identification, and staging. In some embodiments, the technology can be used for initial diagnosis, providing high-resolution, comprehensive imaging to facilitate accurate disease detection. Other embodiments can use the tool for surgical preparation, assisting in detailed planning of procedures by providing deep visualization of disease sites. Furthermore, the diagnosis can serve as a basis for tracking the continued progression of the disease, helping to monitor patient health over time and adjust treatment regimens accordingly. There are embodiments in which the technology can assess the effectiveness of therapies during and after the course of treatment, providing valuable feedback on treatment efficacy. In some embodiments, repeated scans can be performed, which is particularly useful for chronic or long-term conditions that require continuous monitoring. In some embodiments, the imaging agent is a companion diagnostic agent for a therapeutic agent. In some embodiments, the diagnostic tool can be combined with a therapeutic technology (i.e., theranostics), in which the diagnostics and therapeutics are integrated into a single system for a more comprehensive and personalized approach to patient care. In some embodiments, the diagnostic compound can also elicit a therapeutic effect or action. In some embodiments, the peptide imaging agent can also be used to assess the properties of an active pharmaceutical ingredient (API) or drug product, including but not limited to bioavailability, PK, and elimination pathways.

[0105] Some widely used imaging moieties include organic dyes for optical imaging, radionuclides for PET and SPECT imaging, and Gd3+chelators for MR imaging. Functional groups on peptides that can be used for conjugation include, but are not limited to, the epsilon-amino groups on lysine side chains, the guanidino groups on arginine side chains, the carboxyl groups on aspartic or glutamic acids, cysteine thiols, and the phenol on tyrosine. The most common conjugation reactions are carbodiimide / N-hydroxysuccinimidyl (EDC / NHS)-mediated carboxyl and amine coupling, maleimide conjugation to thiol groups, and diazonium modification of the phenol on tyrosine.

[0106] Radiolabeled peptides can also be used as peptide-based imaging agents. Radionuclide-based PET / SPECT imaging can detect imaging agents in the micromolar to picomolar concentration range. Thus, it is possible to minimize the use of peptides to reduce any adverse biological effects. To develop a peptide-based probe for PET / SPECT imaging, a radionuclide should be labeled to the targeting peptide. Several radionuclides for peptide labeling are 99m Tc, 123 I and 111 In and 18 F, 64 Cu and 68Ga. Typically, these radionuclides are attached to the peptide via a chelator or a post-synthetic radiolabeling group. Some widely used chelators include 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), NODAGA (1,4,7-triazacyclononane, 1,5-diglycolic acid-4,7-acetic acid), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetylglycylglycine (MAG3), diaminedithiol (DADT), 2-hydrazinonicotinic acid (HYNIC), N-succinimidyl-4-18F-fluorobenzoate (18FSFB), N-succinimidyl-5-iodo-3-pyridinecarboxylate (SIB), and N-succinimidyl-3-iodobenzoate (SIPC).

[0107] Herein, the compositions of the peptides described in this patent can be labeled with a contrast agent for CT scanning, including but not limited to iron-based, iodine-based, or gold-based contrast agents or other types of imaging moieties. These agents, which contain high atomic number elements such as iodine or gold, exhibit extraordinary X-ray attenuation properties, thereby enabling improved visualization during CT scanning.

[0108] In addition to using a fluorophore instead of a radionuclide, the design of a fluorophore-labeled peptide is similar to a radiolabeled peptide. Various dyes are commercially available (e.g., Cy dyes from GE Healthcare and Alexa Fluor dyes from Invitrogen). While fluorescent imaging has the advantages of high resolution, non-invasive, and safe detection, the use of fluorophores in vivo is limited by light penetration and tissue autofluorescence. The introduction of hybrid derivatives containing both a fluorescent tag and a radiolabel opens new avenues in the field of image-guided surgery.

[0109] In some embodiments, the fluorescent agent is selected from the group consisting of a fluorophore, a fluorescent dye, a dye, a pigment, a fluorescent transition metal, and a fluorescent protein. In some embodiments, the fluorescent agent is selected from the group consisting of Cy5, Cy5.5, Cy2, FITC, TRITC, Cy7, FAM, Cy3, Cy3.5, Texas Red, ROX, HEX, A133, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 633, Alexa Fluor 555, Alexa Fluor 647, DAPI, TMR, R6G, GFP, enhanced GFP, CFP, ECFP, YFP, Citrin, Venus, YPet, CyPet, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine, and Europium.

[0110] Such peptides can include, but are not limited to, SEQ ID NO. 491-SEQ ID NO. 499. Compound 495 variants contain a mini-PEG (polyethylene glycol) linker inserted between the Cy7 labeled cysteine and the subsequent amino acid. This modification can be beneficial for solubility, stability, or bioavailability.

[0111] The peptides described herein can also be optimized into dimeric or multimeric formats to alter receptor targeting capabilities. Similar peptides in dimeric and tetrameric formats have the potential to exhibit higher binding affinities compared to their monomeric counterparts. To optimize these multimeric peptide complexes, different types of linkers can be introduced between the peptide and the imaging moiety. These linkers, which vary in length, flexibility, and hydrophilicity, can significantly impact the overall binding affinity of the peptide. Using this strategy, heterodimers can be constructed using one or more of the peptides described herein that are capable of targeting multiple targets, potentially leading to enhanced imaging results.

[0112] In certain embodiments, the one or more fluorescent agents have excitation and emission wavelengths in the red and near-infrared spectral range. In certain embodiments, the one or more fluorescent agents have excitation and emission wavelengths in the range of 400 nm to 1300 nm, or 440 nm to 1100 nm, or 550 nm to 800 nm, or 600 nm to 900 nm. The use of this portion of the electromagnetic spectrum maximizes tissue penetration and minimizes absorption by physiologically abundant absorbers such as hemoglobin (<650 nm) and water (>1200 nm). In certain embodiments, probe species having excitation and emission wavelengths in other spectra, such as the visible and ultraviolet light spectra, can also be employed. In particular, fluorophores such as certain carbocyanines or polymethine fluorescent dyes or stains can be used as fluorescent agents, e.g., U.S. Patent No. 6,747,159 to Caputo et al. (2004); U.S. Patent No. 6,448,008 to Caputo et al. (2002); U.S. Patent No. 6,136,612 to Della Ciana et al. (2000); U.S. Patent No. 4,981,977 to Southwick et al. (1991); 5,268,486 to Waggoner et al. (1993); U.S. Patent No. 5,569,587 to Waggoner (1996); U.S. Patent No. 5,569,766 to Waggoner et al. (1996); U.S. Patent No. 5,486,616 to Waggoner et al. (1996); U.S. Patent No. 5,627,027 to Waggoner (1997); U.S. Patent No. 5,808,044 to Brush et al. (1998); U.S. Patent No. 5,877,310 to Reddington et al. (1999); U.S. Patent No. 6,002,003 to Shen et al. (1999); U.S. Patent No. 6,004,536 to Leung et al. (1999); U.S. Patent No. 6,008,373 to Waggoner et al. (1999); U.S. Patent No. 6,043,025 to Minden et al. (2000); U.S. Patent No. 6,127,134 to Minden et al. (2000); U.S. Patent No. 6,130,094 to Waggoner et al. (2000); U.S. Patent No. 6,133,445 to Waggoner et al. (2000); U.S. Patent No. 7,445,767 to Licha et al. (2008); U.S. Patent No. 6,534,041 to Licha et al. (2003); U.S. Patent No. 7,547,721 to Miwa et al. (2009);U.S. Patent No. 7,488,468 to Miwa et al. (2009); U.S. Patent No. 7,473,415 to Kawakami et al. (2003); also WO 96 / 17628, EP 0 796 111 B1, EP 1 181 940 B1, EP 0 988 060 B1, WO 98 / 47538, WO 00 / 16810, EP 1113 822 B1, WO 01 / 43781, EP 1 237 583 A1, WO 03 / 074091, EP 1 480 683 B1, WO 06 / 072580, EP 1 833 513 A1, EP 1 679 082 A1, WO 97 / 40104, WO 99 / 51702, WO 01 / 21624, and EP 1 065 250 A1; and Tetrahedron Letters 41, 9185-88 (2000).

[0113] Exemplary fluorescent agents include, for example, the following: Cy5.5, Cy5, Cy7.5, and Cy7 (GE® Healthcare); Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 790, and Alexa Fluor 750 (Invitrogen); VivoTag™ 680, VivoTag™-S680, VivoTag™-S750 (VisEn Medical); Dy677, Dy682, Dy752, and Dy780 (Dyomics®); DyLight® 547 and / or DyLight® 647 (Pierce); HiLyte Fluor™ 647, HiLyte Fluor™ 680, and HiLyte Fluor™ 750 (AnaSpec®); IRDye® 800CW, IRDye® 800RS, and IRDye® 700DX (Li-Cor®); ADS780WS, ADS830WS, and ADS832WS (American Dye Source); XenoLight CF™ 680, XenoLight CF™ 750, XenoLight CF™ 770, and XenoLight DiR (Caliper® Life Sciences); and Kodak® X-SIGHT® 650, Kodak® X-SIGHT 691, Kodak® X-SIGHT 751 (Carestream® Health).

[0114] In some embodiments, the imaging moiety is an MRI agent. In some embodiments, the MRI agent can be Gd (-salt), Ir (-salt), functionalized iron oxide, iron oxide, paramagnetic chemical exchange saturation transfer (CEST) agents,19F active materials, manganese, melanin, or a substance that shortens or lengthens T1 or T2, and combinations thereof. In certain embodiments, the Gd MRI agent can be a compound such as DOTA-Gd, DTPA-Gd, Gd within a polymeric chelator, and Gd immobilized on the layer by negative charge. In certain embodiments, the iron MRI agent can be a compound such as DOTA-Ir, DTPA-Ir, functionalized iron oxide, functionalized iron oxide within a polymeric chelator, and iron immobilized on the layer by negative charge, as described.

[0115] In certain embodiments, the iron oxide MRI agent can be a compound such as small paramagnetic iron oxide (SPIO) or ultrasmall SPIO or superparamagnetic iron oxide nanoparticles (SPION) with or without dextran or other stabilizing layer. Superparamagnetic iron oxide nanoparticles (SPION) are not inherently cell-permeable by themselves. However, their cellular uptake can be improved by surface functionalization with appropriate ligands or peptides that promote internalization by target cells. This can be achieved through various mechanisms such as receptor-mediated endocytosis, macropinocytosis, or direct membrane penetration.

[0116] In some embodiments, functionalization of imaging agents with cell penetrating peptides (CPPs) can enhance their cellular uptake. CPPs are a class of short peptides that can efficiently translocate across the cell membrane and facilitate the internalization of cargo molecules such as nanoparticles, proteins, or nucleic acids. By attaching CPPs to SPIONs or other imaging agents, the cellular permeability of the nanoparticles can be improved, enabling them to enter target cells for various biomedical applications, including but not limited to imaging diagnosis of MRI. In certain embodiments, the paramagnetic CEST MRI agent can be a compound such as lanthanide complexes.

[0117] In some embodiments, the MRI agent can be linked to the layer via a linkage such as a maleimide linkage, NHS ester, click chemistry, or another covalent or non-covalent method or combinations thereof. In some embodiments, the MRI agent can also be loaded without the addition of any exogenous agent, i.e., only the layer or layers and the MRI agent are added.

[0118] In some embodiments, the imaging moiety is an agent for use in PET, which can include but is not limited to 18 F, 64 Cu, 11 C, 13 N, 15 O, 68 Ga, 89 Zr, 82Rb、 14 C 26 Al、 124 I, 76 Br and 52 Fe. In some embodiments, the imaging component is an agent used in SPECT, which may include, but is not limited to, the agent used in SPECT. 99m Tc, 67 Ga、 192 Ir、 111 In、 123 I, 201 Tl、 131 I, 125 I, 133 Xe and 57 Co. In some embodiments, the imaging portion is an alpha-emitting radionuclide, which may include, but is not limited to, At-211, Bi-212, Bi-213, Ra-223, Ac-225, Th-227, and Po-210. In some embodiments, the imaging portion is a beta-emitting radionuclide, which may include, but is not limited to, Cu-67, Y-90, Ag-111, I-131, Pm-149, Sm-153, Ho-166, Lu-177, Re-186, Re-188, Sr89, P-32, and Tl-204.

[0119] In some implementations, the imaging component is an ultrasound contrast agent, such as microbubbles. This can improve the specificity of ultrasound imaging and enable targeted delivery of drugs or other imaging agents, diagnostic agents, or therapeutic agents to endometriosis lesions. Several types of microbubbles exist, including sulfur hexafluoride-filled microbubble contrast agents, perfluorocarbon-filled microbubbles, and nitrogen-filled microbubbles. These agents typically have a shell composed of lipids, proteins, or polymers, and an average diameter of 1–10 micrometers, allowing them to pass through capillaries and circulate in the bloodstream. A commercially available example of a microbubble ultrasound contrast agent is SonoVue® (also known as Lumason® in the United States), which is a sulfur hexafluoride-filled microbubble contrast agent with a phospholipid shell. SonoVue® enhances visualization of vascular and tissue perfusion in a variety of applications, such as echocardiography and characterization of focal liver lesions.

[0120] Microbubble shells can be functionalized with peptides or peptide mimics disclosed herein. This process typically involves conjugating peptides to lipids, proteins, or polymers that form microbubble shells.

[0121] Microbubbles themselves are typically too large to enter cells directly. However, by combining microbubbles with ultrasound energy, cell membrane permeability is enhanced and molecules are facilitated to be transported into cells via sonoporation. By applying ultrasound energy to the target region, microbubbles oscillate or collapse, generating a mechanical force that transiently increases the permeability of the nearby cell membrane. This process, sonoporation, allows the attached molecules (e.g., drugs, genes) to enter the cells. Microbubbles can enhance ultrasound image contrast, allowing real-time visualization and evaluation of the target region by using ultrasound imaging to monitor treatment progress.

[0122] In some embodiments, the provided particles are imaged (or are susceptible to imaging) by an imaging modality selected from the group consisting of MRI, PET, SPECT, CT, X-ray, ultrasound, photoacoustic detection, fluorescence, and / or Raman spectroscopy, and / or combinations thereof.

[0123] In some embodiments, the imaging agent is administered during the scan. In some embodiments, the imaging agent is administered less than 15 min, less than 30 min, less than 60 min, less than 90 min, less than 2 hours, less than 3 hours, less than 4 hours, less than 5 hours, less than 6 hours, less than 7 hours, less than 8 hours, less than 9 hours, less than 10 hours, less than 11 hours, less than 12 hours, less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 2 weeks, less than 3 weeks, or less than 1 month prior to the scan. In some embodiments, the scan can aid in identifying diseased tissue. In some embodiments, the scan can be read as a contrast. In some embodiments, the signal remains in the lesion for a period of time beyond the PK of the drug in the system.

[0124] In some embodiments, the signal can be used to characterize the properties of the API and / or drug product. In some embodiments, the signal can be used to track metabolites. In some embodiments, the signal can be used to determine the release profile at the site of application. In some embodiments, the signal can be used to track the ADME profile of the drug (i.e., bioavailability or root of elimination).

[0125] Linker In some embodiments, the peptide can be conjugated to a linker. The linker moiety can be used to covalently link one or more fluorophores, quenchers, biomodifiers, and non-fluorescent reporters to the enzymatically cleavable oligopeptide or optional biomodifier to produce the agents of the present application. It will be appreciated that the linker, if present, is not subject to particular structural, dimensional, or content limitations. The linker can include, for example, a variety of functional groups that allow assembly of molecules of different structures, such as maleimides, dithiopyridyl groups, thiols, azides, alkenes, or alkynes.

[0126] The linker can be a homofunctional linker or a heterofunctional linker. For example, an amine (NH2)-functionalized moiety can react with a bifunctional crosslinking agent designed to react with an amino group. Particularly useful conjugation reagents that can facilitate the formation of a linker or facilitate the covalent linkage between, for example, a fluorophore and an enzymatically cleavable oligopeptide can include N-hydroxysuccinimide (NHS) esters and / or maleimides. NHS esters can react with amine groups of, for example, a peptide or a fluorophore. Maleimides can react with thiol groups of another molecule. Other particularly useful linker moieties are bifunctional crosslinking agents such as N-succinimidyl 3-(2 pyridyldithio)propionate (SPDP), long-chain SPDP, maleimido benzoic acid-N-hydroxysuccinimide ester (MBS), succinimidyl trans-4-(maleimidomethyl)cyclohexane-l-carboxylate (SMCC), succinimidyl iodoacetate (SIA), and 6-aminohexanoic acid (Ahx).

[0127] In certain embodiments, the linker, if present, can be a derivative of a diamine. Diamine moieties or derivatives can provide linker arms of varying length and chemistry for chemically linking molecules, optionally by derivatization with a carboxylic acid. Non-limiting examples of diamines include ethylenediamine (EDA), propylenediamine, spermidine, spermine, hexanediamine, and diamino acids such as homoarginine, lysine, ornithine, diaminobutyric acid, and diaminopropionic acid. In other embodiments, a moiety of the imaging agent can be chemically linked to a dicarboxylic acid, e.g., succinic acid, glutaric acid, suberic acid, or adipic acid. In one embodiment, the linker is an aminoethylmaleimide.

[0128] In certain embodiments, the linker can be formed from an azide moiety that can react with a substituted alkyne in an azide-alkyne Huisgen [3+2] cycloaddition. In certain embodiments, the azide or alkyne linker can link a polyethylene glycol (PEG) moiety to, for example, an enzymatically cleavable oligopeptide. Other contemplated linkers include propargylglycine, valeroyl, pentynoic acid, propynoic acid, and / or propargylamine moieties.

[0129] In some embodiments, polyethylene glycol (PEG) modification can be used as a linker. PEGylation can enhance peptide solubility, improve handling and formulation. In addition, PEGylation can confer increased stability by preventing enzymatic degradation, thereby prolonging peptide half-life. Cellular uptake can be improved due to evasion of increased endocytosis and efflux transporters, while overall toxicity can be reduced. PEGylation can also enhance water solubility of the peptide while reducing its renal clearance. The pharmacokinetics of the agent can be adjusted based on PEG chain length, allowing for tailored release rates, biodistribution, and elimination kinetics. In some embodiments, direct conjugation linkers can be used, in which the imaging molecule is directly attached to the peptide. Another embodiment includes the use of peptide linkers, composed of short sequences of amino acids, providing biodegradability and flexibility designed with specific properties. In other embodiments, carbohydrate-based linkers can be utilized, which exhibit biocompatibility and ease of synthesis, with the possibility of modification to adjust their properties. Another embodiment can use ester and amide linkers, in which amide linkers provide significant stability and resistance to decomposition in vivo, and ester linkers are engineered to cleave under specific conditions. Further embodiments involve disulfide bond linkers, which are designed to be cleaved by glutathione, a reducing agent found in cells, enabling selective release of the imaging molecule in certain body regions. Heterobifunctional linkers with two different functional groups to allow attachment of two different types of molecules are another embodiment. Enzymatically cleavable linkers, which are designed to cleave in response to specific enzymes, can also be used, providing a mechanism for controlled release of the imaging molecule. These linkers allow for optimization of desired biodistribution and pharmacokinetics, as well as the intended use of the imaging agent. These embodiments provide innovative strategies in the design and application of peptide imaging agents, enhancing the efficacy and adaptability of countless imaging, clinical, and diagnostic applications.

[0130] In certain embodiments, the fluorophore, quencher, or other reporter is directly linked to the imaging agent using a reactive NHS ester group on the fluorophore, quencher, or reporter that reacts with an amine group on the enzymatically cleavable oligopeptide. In certain other embodiments, a carboxylic acid group on the fluorophore, quencher, or other reporter can be activated in situ by activators known in the art, such as 2-(1 Hbenzotriazol-1 -yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1 -ethyl-3-(3'-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-disuccinimidyl carbonate (DSC). In other embodiments, a reporter comprising a thiol or thiol group can be chemically linked to the agent via a bifunctional crosslinking agent having a second moiety that can react with the thiol (thiol) group. Such crosslinking agents include, for example, SPDP, long chain SPDP, SIA, MBS, SMCC, and other crosslinking agents well known in the art, as described above.

[0131] Useful linker moieties include both natural and unnatural amino acids, oligopeptides (e.g., linear or cyclic oligopeptides), and nucleic acids.

[0132] The linker can be a peptide or a peptide moiety, which optionally includes a proteolytic or non-proteolytic cleavage site such as an ester linkage that can be cleaved due to a change in pH at the site of interest.

[0133] Image-guided surgery Disclosed herein are methods of image-guided surgery using the disclosed imaging agents. In some embodiments, multiple technologies are used for image-guided surgery, enabling enhanced diagnosis, precision, and accuracy during surgical procedures. In some embodiments, surgeons can use the disclosed imaging agents to diagnose endometriosis and stage cancer. In some embodiments, surgeons can use the disclosed imaging agents to detect the location of endometriosis lesions or cancerous tumors on an organ system. In some embodiments, surgeons can use the disclosed imaging agents to perform identification and / or staging of disease. In some embodiments, surgeons can use the disclosed imaging agents to aid in resection of lesions or tumors.

[0134] In some embodiments, surgeons can use the disclosed imaging agents to aid in resection of lesions or tumors. In some embodiments, this includes fluorescence-guided surgery (FGS). FGS uses fluorescent probes or dyes that are ingested or injected into the patient and then accumulate in the tumor. When exposed to light of a specific wavelength, these probes or dyes emit fluorescence that can be detected by a specialized camera system and viewed by the surgeon, aiding in visualization of the tumor. Imaging systems for visualizing fluorescence in FGS typically include a light source to excite the fluorescent compound, a camera to capture the emitted fluorescence, and a display monitor for the surgeon to view the image. For example, the “Da Vinci Surgical System” has been upgraded to include a near-infrared fluorescence imaging system for real-time image-guided surgery. The SPY Elite system, Artemis and Artemis Pro (Quest Medical Imaging), and handheld devices such as Fluobeam (Fluoptics), PDE-Neo by Hamamatsu, Visee PDT Imaging System (Visee Medical): for visualization of fluorescence during photodynamic therapy (PDT). In some embodiments, a handheld probe that is part of a surgical microscope system (Leica FL560 (Leica Microsystems)) can be used for fluorescence visualization in areas that can be difficult to reach using a microscope.

[0135] In some embodiments, the disclosed imaging agent can also be visualized using instruments such as the LightPath Imaging System (Lightpoint Medical), with a handheld device designed to detect Cerenkov luminescence emitted by certain types of radiotracers, including carbon-11 (…). 11 C) Nitrogen-13 ( 13 N), Oxygen-15 ( 15 O), Copper-64 ( 64 Cu), Gallium-68 ( 68 Ga), Fluorine-18 ( 18 F).

[0136] This document describes a method for identifying, visualizing, diagnosing, and surgically removing diseased tissue. The method involves applying an imaging agent to a patient, which facilitates visualization of the diseased tissue by the naked eye or with machine assistance. In some embodiments, images of areas of luminescent diseased tissue are captured and acquired prior to surgery. In some embodiments, the process is performed in real time. In some embodiments, the captured images are presented to the surgeon. In some embodiments, the images are still images or a series of still images. In some embodiments, the images are video. The images are then presented to the surgeon during surgery using a reader device, allowing the surgeon to visually identify the luminescent diseased tissue in the patient. The surgeon can directly observe the identified luminescent tissue or with the assistance of a specialized visualization machine. Based on the identified luminescent diseased tissue, the surgeon can perform surgical diagnosis, staging, and / or resection of the diseased tissue accordingly.

[0137] In some embodiments, the agent is visualized using magnetic resonance imaging (MRI). In some embodiments, computed tomography (CT) can be used. In other embodiments, imaging methods such as X-rays and single-photon emission computed tomography (SPECT) can be employed. In other embodiments, ultrasound imaging can be used. In some embodiments, the peptide can be used for optical imaging, which uses light to probe the molecular composition of tissue, thereby facilitating surgery on tumors or targets close to the surface. In some embodiments, positron emission tomography (PET) is used to guide specific surgical procedures. In some embodiments, PET is used in combination with CT.

[0138] In some implementations, the agent is visualized using an MRI machine with a lower magnetic field strength, which is cheaper but ineffective against gadolinium contrast agents.

[0139] In some embodiments, the agent is visualized with 0.064 T portable MRI machines. These machines are designed for point-of-care use, making MRI technology more accessible and affordable. They can be used at the patient’s bedside and are particularly useful in environments where traditional MRI accessibility is limited. While offering less detail due to their low field strength, they provide significant value in specific clinical situations, such as stroke diagnosis in emergency situations.

[0140] In some embodiments, the agent is visualized with low-field MRI machines (0.2 T - 0.4 T). These machines are suitable for larger patients or those with claustrophobia due to their open design. These machines have lower costs but produce less detailed images. They are typically used for extremity imaging, with some capable of whole-body scanning.

[0141] In some embodiments, the agent is visualized with 1.5 T MRI machines. These machines are the standard for clinical imaging, offering a balance between image quality, speed, and cost. They are versatile, suitable for a wide range of diagnostic purposes, including brain, spine, and musculoskeletal imaging.

[0142] In some embodiments, the agent is visualized with 3 T MRI machines. These machines offer higher image quality and detail, making them useful for advanced imaging needs, such as detailed brain, musculoskeletal studies, and high-resolution imaging. While more expensive, they can improve patient throughput with faster imaging times.

[0143] In some embodiments, the agent is visualized with ultra-high field MRI machines (7 T and above). These machines are primarily used for research, with scanners offering extraordinary detail for medical research. Beneficial for imaging of small structures and functional MRI, but not commonly used in standard clinical practice due to their high cost and technical requirements.

[0144] In some embodiments, the agent is visualized with open MRI machines. These machines are designed to reduce claustrophobia and accommodate larger patients, with lower field strength (approximately 0.5 T) resulting in less detailed images than closed-bore machines but offering a more comfortable experience for certain patients.

[0145] In some embodiments, the agent is visualized with front-open MRI machines. These machines are similar to open MRI but are designed for increased comfort and accessibility, particularly for examinations requiring specific body positions. They offer an option for patients with claustrophobia or mobility issues.

[0146] MRI machines can generally use contrast agents to enhance image quality, with the choice of machine depending on imaging needs, patient considerations, and cost effectiveness. 1.5T and 3T machines are most commonly accessible for clinical use outside of specialized imaging centers due to their versatility as well as the balance of image quality, speed, and cost.

[0147] In some embodiments, the agent is visualized by a whole body scan (i.e. not just a pelvic scan) to diagnose all subtypes and stages of endometriosis lesions on any organ system. This can provide a deeper understanding of disease progression and prognosis for both the patient and the clinical provider for data-driven care. For example, endometriosis on the bladder can present itself as a painful UTI, while endometriosis on the appendix can present itself as a painful appendicitis. Critically, this MRI approach can also enable detection and characterization of malignancies, providing critical information to patients so that they can receive appropriate care. For example, but not limited to, the whole body scan can be used to detect endometriosis lesions in the appendix, brain, spine, thyroid, lung, liver, gallbladder, pancreas, spleen, kidney, adrenal gland, bladder, ovary, uterus, intestine, or any combination thereof.

[0148] In one embodiment, the present disclosure discloses the utilization of the imaging agent during endometriosis surgery. In certain embodiments, the agent is introduced into the peritoneal cavity at the same time as the surgical procedure. In alternative embodiments, administration of the agent occurs prior to the surgical intervention, not less than 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks prior to the surgery.

[0149] Further, in additional embodiments, the agent can be administered through alternative routes, encompassing but not limited to intravenous (IV) route, subcutaneous (SC) route, vaginal route, rectal route, oral route, and intramuscular (IM) route. By employing this approach, the surgeon gains the ability to visualize endometriosis lesions with remarkable precision, facilitating their identification in terms of size, location, and disease staging. This visualization can be achieved by the naked eye or by utilizing handheld tools or imaging devices. The surgeon can then proceed with meticulous resection of the endometriosis lesions with this enhanced visual information, while minimizing the risk of unintentionally resecting healthy tissue. This technology represents a significant advancement in endometriosis surgery, enabling improved surgical outcomes and patient care.

[0150] This can be applied to surgeries involving diseases with dysregulation of beta-catenin, including but not limited to colorectal cancer, hepatocellular carcinoma, endometrial cancer, ovarian cancer, medulloblastoma, glioblastoma, melanoma, and Alzheimer’s disease. In some embodiments, these agents can be useful for diagnosing, imaging, and treating gynecological conditions, including endometriosis, uterine fibroids, adenomyosis, ovarian cysts, polycystic ovary syndrome (PCOS), endosalpingiosis, and cervical, uterine, and vaginal polyps and / or symptoms thereof.

[0151] Pharmaceutical compositions Disclosed herein are agents comprising an amino acid sequence of any one of SEQ ID NO: 1- SEQ ID NO: 499 or a peptide and peptidomimetic having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1- SEQ ID NO: 499 in a pharmaceutically acceptable carrier.

[0152] In some embodiments, the agents and / or peptides described herein are formulated into pharmaceutical compositions. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active agent and / or peptide into a preparation for pharmaceutical use. Suitable preparations depend on the chosen route of administration. A summary of pharmaceutical compositions described herein can be found in, for example, Remington: The Science and Practice of Pharmacy, Nineteenth Ed. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H.A and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), the disclosures of which are incorporated herein by reference for such disclosure.

[0153] In some embodiments, the agents and / or peptides described herein are administered alone or in combination with a pharmaceutically acceptable carrier, excipient or diluent in a pharmaceutical composition. Administration of the agents and / or peptides and compositions described herein can be effected by any method that enables delivery of the agents and / or peptides to the site of action. These methods include, though are not limited to, delivery via enteral routes (including oral, gastric or duodenal feeding tubes, rectal suppositories and rectal enemas), parenteral routes (injection or infusion, including intra-arterial, intra-cardiac, intra-dermal, intra-duodenal, intra-medullary, intra-muscular, intra-osseous, intra-peritoneal, intra-thecal, intra-vascular, intra-venous, intra-vitreous, epidural and subcutaneous), inhalation, transdermal, transmucosal, sublingual, buccal and topical (including epicutaneous, dermal, enema, eye drop, ear drop, intranasal, vaginal and intra-uterine) administration, although the most appropriate route can depend, for example, on the condition and disorder of the recipient. By way of example only, the agents and / or peptides described herein can be administered locally to an area in need of treatment by, for example, local infusion during surgery, topical application such as a cream or ointment, injection, catheter, implant or insertion device. Administration can also be by direct injection at the site of diseased tissue or organ.

[0154] In some embodiments, the pharmaceutical composition is in the form of a solution. In some embodiments, the formulation utilizes a buffer such as sodium acetate, histidine buffer. In some embodiments, co-solvents of NMP, DMSO, polypropylene glycol are used. In some embodiments, aggregation inhibitors such as PS80 are used. In some embodiments, sodium oleate, 2-phenoxyethanol, nicotinamide, HPBCD and trehalose are used as excipients. In some embodiments, the formulation is an aqueous suspension. In some embodiments, NaCMC, PS80, PBS and trehalose are utilized as excipients.

[0155] In some embodiments, the pharmaceutical composition is an oily suspension. In some embodiments, the oil is cottonseed oil, corn oil, olive oil, sesame oil and / or castor oil. In some embodiments, the formulation is a water-in-oil or oil-in-water emulsion. In some embodiments, the formulation consists of a mixture of cottonseed oil:soybean oil:olive oil, corn oil and cottonseed oil, sesame oil and cottonseed oil. In some embodiments, emulsifiers are used (including but not limited to PS80, PS20, Kolliphor EL, lecithin or some combination). In some embodiments, NMP, ethanol, propylene glycol and DMSO can be used as solvents.

[0156] In some embodiments, the pharmaceutical compositions suitable for oral administration are presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. In some embodiments, the active ingredient is presented as a bolus, electuary or paste.

[0157] Pharmaceutical compositions that can be used orally include tablets, push-fit capsules, and gelatin capsules. Tablets can be compressed or molded, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, inert diluent, or lubricating, surface- active, or dispersing agent. Molded tablets can be made by molding, in a suitable machine, a mixture of the powdered peptide and / or active ingredient and inert liquid diluent, e.g. moistened by a solution of a mixture of compounds of the present application. In some embodiments, tablets are coated or scored and are formulated to provide slow or controlled release of the active ingredient therein. All formulations for oral administration should provide at least 0.1 mg per dose. Push-fit capsules can contain the active ingredients in admixture with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredient and / or peptide can be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In some embodiments, stabilizers are added. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings, e.g., for identification or to characterize different combinations of active ingredient and / or peptide doses.

[0158] In some embodiments, the pharmaceutical compositions are formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, including added preservatives. Compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Compositions can be provided in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.

[0159] Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous sterile injection solutions of the active agent and / or peptide which can contain anti-oxidants, buffers, bactericides and solutes which render the formulation isotonic to the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension also can contain suitable stabilizers or agents which increase the solubility of the active agent and / or peptide to allow for the preparation of highly concentrated solutions.

[0160] Pharmaceutical compositions also can be formulated as depot preparations. Such long-acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the agent and / or peptide can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0161] For buccal or sublingual administration, the compositions can take the form of tablets, lozenges, hard or soft gelules or gel. Such compositions can comprise the active ingredient in a flavored basis such as sucrose and acacia or tragacanth.

[0162] Pharmaceutical compositions can also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter, polyethylene glycol, or other glycerides. When in the form of suppositories, the suppository base will melt and / or become water miscible at body temperature to allow the components to become in contact with the mucosa for a sufficient period of time to have an imaging, diagnostic, and / or therapeutic effect. The weight percent of the suppository base depends on the size of the body orifice of the human and / or animal, the dosage composition required to have an imaging, diagnostic, and / or therapeutic effect, and its physicochemical properties that allow it to remain solid at room temperature or below. In some embodiments, the suppository comprises about 50% to greater than 99% or about 75% to greater than 99% by weight of the suppository base. In some embodiments, the suppository comprises about 75% to about 98% by weight of polyethylene glycol. In some embodiments, the suppository comprises about 2% to about 25% by weight of polysorbate. The suppository base has a molecular weight in the range of about 400 to about 5000 or about 950 to about 3700 (US 2009 / 0311290, which is incorporated herein by reference for such disclosure).

[0163] In some cases, the pharmaceutical composition comprises an absorption enhancer, such as sodium caprate. Some such cases include compositions and dosage forms for use in the rectum or vagina as described herein.

[0164] The pharmaceutical composition can be administered topically or rectally, i.e., by non-systemic administration. This includes external application to the skin or mouth, and instillation into the rectum or vagina, such that the agent and / or peptide does not enter the bloodstream significantly. In contrast, systemic administration refers to oral, intravenous, intraperitoneal, and intramuscular administration.

[0165] Pharmaceutical compositions suitable for topical administration include liquid or semi-liquid preparations suitable for penetration of the skin, such as solutions, lotions, shake lotions, creams, ointments, gels, foams, transdermal patches, powders, solids, sponges, tapes, vapors, pastes, tinctures, microparticles, microcapsules, nanoparticles, liposomes, or emulsions, including those suitable for delivery to the vagina or rectum. For topical administration, the active ingredient can comprise 0.001% w / w to 10% w / w, e.g., 1% w / w to 2% w / w of the formulation by weight.

[0166] Pharmaceutical compositions for administration by inhalation are conveniently delivered from an insufflator, nebulizer, pressurized pack or other convenient means of aerosol spray dispenser. Pressurized packs can comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized spray, the dosage unit can be determined by providing a valve to deliver a metered amount. Alternatively, for administration by inhalation or insufflation, the pharmaceutical preparation can take the form of a dry powder composition, for example a powder mix of the agent and / or peptide and a suitable powder base such as lactose or starch. The powder composition can be presented in unit dosage form, for example in capsules, cartridges, gelatin or blister packs, from which the powder can be administered with a device such as a inhaler or insufflator.

[0167] In some embodiments, the agents disclosed herein are formulated in such a way as to achieve delivery of the agent and / or peptide to a specific region of the gastrointestinal tract. For example, the agents and / or peptides disclosed herein are formulated with bioadhesive polymers, pH sensitive coatings, time dependent, biodegradable polymers, microflora-activated systems, and the like for oral delivery to achieve delivery of the agent and / or peptide to a specific region of the gastrointestinal tract.

[0168] In some embodiments, the agents and / or peptides disclosed herein are formulated in such a way as to achieve delivery of the agent and / or peptide to a specific region of the urogenital or anorectal mucosa. For example, the agents and / or peptides disclosed herein are formulated with bioadhesive polymers, pH sensitive coatings, time dependent, biodegradable polymers, microflora-activated systems, and the like for intravaginal delivery to achieve delivery of the agent and / or peptide to a specific region of the urogenital system. In some embodiments, the agents and / or peptides disclosed herein are formulated to provide controlled release of the agent and / or peptide. Controlled release refers to the release of the agents and / or peptides described herein from the dosage form in which they are incorporated according to a desired profile over an extended period of time. Controlled release profiles include, for example, sustained release, extended release, pulsatile release, and delayed release profiles. Controlled release compositions allow for the delivery of the agent to a subject according to a predetermined profile over an extended period of time as compared to immediate release compositions. Such release rates can provide imaging and / or therapeutically effective levels of the agent over an extended period of time and thereby provide a pharmacological response for a longer period of time while minimizing side effects as compared to conventional fast release dosage forms. Such longer periods of response provide inherent benefits that are not achievable with corresponding short-acting immediate release products.

[0169] Methods of delivering intact therapeutics, imaging agents, and / or diagnostic agents to specific regions of the genitourinary system (such as the vagina) or the gastrointestinal tract (e.g., such as the colon) include: (i) coating with a polymer: intact molecules can be delivered to the colon without absorption in the upper intestine by coating the drug molecules with a suitable polymer that degrades only in the colon. Additionally, coating with a polymer can provide protection or a controlled release profile for vaginal formulations; (ii) coating with a pH sensitive polymer: enteric, colonic, and vaginal targeting delivery systems can be based on the coating of tablets or pellets filled into conventional hard gelatin capsules. The most commonly used pH dependent coating polymers are methacrylic acid copolymers, commonly known as Eudragit® S, more specifically Eudragit® L and Eudragit® S. Eudragit® L100 and S 100 are copolymers of methacrylic acid and methyl methacrylate; (iii) coating with a biodegradable polymer; (iv) embedding in a matrix; (v) embedding in a biodegradable matrix and hydrogel; (vi) embedding in a pH sensitive matrix; (vii) time release systems; (viii) redox sensitive polymers; (ix) bioadhesive systems; (x) coating with microparticles; and (xi) osmotic controlled drug delivery.

[0170] Another method for vaginal and colonic targeted drug delivery or controlled release systems includes embedding the drug in a polymeric matrix to trap it and release it in the vagina or colon. These matrices can be pH sensitive or biodegradable. Matrix-based systems, such as multi-matrix (MMX)-based delayed-release tablets, ensure the release of the drug in the vagina or colon.

[0171] The compositions and methods described herein can include delivery of therapeutic, imaging, and / or diagnostic agents via a variety of dosage forms and devices, including those described above and below. Many of the dosage forms described below provide several advantages. These can include local delivery of therapeutic, imaging, and / or diagnostic agents and / or peptides. Local delivery can reduce side effects that can sometimes be associated with systemic delivery and off-target effects. In some cases, the dosage forms and devices described herein can increase patient compliance, including, for example, by providing long-term or continuous delivery of the agents and / or peptides described herein. In some cases, these dosage forms and devices can also increase tolerability and safety.

[0172] The peptides and compositions described herein can be incorporated into intravaginal rings for delivery. Delivery of therapeutic, imaging, and / or diagnostic agents via intravaginal rings (IVRs) allows for local delivery, increasing the safety and tolerability of the therapeutic, imaging, and / or diagnostic agents. Additionally, IVRs can provide a variety of advantages: bypassing gastrointestinal absorption and liver / kidney first pass metabolism, lower effective dose, continuous delivery and / or controlled release profile, extended time between doses, reduced side effects, low serum drug concentration, patient self-administration, and improved patient satisfaction. IVRs can successfully deliver drugs, including hydrophilic agents and macromolecular agents, to organs affected by EMS, including deep infiltrating EMS (DIE). The biodistribution of the intravaginal ring delivery system is via tissue absorption rather than serum, and can reach all organs affected by EMS in the peritoneal cavity.

[0173] Intravaginal rings are generally composed of an inert elastomer ring coated with another layer of elastomer containing the drug to be delivered. The ring can be easily inserted, left in place for the desired period of time, and then removed by the user. The ring can be solid or hollow, containing the therapeutic, imaging, and / or diagnostic components, or it can be a porous material from which the drug is released. The ring can optionally include a third, outer, rate controlling elastomer layer that is free of drug. Optionally, the third ring can contain a second drug for a dual release ring. The drug can be incorporated throughout the silicone elastomer ring in polyethylene glycol to act as a reservoir for the drug to be delivered. In some cases, the IVR can include a silicone resin, compressed tablet, or lyophilized gel.

[0174] Vaginal pessaries, cups, strips, tablets, and suppositories are other examples of drug delivery systems that can be used in the present disclosure. These systems have been used to deliver vaginal contraceptives and have been extensively described in the literature.

[0175] Another example of a delivery system is a vaginal sponge and foam. As described in the literature, the desired pharmaceutical agent can be incorporated into a silicone matrix coated on a cylindrical, drug-free polyurethane vaginal sponge.

[0176] In some embodiments, the IVR comprises a peptide or composition as described herein that is formulated as a suppository, a solution, a lotion, a shake lotion, a cream, an ointment, a gel, a foam, a transdermal patch, a powder, a solid, a sponge, a tape, a paste, a tincture, an emulsion, a microparticle, a microcapsule, a nanoparticle, a liposome, or a capsule comprising a microparticle, a microcapsule, a nanoparticle, or a liposome. IVRs have been further modified to include a variety of delivery vehicles, such as silicone inserts, compressed tablets, or lyophilized gels, to optimize the release profile of hydrophilic agents or high molecular weight agents such as peptides, proteins, or antibodies (Morrow, et al, Eur J Pharm Biopharm,2011 January; 77(1):3-10, which is incorporated herein by reference for such disclosure). The IVR can also contain one or more absorption enhancers, such as sodium caprate.

[0177] In some embodiments, the IVRs described herein are formulated to contain about 0.01 mg to about 5000 mg of the agent and / or peptide. In some embodiments, the IVR can contain about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 150 mg, about 200 mg, about 400 mg, about 600 mg, about 800 mg, about 1000 mg, about 1200 mg, about 1400 mg, about 1600 mg, about 1800 mg, about 2000 mg, about 2500 mg, about 3000 mg, about 3500 mg, about 4000 mg, about 4500 mg, or about 5000 mg of the agent and / or peptide.

[0178] In some embodiments, the IVRs described herein are formulated to deliver about 0.01 mg to about 1000 mg of the agent and / or peptide per day. In some embodiments, the IVR is formulated to deliver about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.5 mg, about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 150 mg, about 200 mg, about 400 mg, about 600 mg, about 800 mg, or about 1000 mg of the agent and / or peptide per day.

[0179] The peptides and compositions described herein can be incorporated into a tampon device for delivery. The tampon device generally comprises a vaginal tampon having a proximal end and a distal end. A cup-shaped porous foam portion at the distal end fits around the cervix and contains a peptide or composition for delivery as described herein. The device can also include a non-absorbent axial tube with a distal opening and extending through the porous foam cup into the tampon for conducting blood flow to the absorbent material. Optionally, a retrieval string or tape connected to the tampon device is also included. The absorbent vaginal tampon contains a peptide or composition as described herein, or can be coated with a peptide or composition as described herein and used as a medicated tampon for delivery.

[0180] The peptides and compositions described herein can be incorporated into a solid for local delivery. The solid can be in the form of a vaginal suppository or a vaginal or rectal suppository. The solid dosage form can melt upon reaching body temperature. Alternatively, the solid can retain its structure and release the incorporated compositions as described herein. The solid can be a pessary designed to provide a support within the vagina. A vaginal sponge can be embedded with the compositions described herein for intravaginal delivery.

[0181] The peptides and compositions described herein can be incorporated into a topical formulation for delivery. A topical medicament is applied to a body surface, such as the skin or mucosa. In some cases, the body surface includes, but is not limited to, epithelial tissue, mucosal tissue, peritoneum, perimetrium, and endometrium. The agent and / or peptide is absorbed through the body surface to achieve local or systemic effects. The topical medicament is optionally formulated in the following categories: topical solution; lotion; shake lotion; cream; ointment; gel; foam; transdermal patch; powder; solid; sponge; tape; vapor; paste; or tincture. The topical solution can be applied as an irrigant, spray, or drop, typically having a low viscosity, containing water or alcohol in the base. The lotion can be thicker and more lubricating than a solution. It is usually an oil mixed in water, and can have less alcohol than a solution. The shake lotion is a mixture that is divided into two or three parts over time. It can be an oil mixed with a water-based solution that needs to be shaken into suspension before use. The cream is an emulsion of oil and water in about equal proportions. The ointment is a uniform, viscous, semisolid preparation, most commonly a greasy, viscous, oily substance (oil 80%-water 20%) with a high viscosity. The ointment can include a hydrocarbon base, an absorbent base, a water-soluble base, an emulsifying base, or a vegetable oil, or any combination thereof. The ointment is formulated in a base that can include, but is not limited to, a hydrocarbon base such as hard paraffin, soft paraffin, microcrystalline wax, or petrolatum; an absorbent base such as lanolin or beeswax; a water-soluble base such as macrogols 200, 300, or 400; an emulsifying base such as emulsifying wax or bromo- tri-methylammonium; or a vegetable oil such as olive oil, coconut oil, sesame oil, almond oil, or peanut oil.

[0182] The gel is a semisolid emulsion. Non-limiting examples of useful emulsifiers include acrylic polymers (such as Carbopol brand thickening agents, e.g., Carbopol 934P, manufactured by Voveon, Inc.), polyoxyethylene-10-stearyl ether, polyoxyethylene-20-stearyl ether, cetostearyl alcohol, cetyl alcohol, cholesterol, diethylene glycol stearate, glycerol monostearate, glyceryl stearate, polyglyceryl-3-oleate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lanolin, polyoxyethylene lauryl ether, methylcellulose, polyoxyethylene stearate, polysorbate, propylene glycerol monostearate, sorbitan esters, stearic acid, or a mixture of two or more thereof.

[0183] The amount of emulsifier in the topical gel formulation can range from about 1 wt% to about 40 wt%, and in some embodiments, from about 5 wt% to about 30 wt%, based on the total weight of the topical formulation.

[0184] The gel formulations as described herein can include one or more gelling agents. Non-limiting examples of useful gelling agents include carboxylic acid polymers, including acrylic acid polymers crosslinked with a crosslinking agent, such as the Carbopol brand thickening agents, cetyl stearyl alcohol, hydroxymethylcellulose, polyoxyethylene polyoxypropylene copolymer, sodium carboxymethylcellulose, polyvinylpyrrolidone, or a mixture of two or more thereof.

[0185] The amount of gelling agent in the topical gel formulation can range from about 0.1 wt% to about 10 wt%, and in some embodiments, from about 0.1 wt% to about 1 wt%, based on the total weight of the topical gel formulation.

[0186] The gel formulations described herein can also include one or more basifying agents, such as sodium hydroxide, as a gelling activator, in an amount less than about 2 wt%.

[0187] The formulations can include one or more additional excipients well known in the art, such as water and thickening agents such as colloidal silicon dioxide.

[0188] Thermoreversible gels are liquid formulations that become gels after insertion into the rectum or vagina. Thermoreversible gels allow for easier administration and positioning than conventional suppositories or pessaries, and can prevent leakage of the dosage form. It is formulated as a solution of polymers consisting of thermoreversible polymers (e.g., poloxamers, in combination with a mucoadhesive polymer capable of gelling attachment to the mucosa). The in situ thermoreversible liquid gel formulation, also known as a thermoreversible “liquid suppository,” is a liquid at low temperature (<10C) and becomes a gel at body temperature.

[0189] Active agents can be incorporated into surgical tape for occlusive dressings. Medications can be applied as an ointment or gel, reaching the mucosa by evaporation. A paste combines oil, water, and powder. Tinctures usually contain a high percentage of alcohol for application to the skin.

[0190] The peptides and compositions described herein can be incorporated into transdermal patches for delivery. Transdermal patches provide controlled release of a drug through a porous membrane covering a drug reservoir or by body heat melting a thin layer of drug embedded in the patch adhesive.

[0191] The compositions described herein can be incorporated into a film for delivery. A film is a thin, small polymeric formulation that can be easily inserted into the vaginal canal without the need for an applicator and does not cause discomfort. Vaginal films are easier to apply than other types of vaginal formulations such as pessaries, foams, and gels (Rohan L C et al., AAPS Journal 2009; 11 :78-87, which is incorporated herein by reference for such disclosure).

[0192] The peptides and compositions described herein can be incorporated into an intrauterine device (more commonly a small T-shaped device) that is inserted into the uterus for delivery. IUDs typically contain copper, progestin, or levonorgestrel. The compositions as described herein are incorporated into the IUD device for slow release over time.

[0193] The peptides and compositions as described herein can be formulated for systemic delivery. Parenteral injections can be formulated for bolus injection or continuous infusion. Pharmaceutical compositions can be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the peptides described herein in water-soluble form. Suspensions of the peptides described herein can be prepared as oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Suspensions can also contain suitable stabilizers or agents that increase the solubility and / or decrease the aggregation of such peptides described herein to allow preparation of highly concentrated solutions. Alternatively, the peptides described herein can be lyophilized or in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In some embodiments, the purified peptides are administered intravenously.

[0194] The peptides and compositions described herein can be formulated for oral delivery. Pharmaceutical preparations that can be used orally include tablets, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Tablets can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, inert diluent, or lubricating, surface- active, or dispersing agent. Molded tablets can be made by molding, in a suitable machine, a mixture of the powdered peptide and / or active ingredient and inert liquid diluent, after which the mixture can harden. In some embodiments, the tablets are coated or scored and prepared with coatings and shells, such as enteric coatings and other coatings. Formulations for oral administration can be dosed in a manner suitable for such administration. Push-fit capsules can contain the active ingredients in admixture with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active agent and / or peptide can be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In some embodiments, stabilizers are added. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to tablets or to dragee coatings, for example, to identify or to characterize different combinations of active agent and / or peptide doses.

[0195] Additional pharmaceutical approaches to targeted delivery of therapeutic agents to specific regions of the gastrointestinal tract are known. Chourasia MK, Jain S K, Pharmaceutical approaches to colon targeted drug delivery systems., J Pharm Sci. 2003 January-April; 6(1):33-66. Patel M, Shah T, Amin A. Therapeutic opportunities in colon-specific drug-delivery systems Crit Rev Ther Drug Carrier Syst. 2007; 24(2): 147-202. Kumar P, Mishra B. Colon targeted drug delivery systems—an overview. Curr Drug Deliv. 2008 July; 5(3): 186-98. Van den Mooter G. Colon drug delivery. Expert Opin Drug Deliv. 2006 January; 3(1): 111-25. Seth Amidon, Jack E. Brown, and Vivek S. Dave, Colon-Targeted Oral Drug Delivery Systems: Design Trends and Approaches, AAPS Pharm SciTech. 2015 August; 16(4): 731-741. Each of these references is incorporated herein by reference for such disclosure.

[0196] It will be appreciated that, in addition to the ingredients especially mentioned above, the agents and / or peptides and compositions described herein can include other agents conventional in the art taking into account the type of formulation in question. For example, agents suitable for oral administration can include flavoring agents.

[0197] The peptides and compositions described herein can include additional excipients to improve the performance of the imaging, diagnostic, and / or therapeutic agents. The compositions can include absorption enhancers or penetration enhancers to improve absorption and membrane penetration across the epidermis or mucosal surfaces. Enhancers formulated in the compositions described herein can include, but are not limited to, sulfoxides such as dimethyl sulfoxide (DMSO); laurocapram (1-dodecylazacycloheptan-2-one); pyrrolidones such as n-methyl-2-pyrrolidone; terpenes and terpenoids; essential oils; oxazolidinones such as 4-decyl-oxazolidin-2-one; urea; cyclopentadecalactone; N-[8-(2-hydroxybenzoyl)amino]octanoic acid sodium salt (SNAC); 8-(N-2-hydroxy-5-chloro-benzoyl)-amino-octanoic acid (5-CNAC); medium-chain fatty acids, salts, and derivatives; sodium decanoate; sodium octanoate; protease inhibitors and omega-3 fatty acids; liquid mixed micellar sprays; lipid-polymer micelles; alkylglycosides; chitosan; dodecyl-2-N,N-dimethylaminopropionate (DDAIP); cellular membrane lipid components; nanoparticles; liposomes, ligands; and lipophilic modifications.

[0198] The peptides and compositions described herein can be formulated as solutions, lotions, shake lotions, creams, ointments, gels, foams, mucoadhesive compositions, emulsions, liposomes, coatings, cores, matrices, lyophilizates.

[0199] Methods of administration In some embodiments, the agents and / or peptide compositions described herein are used in the preparation of a medicament for imaging, diagnosis, and / or treatment of a disease, condition, or symptom in a mammal that would benefit from administration of a beta-catenin agent and / or peptide. Methods for treating any of the diseases or conditions described herein in a mammal in need of such treatment include administering to the mammal an imaging and / or therapeutically effective amount of a pharmaceutical composition comprising at least one agent and / or peptide described herein.

[0200] Diseases, conditions, or symptoms that can benefit from treatment with a composition as described herein include, but are not limited to, endometriosis, endometriosis lesions, endometrioma, superficial endometriosis implants, deep infiltrating endometriosis, chronic pain, central sensitization, myofascial pain, adnexal masses, infertility, dysmenorrhea, genetic predisposition, non-menstrual pelvic-abdominal pain, dyspareunia, bowel symptoms (diarrhea, cramping, constipation), defecation pain (dyschezia), ovarian masses or tumors, bladder pain symptoms, and dysuria.

[0201] In certain embodiments, compositions comprising one or more of the agents and / or peptides described herein are administered for prophylactic and / or therapeutic treatment. In certain therapeutic, imaging, and diagnostic applications, compositions are administered to a patient already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of at least one symptom of the disease or condition. The effective amount for this use depends on the severity and course of the disease or condition, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating physician. Imaging and therapeutically effective amounts are optionally determined by methods including, but not limited to, dose escalation and / or dose ranging clinical trials.

[0202] In prophylactic applications, compositions comprising the agents and / or peptides described herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition. Such an amount is defined to be a "prophylactically effective amount or dose." In this use, the precise amounts also depend on the health status, weight, etc., of the patient. When used in patients, effective amounts for such use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status, and response to the drugs, and the judgment of the treating physician. In one aspect, prophylactic treatment includes administration of a pharmaceutical composition comprising the agents and / or peptides described herein to a mammal previously experiencing at least one symptom of the treated disease and is now in remission, to prevent recurrence of symptoms of the disease or condition.

[0203] In certain embodiments where the patient's status does not improve, the agents and / or peptides are administered chronically, i.e., for extended periods of time (including for the duration of the patient's life), according to the judgment of the physician, to ameliorate or otherwise control or limit the symptoms of the patient's disease or condition.

[0204] In certain embodiments where the patient's status does improve, the dosage of the administered drug is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). In particular embodiments, the length of the drug holiday is between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or more than 28 days. The reduction in dosage during the drug holiday is, by way of example only, from 10% to 100%, including by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

[0205] Once the patient's condition improves, a maintenance dose is administered as needed. Subsequently, in certain implementations, the dose or frequency of administration is reduced, or both are reduced, depending on the symptoms, to maintain the level of disease, disorder, or condition improvement. However, in some implementations, the patient requires long-term intermittent treatment following any recurrence of symptoms.

[0206] The amount of a given agent corresponding to such a quantity varies depending on factors such as the specific agent and / or peptide, the disease condition and its severity, and the identity of the subject or host requiring treatment (e.g., weight, sex), but is still determined based on the specific circumstances surrounding the case, including, for example, the specific agent administered, the route of administration, the condition of treatment, and the subject or host being treated.

[0207] Typically, the dosage for treatment in adults ranges from about 0.01 mg to about 5000 mg per day. In some aspects, the dosage for treatment in adults is from about 0.01 mg to about 1000 mg per day. In some embodiments, the desired dosage is conveniently presented as a single dose or as fractions administered simultaneously or at appropriate intervals, for example as two, three, four or more sub-dose per day. In other embodiments, the compositions described herein are formulated to extend release over a period of time, such as hours, days or months. In some embodiments, the composition is formulated for delivery over the following durations: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, or 12 years.

[0208] In some embodiments, the daily dose of the agent and / or peptide described herein is from about 0.01 mg / kg / body weight to about 50 mg / kg / body weight. In some embodiments, the amount of active substance in the daily dose or dosage form is below or above the range indicated herein, based on numerous variables relating to individual treatment regimens. In many embodiments, the daily dose and unit dose vary depending on numerous variables, including but not limited to the activity of the agent and / or peptide used, the disease or condition to be treated, the route of administration, the individual subject's needs, the severity of the disease or condition being treated, and the practitioner's judgment.

[0209] The toxicity and efficacy of such imaging and treatment protocols are assessed through standard pharmaceutical procedures in cell cultures or laboratory animals, including but not limited to LD50. 50 IC 50 EC 50 and ED 50 The therapeutic effect is determined by the dose ratio between toxicity and therapeutic effect, which is called the therapeutic index, and the therapeutic index is expressed as LD50. 50 and ED 50 The ratio between. In some embodiments, data obtained from cell culture assays and animal studies are used to formulate therapeutically effective daily dose ranges and / or therapeutically effective unit doses for use in mammals (including humans). In some embodiments, the daily dose of the agents and / or peptides described herein is within the range of ED (expiratory power reserve) with minimal toxicity. 50 Within the cyclic concentration range. In some embodiments, the daily dose range and / or the amount of unit dose varies within this range, depending on the dosage form and route of administration used.

[0210] Any of the foregoing aspects also have additional embodiments, wherein the effective amount of the agent and / or peptide described herein is: (a) administered systemically to a mammal; and / or (b) administered orally to a mammal; and / or (c) administered intravenously to a mammal; and / or (d) administered to a mammal by injection; and / or (e) administered superficially to a mammal; and / or (f) administered non-systemically or locally to a mammal.

[0211] Any of the foregoing aspects also includes additional embodiments comprising a single-dose effective amount of the agent and / or peptide, including wherein (i) the agent and / or peptide is administered once daily; or (ii) the agent and / or peptide is administered multiple times to a mammal over a daily span. Additional aspects also include embodiments wherein the agent and / or peptide is administered continuously over a period of time.

[0212] Any of the foregoing aspects also includes additional embodiments comprising multiple administrations of an effective amount of the agent and / or peptide, including wherein (i) the agent and / or peptide is administered continuously or intermittently as a single dose; (ii) the time between multiple administrations is every 6 hours; (iii) the agent and / or peptide is administered to a mammal every 8 hours; (iv) the agent and / or peptide is administered to a mammal every 12 hours; and (v) the agent and / or peptide is administered to a mammal every 24 hours.

[0213] Any of the foregoing aspects also includes additional embodiments of agents and / or peptides incorporated into a delivery medium for sustained delivery of a provider. In some embodiments, delivery lasts for approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, or 12 years. In some implementations, agents and / or peptides are incorporated into transdermal patches, tapes, suppositories, vaginal suppositories, tampons, vaginal rings, vaginal strips, vaginal capsules, vaginal tablets, vaginal pessaries, vaginal cups, vaginal sponges, or intrauterine devices.

[0214] In another or alternative embodiment, the method includes a withdrawal period, wherein the administration of the agent and / or peptide is temporarily suspended or the dose of the administered agent and / or peptide is temporarily reduced; at the end of the withdrawal period, the administration of the agent and / or peptide is resumed. In one embodiment, the length of the withdrawal period is from 2 days to 1 year.

[0215] In certain circumstances, it is appropriate to administer at least one of the agents and / or peptides described herein in combination with one or more other therapeutic agents. Exemplary additional therapeutic agents may include, but are not limited to, hormonal therapeutic agents, including contraceptives, combination contraceptives, selective progesterone receptor antagonists, selective progesterone receptor agonists, gonadotropin-releasing hormone receptor antagonists, gonadotropin-releasing hormone receptor agonists, antiretroviral agents, anti-vesicle agents, anti-inflammatory agents, nonsteroidal anti-inflammatory agents, or any combination thereof.

[0216] In one embodiment, the therapeutic efficacy of one of the agents and / or peptides described herein is enhanced by the administration of an adjuvant (i.e., the adjuvant itself has minimal therapeutic benefit, but when combined with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced). Alternatively, in some embodiments, the benefit experienced by the patient is increased by administering one of the agents and / or peptides described herein together with another agent (which also includes treatment regimens) that also has therapeutic benefit.

[0217] In one particular implementation, the agents and / or peptides described herein are administered in combination with a second therapeutic agent. In some cases, the agents and / or peptides described herein, along with the second therapeutic agent, modulate different aspects of the treated disease, disorder, or condition, thereby providing a greater overall benefit than the administration of either therapeutic agent alone. In any case, regardless of the treated disease, disorder, or condition, the overall benefit experienced by the patient may be additive between the two therapeutic agents, or the patient may experience a synergistic benefit.

[0218] In some embodiments, different effective doses of the disclosed agents and / or peptides may be used to formulate pharmaceutical compositions and / or for treatment regimens when administered in combination with one or more other agents (such as additional therapeutically effective drugs, adjuvants, etc.). The effective doses of the drugs and other agents used in the combination treatment regimen are optionally determined by methods similar to those described above for the active substances themselves. Furthermore, the preventative / treatment methods described herein cover the use of rhythmic dosing, i.e., providing more frequent, lower doses to minimize toxic side effects. In some embodiments, the combination treatment regimen covers a treatment regimen in which the administration of the agents and / or peptides described herein is initiated before, during, or after treatment with the second agent described herein, and continues until any time during or after treatment with the second agent. It also includes treatment in which the agents and / or peptides described herein, along with the second agent used in combination, are administered simultaneously or at different times and / or at decreasing or increasing intervals during the treatment period. Combination treatment also includes periodic treatments that are started and stopped at different times to aid in the clinical management of the patient.

[0219] It should be understood that dosage regimens for imaging, diagnosis, treatment, prevention, or improvement of one or more conditions seeking relief can be modified based on a variety of factors, such as the subject's disease, disorder, or condition; the subject's age, weight, sex, diet, and medical condition. Therefore, in some cases, the actual dosage regimen used differs, and in some implementations, it deviates from the dosage regimen described herein.

[0220] For the combination therapies described herein, the dosage of the co-administered agents and / or peptides varies depending on the type of co-administered medication, the specific medication used, the disease or condition being treated, etc. In another embodiment, when co-administered with one or more other therapeutic agents, the agents and / or peptides provided herein are administered simultaneously or sequentially with one or more other therapeutic agents.

[0221] In combination therapy, multiple therapeutic agents (one of which is one of the agents and / or peptides described herein) are administered in any order or even simultaneously. If administered simultaneously, by way of example only, multiple therapeutic agents are provided in a single form, a uniform form, or multiple forms (e.g., as a single pill or as two separate pills).

[0222] The agents and / or peptides described herein, as well as combination therapies, are administered before, during, or after the onset of a disease or condition, and the timing of administration of compositions containing agents and / or peptides varies. Thus, in one embodiment, the agents and / or peptides described herein are used as preventative agents and are administered continuously to a subject with a developmental condition or predisposition to prevent the occurrence of the disease or condition. In another embodiment, the agents and / or peptides and compositions are administered to the subject during or as soon as possible after the onset of symptoms. In a particular embodiment, the agents and / or peptides described herein are administered as soon as practicable after the onset of a disease or condition is detected or suspected, and the duration of treatment for the disease is as long as necessary. In some embodiments, the duration of treatment varies, and the treatment length is adjusted to suit the specific needs of each subject. For example, in a particular embodiment, the agents and / or peptides described herein, or formulations containing such agents and / or peptides, are administered for at least 2 weeks, about 1 month, to about 5 years.

[0223] The peptides and compositions described herein can be administered in combination with other therapeutic agents. In the embodiments described herein, the cyclic peptide compositions can be administered in combination with a drug for treating osteoporosis, including but not limited to alendronate, ibandronate, risedronate, zoledronic acid, denosumab, calcitonin, estrogen, raloxifene, bazodoxifene, teriparatide, abaloparatide, or any combination thereof.

[0224] Implementation Plan Implementation Scheme 1. An imaging agent comprising a peptide or peptide mimic conjugated to an imaging portion via a linker, wherein: (a) The peptide or peptide mimic comprises an amino acid sequence according to the following formula: R-X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 - X 13 -X 14 -X 15 -X 16 , in: R is NH2, acetylated, stearic acid, palmitic acid, myristic acid, lauric acid, C1-C8 hydrocarbon, C1-C8 fatty acid, or absent; X1 is M, G, β-alanine, leucine, valine, or absent; X2 is W, N-methyl-W, R, Y, F, citrulline, or K. X3 is P, W, N-methyl-W, N-ethyl-W, N-methyl-A, N-ethyl-A, L, Pip, Aib, Y, or F; X4 is E, Q, N, or D; X5 is S, α-methyl-S, K, D, Orn, T, or E; X6 is I, Chg, H, or L; X7 is either L or I; X8 is D, N, E, or Q; X9 is D, E, K, Q, or Orn; X 10 It is H or methyl-H; X 11 It is V, α-methyl-V, Chg, L, I, or valine; X 12 It is Q, Aib, S, R, or N; X 13 It is R, K, citrulline, Orn, D, or E; X 14 It is V, I, L or valine; X 15 It is W, Y, or F; and X 16 It is R, G, or does not exist; and (b) The connector is absent or the connector is conjugated to the imaging portion and the peptide or peptide mimic.

[0225] Implementation Scheme 2. An imaging agent comprising a peptide or peptide mimic conjugated to an imaging portion via a linker, wherein: (a) The peptide or peptide mimic comprises an amino acid sequence according to the following formula: X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 - X 13 -X 14 -X 15 -X 16 , in: X1 is M or does not exist; X2 is S, I, G, T, A, L, or does not exist; X3 is R, K, or does not exist; X4 is a positively charged amino acid, citrulline, Orn, D, E, 8-aminooctanoic acid, or an aminocarboxylic acid with between 4 and 12 carbon atoms; X5 is M, ortholeucine, Orn, D, E, K, H, R, K, 8-aminooctanoic acid, an aminocarboxylic acid with between 4 and 12 carbon atoms, or is absent; X6 is W, Y, F, or N-methyl-A; X7 is F, I, L, Chg, Cha, or Tie; X8 is L, I, or A; X9 is L, I, or A; X 10 It is C, S, A, Abu, C(me) or S(Bzl); X 11 It is F, H, A, K, E, Chg, Cng, or Orn; X 12 It is W, Y, A, or F; and X 13 It is G, GABA, or does not exist; X 14 It is V, I, L, or valine, or it is absent; X 15 It is W, Y, F, or does not exist; X 16 It is R, G, or does not exist. (b) The connector is absent or the connector is conjugated to the imaging portion and the peptide or peptide mimic.

[0226] Implementation Scheme 3. The imaging agent according to Implementation Scheme 1 or 2, wherein the imaging component is a fluorescent molecule, a magnetic resonance imaging (MRI) contrast agent, a radionuclide contrast agent, a microbubble contrast agent, a computed tomography (CT) contrast agent, or a photoacoustic contrast agent.

[0227] Implementation Scheme 4. The imaging agent according to Implementation Scheme 1 or 2, wherein the imaging component is a fluorophore, fluorescent dye, dye, pigment, fluorescent transition metal, or fluorescent protein.

[0228] Implementation Scheme 5. The imaging agent according to Implementation Scheme 1 or 2, wherein the imaging portion is a paramagnetic ion complex or superparamagnetic magnetite particles.

[0229] Implementation Scheme 6. The imaging agent according to Implementation Scheme 1 or 2, wherein the imaging component is a gadolinium-based contrast agent, a manganese-based contrast agent, an iron oxide contrast agent, an iron-platinum contrast agent, or an iron complex.

[0230] Implementation Scheme 7. The imaging agent according to Implementation Scheme 1 or 2, wherein the imaging portion comprises at least one selected from 18 F, 64 Cu、 68 Ga and 99m Radionuclides in the Tc group.

[0231] Implementation Scheme 8. The imaging agent according to Implementation Scheme 1 or 2, wherein the imaging portion comprises superparamagnetic iron oxide nanoparticles (SPION) or DOTA-Fe(III) complex.

[0232] Implementation Scheme 9. The imaging agent according to Implementation Scheme 1 or 2, wherein the imaging portion is gold nanoparticles or an iodide compound.

[0233] Implementation Scheme 10. The imaging agent according to any one of Implementation Schemes 1-9, wherein the linker comprises an amino acid, –NH(CH2). n C(O)O–、–(OCH2CH2) m –、–O–、–NH–、C1-C6 alkylene、C1-C6 alkylhalide、C1-C6 alkoxy、C1-C6 heteroalkylene、C2-C6 alkenyl、C2-C6 alkyneyl、alkylamino、alkylthio、alkylcarbonyl、alkoxycarbonyl、alkylsulfonyl、C3-C 10 Cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-C 10 One or more of aryl or 5- to 10-membered heteroaryl groups or combinations thereof, wherein each is a C1-C6 alkylene group, a C1-C6 haloalkylene group, a C1-C6 alkoxy group, a C1-C6 heteroalkylene group, a C2-C6 alkenyl group, a C2-C6 alkyneyl group, an alkylamino group, an alkylthio group, an alkylcarbonyl group, an alkoxycarbonyl group, an alkylsulfonyl group, or a C3-C... 10 Cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-C 10 Aryl and 5- to 10-membered heteroaryl groups are optionally and independently bounded by 1, 2, or 3 R groups. 9 Replace; of which: n is an integer from 1 to 10 independently; m is an integer independently from 1 to 10; and R 9 Independently, it is a halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 heteroalkyl, –C(O)H, –C(O)OH, –CN, C3-C 10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-C 10Aryl, 5- to 10-membered heteroaryl, -C(O)(C1-C4 alkyl), -C(O)O(C1-C4 alkyl), –C(O)NH2, –C(O)NH(C1-C4 alkyl), –C(O)N(C1-C4 alkyl)2, -NH2, -NH(C1-C4 alkyl), -N(C1-C4 alkyl)2, -NH(C2-C4 alkylene)OH, -NH(C2-C4 alkylene)-O-(C1-C4 alkyl), -OH, -O(C1 -C4 alkyl), -O(C1-C4 haloalkyl), -O(C2-C4 alkylene)-NH2, -O(C2-C4 alkylene)-NH-(C1-C4 alkyl), -O(C2-C4 alkylene)-N-(C1-C4 alkyl)2, -O(C1-C4 alkylene)-C(O)OH, -O(C1-C4 alkylene)-C(O)O-(C1-C4 alkyl), -O(C2-C4 alkenyl), -O(C1-C4 alkylene)-(C6-C 10 aryl), -O(C1-C4 alkylene)-(5- to 10-membered heteroaryl), -O(C6-C 10 aryl), -SH, S(O)2OH, -S(O)2 (C1-C4 alkyl), -S(O)2NH2, -S(O)2NH (C1-C4 alkyl) or –S(O)2N (C1-C4 alkyl)2; or two R 9 Together with the atoms to which they are attached, they form C3-C 10 Cycloalkyl or 3- to 10-membered heterocyclic alkyl rings.

[0234] Implementation Scheme 11. The imaging agent according to Implementation Scheme 10, wherein the linker comprises cysteine ​​and NH(CH2). n C(O)-、-C(O)(CH2) n NH-, -(OCH2CH2) m -、-O-、-NH-、C1-C6 alkylene、C1-C6 alkoxy、C1-C6 heteroalkylene、C3-C 10 Cycloalkylene or 3- to 10-membered heterocycloalkylene, or combinations thereof, wherein each C1-C6 alkylene, C1-C6 alkoxy, C1-C6 heteroalkylene, C3-C 10 Cycloalkylene and 3- to 10-membered heterocyclic alkylene groups are optionally independently bound by 1, 2, or 3 R groups. 9 Replace, where n, m and R 9 Defined in Implementation Scheme 10.

[0235] Implementation Scheme 12. The imaging agent according to any one of Implementation Schemes 1-11, wherein the connector is in contact with the peptide or peptide mimicry at X1 to X 16 At least one of the amino acids is up-conjugated.

[0236] Implementation Scheme 13. The imaging agent according to any one of Implementation Schemes 1-11, wherein the linker is conjugated to the N-terminus of the peptide or peptide mimic.

[0237] Implementation Scheme 14. The imaging agent according to Implementation Scheme 13, wherein the peptide or peptide mimic further comprises -NH2 at the C-terminus.

[0238] Implementation Scheme 15. The imaging agent according to any one of Implementation Schemes 1-12, wherein the linker is conjugated to the C-terminus of the peptide or peptide mimic.

[0239] Implementation Scheme 16. The imaging agent according to Implementation Scheme 15, wherein the peptide or peptide mimic further comprises a C1-C8 alkyl-C(O)- at the N-terminus.

[0240] Implementation Scheme 17. The imaging agent according to Implementation Scheme 15, wherein the peptide or peptide mimic further comprises CH3(CH2) at its N-terminus. p C(O)-, where p is an integer from 0 to 7.

[0241] Implementation Scheme 18. The imaging agent according to Implementation Scheme 15, wherein the peptide or peptide mimic further comprises CH3(CH2)6C(O)- at the N-terminus.

[0242] Implementation Scheme 19. The imaging agent according to any one of Implementation Schemes 1-12, wherein the peptide or peptide mimic comprises the amino acid sequence SEQ ID NO: 496 or SEQ ID NO: 497.

[0243] Implementation Scheme 20. The imaging agent according to any one of Implementation Schemes 1-21, wherein the imaging component is iron oxide nanoparticles.

[0244] Implementation Scheme 21. The imaging agent according to any one of Implementation Schemes 1-21, wherein the imaging portion is iron chelated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0245] Implementation Scheme 22. An imaging agent according to any one of Implementation Schemes 1-21, wherein the imaging component is a fluorescent molecule, an MRI contrast agent, a radionuclide, a microbubble, a CT contrast agent, or a photoacoustic contrast agent.

[0246] Implementation Scheme 23. The imaging agent according to any one of Implementation Schemes 1-21, wherein the imaging portion is a fluorophore, fluorescent dye, dye, pigment, fluorescent transition metal, or fluorescent protein.

[0247] Implementation Scheme 24. The imaging agent according to any one of Implementation Schemes 1-21, wherein the imaging portion is superparamagnetic iron oxide nanoparticles (SPION) or a gadolinium complex.

[0248] Implementation Scheme 25. The imaging agent according to any one of Implementation Schemes 1-21, wherein the imaging portion is 18 F, 64 Cu、 68 Ga or 99m Tc radionuclides.

[0249] Implementation Scheme 26. The imaging agent according to any one of Implementation Schemes 1-21, wherein the imaging portion is gold nanoparticles or an iodide compound.

[0250] Implementation Scheme 27. The imaging agent according to any one of Implementation Schemes 1-26, wherein the peptide or peptide mimic is cyclic or bicyclic.

[0251] Implementation Scheme 28. The imaging agent according to Implementation Scheme 27, wherein the peptide or peptide mimic is cyclized with Cys-Cys disulfide bonds.

[0252] Implementation Scheme 29. The imaging agent according to Implementation Scheme 27, wherein the peptide or peptide mimic is cyclized with an amide bond.

[0253] Implementation Scheme 30. The imaging agent according to Implementation Scheme 29, wherein the amide bond is a head-to-tail amide bond between the N-terminus and the C-terminus.

[0254] Implementation Scheme 31. The imaging agent according to Implementation Scheme 29, wherein the amide bond is a head-to-side chain amide bond between the N-terminus and the internal COOH.

[0255] Implementation Scheme 32. The imaging agent according to Implementation Scheme 29, wherein the amide bond is a side-chain to tail amide bond between the internal NH2 and the C-terminus.

[0256] Implementation Scheme 33. The imaging agent according to Implementation Scheme 27, wherein the amide bond is a side-chain to side-chain amide bond between the internal NH2 and the internal COOH.

[0257] Implementation Scheme 34. The imaging agent according to Implementation Scheme 27, wherein the peptide or peptide mimic uses hydrocarbon pinning cyclization.

[0258] Implementation Scheme 35. The imaging agent according to Implementation Scheme 27, wherein the peptide or peptide mimic is cyclized using click chemistry.

[0259] Implementation Scheme 36. The imaging agent according to any one of Implementation Schemes 1 to 35, wherein the peptide is less than 81 amino acid residues.

[0260] Implementation Scheme 37. The imaging agent according to any one of Implementation Schemes 1 to 36, wherein the peptide comprises one or more non-natural amino acids.

[0261] Implementation Scheme 38. The imaging agent according to Implementation Scheme 36, wherein one or more non-natural amino acids are N-methyl amino acids.

[0262] Implementation Scheme 39. A method for detecting endometriosis lesions in a subject, comprising: (a) administering to the subject a formulation comprising the imaging agent described in any one of embodiments 1 to 38 or 68 to 85; and (b) Image the subject to determine the presence of the image portion.

[0263] Implementation Scheme 40. The method according to Implementation Scheme 39 further includes: (c) administering a therapeutically effective amount of a second β-linkin-binding peptide or peptide mimic to the subject; and (d) Wait at least 1 week, and then repeat steps (a) and (b).

[0264] Implementation Scheme 41. The method according to Implementation Scheme 39 or 40, wherein the imaging in (b) further includes creating an image and displaying the image.

[0265] Implementation Scheme 42. The method according to Implementation Scheme 41, wherein the image is a video, a still image, a real-time video, or any combination thereof.

[0266] Implementation Scheme 43. The method according to Implementation Scheme 42, wherein the subject or the image is visualized or displayed due to the imaging agent during surgery performed on the subject.

[0267] Implementation Scheme 44. The method according to Implementation Scheme 43, wherein the imaging agent is administered during or before surgery for the diagnosis, staging and / or resection of endometriosis.

[0268] Implementation Scheme 45. The method according to any one of Implementation Schemes 36 to 44, wherein the preparation is administered vaginally or intravenously.

[0269] Implementation Scheme 46. The method according to any one of Implementation Schemes 36 to 45, wherein the imaging in (b) includes in vivo fluorescence imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound imaging, computed tomography (CT), or photoacoustic imaging.

[0270] Implementation Scheme 47. The method according to any one of Implementation Schemes 36 to 46, wherein the subject is a human.

[0271] Implementation Scheme 48. The method according to Implementation Scheme 47, wherein the imaging includes whole-body imaging.

[0272] Implementation Scheme 49. The method according to any one of Implementation Schemes 36-48, wherein the subject is a cultured cell line.

[0273] Implementation Scheme 51. The method according to any one of Implementation Schemes 36 to 48, wherein the subject is an ex vivo tissue extracted from an animal.

[0274] Implementation Scheme 52. The method according to any one of Implementation Schemes 36 to 51, wherein the second β-linkin-binding peptide or peptide mimic comprises an amino acid sequence according to the following formula: R-X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 - X 13 -X 14 -X 15 -X 16 , in: R is NH2, acetylated, stearic acid, palmitic acid, myristic acid, lauric acid, C1-C8 hydrocarbon, C1-C8 fatty acid, or absent; X1 is M, G, β-alanine, leucine, valine, or absent; X2 is W, N-methyl-W, R, Y, F, citrulline, or K. X3 is P, W, N-methyl-W, N-ethyl-W, N-methyl-A, N-ethyl-A, L, Pip, Aib, Y, or F; X4 is E, Q, N, or D; X5 is S, α-methyl-S, K, D, Orn, T, or E; X6 is I, Chg, H, or L; X7 is either L or I; X8 is D, N, E, or Q; X9 is D, E, K, Q, or Orn; X 10 It is H or methyl-H; X 11 It is V, α-methyl-V, Chg, L, I, or valine; X 12 It is Q, Aib, S, R, or N; X 13It is R, K, citrulline, Orn, D, or E; X 14 It is V, I, L or valine; X 15 It is W, Y, or F; and X 16 It is R, G, or does not exist.

[0275] Implementation Scheme 53. The method according to Implementation Scheme 52, wherein the second β-linkin-binding peptide or peptide mimic has the amino acid sequence Oct-W-NMeA-Q-Orn*-ILDE*HChg-QRVWG-NH2 (SEQ ID NO: 491).

[0276] Implementation Scheme 54. The method according to any one of Implementation Schemes 36 to 51, wherein the second β-linkin-binding peptide or peptide mimic comprises an amino acid sequence according to the following formula: X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 - X 13 -X 14 -X 15 -X 16 , in: X1 is M or does not exist; X2 is S, I, G, T, A, L, or does not exist; X3 is R, K, or does not exist; X4 is a positively charged amino acid, citrulline, Orn, D, E, 8-aminooctanoic acid, or an aminocarboxylic acid with between 4 and 12 carbon atoms; X5 is M, ortholeucine, Orn, D, E, K, H, R, K, 8-aminooctanoic acid, an aminocarboxylic acid with between 4 and 12 carbon atoms, or is absent; X6 is W, Y, F, or N-methyl-A; X7 is F, I, L, Chg, Cha, or Tie; X8 is L, I, or A; X9 is L, I, or A; X 10 It is C, S, A, Abu, C(me) or S(Bzl); X 11 It is F, H, A, K, E, Chg, Cng, or Orn; X 12 It is W, Y, A, or F; X 13It is G, GABA, or does not exist; X 14 It is V, I, L, or valine, or it is absent; X 15 It is W, Y, F or does not exist; and X 16 It is R, G, or does not exist.

[0277] Implementation Scheme 55. The method according to any one of Implementation Schemes 39 to 54, wherein the peptide binds to cytoplasmic beta-binin to inhibit the translocation of beta-binin to the cell nucleus.

[0278] Implementation Scheme 56. The method according to any one of Implementation Schemes 39 to 54, wherein the peptide binds to cytoplasmic beta-binin to maintain or increase membrane-bound beta-binin.

[0279] Implementation Scheme 57. The method according to any one of Implementation Schemes 39 to 54, wherein the peptide binds to cytoplasmic beta-binin to prevent beta-binin from acting as a transcription factor for oncogenes, matrix metalloproteinase 9 (MMP9) or chloride C3 channels (ClC-3).

[0280] Implementation Scheme 58. The method according to any one of Implementation Schemes 39 to 54, wherein the peptide binds to cytoplasmic beta-binding protein to prevent transformation, invasion, migration, fibrosis, or any combination thereof of EMS cells.

[0281] Implementation Scheme 59. The method according to any one of Implementation Schemes 39 to 54, wherein the peptide binds to cytoplasmic beta-binin to prevent beta-binin from binding to estrogen receptor (ESR1).

[0282] Implementation Scheme 60. The method according to any one of Implementation Schemes 39 to 54, wherein the peptide binds to cytoplasmic beta-binin and the membrane activity of beta-binin is not reduced.

[0283] Implementation Scheme 61. The method according to any one of Implementation Schemes 39 to 54, wherein the peptide binds to cytoplasmic beta-nectin, and the beta-nectin-E-cadherin binding is not reduced.

[0284] Implementation Scheme 62. The method according to any one of Implementation Schemes 39 to 54, wherein the peptide inhibits the activity of oncogenic transcription factors.

[0285] Implementation Scheme 63. The method according to any one of Implementation Schemes 39 to 54, wherein the amount of nuclear β-linkin in the cells of the subject is reduced by at least 5%.

[0286] Implementation Scheme 64. The method according to Implementation Scheme 62, wherein the reduction of nuclear beta-binin is relative to the cells of a control subject who has not been administered an effective amount of the therapeutic composition.

[0287] Implementation Scheme 65. The method according to Implementation Scheme 62, wherein the reduction of nuclear beta-binin is relative to cells of the subject collected prior to the development of the condition in the subject.

[0288] Implementation Scheme 66. The method according to Implementation Scheme 62, wherein the reduction of nuclear β-linkin is relative to cells collected from the subject at different time points.

[0289] Implementation Scheme 67. The method according to any one of Implementation Schemes 39 to 66, wherein the therapeutically effective amount is about 0.01 mg to about 1000 mg.

[0290] Implementation Scheme 68. An imaging agent according to Formula III: labeled-linker-peptide (Formula III); wherein: (a) The marker is an imaging portion; (b) The peptide comprises the amino acid sequence according to formula IV: X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 - X 13 -X 14 -X 15 -X 16 (Formula IV), where: X1 is M, G, β-alanine, leucine, valine, or absent; X2 is W, N-methyl-W, R, Y, F, citrulline, or K. X3 is P, W, N-methyl-W, N-ethyl-W, N-methyl-A, N-ethyl-A, L, Pip, Aib, Y, or F; X4 is E, Q, N, or D; X5 is S, α-methyl-S, K, D, Orn, T, or E; X6 is I, Chg, H, or L; X7 is either L or I; X8 is D, N, E, or Q; X9 is D, E, K, Q, or Orn; X 10 It is H or methyl-H; X 11 It is V, α-methyl-V, Chg, L, I, or valine; X12 It is Q, Aib, S, R, or N; X 13 It is R, K, citrulline, Orn, D, or E; X 14 It is V, I, L or valine; X 15 It is W, Y, or F; and X 16 It is R, G, or does not exist; and (c) The adapter is absent or the adapter is conjugated with the tag and the peptide.

[0291] Implementation Scheme 69. The imaging agent according to Implementation Scheme 68, wherein the label is a fluorescent molecule, a magnetic resonance imaging (MRI) contrast agent, a radionuclide contrast agent, a microbubble contrast agent, a computed tomography (CT) contrast agent, or a photoacoustic contrast agent.

[0292] Implementation Scheme 70. The imaging agent according to Implementation Scheme 68, wherein the label is a fluorophore, fluorescent dye, dye, pigment, fluorescent transition metal, or fluorescent protein.

[0293] Implementation Scheme 71. The imaging agent according to Implementation Scheme 68, wherein the marker is a paramagnetic ion complex or a superparamagnetic magnetite particle.

[0294] Implementation Scheme 72. The imaging agent according to Implementation Scheme 68, wherein the label is a gadolinium-based contrast agent, a manganese-based contrast agent, an iron oxide contrast agent, an iron-platinum contrast agent, or an iron complex.

[0295] Implementation Scheme 73. The imaging agent according to Implementation Scheme 68, wherein the marker comprises at least one selected from 18 F, 64 Cu、 68 Ga and 99m Radionuclides in the Tc group.

[0296] Implementation Scheme 74. The imaging agent according to Implementation Scheme 68, wherein the label comprises superparamagnetic iron oxide nanoparticles (SPION) or DOTA-Fe(III) complexes.

[0297] Implementation Scheme 75. The imaging agent according to Implementation Scheme 68, wherein the marker is a gold nanoparticle or an iodide compound.

[0298] Implementation Scheme 76. The imaging agent according to any one of Implementation Schemes 68-75, wherein the label comprises an amino acid, –NH(CH2). n C(O)O–、–(OCH2CH2) m–、–O–、–NH–、C1-C6 alkylene、C1-C6 haloalkylene、C1-C6 alkoxy、C1-C6 heteroalkylene、C2-C6 alkenyl、C2-C6 alkyneyl、alkylamino、alkylthio、alkylcarbonyl、alkoxycarbonyl、alkylsulfonyl、C3-C 10 Cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-C 10 One or more of aryl or 5- to 10-membered heteroaryl groups or combinations thereof, wherein each is a C1-C6 alkylene group, a C1-C6 haloalkylene group, a C1-C6 alkoxy group, a C1-C6 heteroalkylene group, a C2-C6 alkenyl group, a C2-C6 alkyneyl group, an alkylamino group, an alkylthio group, an alkylcarbonyl group, an alkoxycarbonyl group, an alkylsulfonyl group, or a C3-C... 10 Cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-C 10 Aryl and 5- to 10-membered heteroaryl groups are optionally and independently bounded by 1, 2, or 3 R groups. 9 Replace; of which: n is an integer independently from 1 to 10; m is an integer independently from 1 to 10; and R 9 Independently, it is a halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 heteroalkyl, -C(O)H, -C(O)OH, -CN, C3-C 10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-C 10 Aryl, 5- to 10-membered heteroaryl, -C(O)O(C1-C4 alkyl), -C(O)O(C1-C4 alkyl), -C(O)NH2, -C(O)NH(C1-C4 alkyl), -C(O)N(C1-C4 alkyl)2, -NH2, -NH(C1-C4 alkyl), -N(C1-C4 alkyl)2, -NH(C2-C4 alkylene)-OH, -NH(C2-C4 alkylene)-O-(C1-C4 alkyl), –OH, -O( C1-C4 alkyl), -O(C1-C4 haloalkyl), -O(C2-C4 alkylene)-NH2, -O(C2-C4 alkylene)-NH-(C1-C4 alkyl), -O(C2-C4 alkylene)-N-(C1-C4 alkyl)2, -O(C1-C4 alkylene)-C(O)OH, -O(C1-C4 alkylene)-C(O)O-(C1-C4 alkyl), -O(C2-C4 alkenyl), -O(C1-C4 alkylene)-(C6-C 10 aryl), -O(C1-C4 alkylene)-(5- to 10-membered heteroaryl), -O(C6-C 10 Aryl), -SH, S(O)2OH, -S(O)2 (C1-C4 alkyl), -S(O)2NH2, –S(O)2NH (C1-C4 alkyl) or –S(O)2N (C1-C4 alkyl)2; or two R 9 Together with the atoms to which they are attached, they form C3-C 10 Cycloalkyl or 3- to 10-membered heterocyclic alkyl rings.

[0299] Implementation Scheme 77. The imaging agent according to Implementation Scheme 76, wherein the linker comprises cysteine ​​and NH(CH2). n C(O)-、-C(O)(CH2) n NH-, -(OCH2CH2) m -、-O-、-NH-、C1-C6 alkylene、C1-C6 alkoxy、C1-C6 heteroalkylene、C3-C 10 Cycloalkylene or 3- to 10-membered heterocyclic alkylene, or combinations thereof, wherein each C1-C6 alkylene, C1-C6 alkoxy, C1-C6 heteroalkylene, C3-C 10 Cycloalkylene and 3- to 10-membered heterocyclic alkylene groups are optionally independently bound by 1, 2, or 3 R groups. 9 Replace, where n, m and R 9 Defined in implementation scheme 76.

[0300] Implementation Scheme 78. The imaging agent according to any one of Implementation Schemes 68-77, wherein the linker is in contact with the peptide at X1 to X... 16 At least one of the amino acids is up-conjugated.

[0301] Implementation Scheme 79. The imaging agent according to any one of Implementation Schemes 68-77, wherein the linker is conjugated to the N-terminus of the peptide.

[0302] Implementation Scheme 80. The imaging agent according to Implementation Scheme 79, wherein the peptide further comprises -NH2 at its C-terminus.

[0303] Implementation Scheme 81. The imaging agent according to any one of Implementation Schemes 68-77, wherein the linker is conjugated to the C-terminus of the peptide.

[0304] Implementation Scheme 82. The imaging agent according to Implementation Scheme 80, wherein the peptide further comprises C1-C8 alkyl-C(O)- at its N-terminus.

[0305] Implementation Scheme 83. The imaging agent according to Implementation Scheme 80, wherein the peptide further comprises CH3(CH2) at its N-terminus. p C(O)-, where p is an integer from 0 to 7.

[0306] Implementation Scheme 84. The imaging agent according to Implementation Scheme 80, wherein the peptide further comprises CH3(CH2)6C(O)- at its N-terminus.

[0307] Implementation Scheme 85. An imaging agent according to any one of Implementation Schemes 68-84, wherein the amino acid sequence is cyclized with an amide bond, and wherein the amide bond is (1) a head-to-tail amide bond between the N-terminus and the C-terminus, (2) a head-to-side chain amide bond between the N-terminus and the internal COOH, (3) a side chain-to-tail amide bond between the internal NH2 and the C-terminus, or (4) a side chain-to-side chain amide bond between the internal NH2 and the internal COOH, and wherein the amide bond is linked to an amino acid not connected to the adapter or the tag.

[0308] Various embodiments of this disclosure have been described. Nevertheless, it should be understood that various changes can be made without departing from the spirit and scope of this disclosure. Therefore, other embodiments are within the scope of the appended claims. Example

[0309] Example 1: Peptides conjugated to imaging components can be used as imaging agents to visualize in vivo diseases and API / drug product characteristics. The series of figures presented aims to demonstrate the potential of peptides in advanced biomedical applications. Figure 1 In the examples related to endometriosis, compounds 492, 494, and 493 were used in animals 2-4, respectively, to demonstrate that endometriosis lesions in animals can be visualized using these imaging and diagnostic agents as valuable tools for imaging subjects to detect the presence of diseased tissue. The same mice sent for necropsy showed ex vivo analysis similar to their in vivo imaging.

[0310] C57Bl / 6 albino mice were imaged using spectral imaging (Living Image 4.7.3, IVIS Spectrum, Perkin Elmer). Mice were induced to sleep using isoflurane following standard procedure. Spectral emission scans were performed from 757 nm to 781 nm using adaptive fluorescence background subtraction and spectral unmixing, with Cy7 epi-illumination. Each time point was normalized by setting a threshold for endometriotic mice and applying it to the threshold for healthy mice.

[0311] These agents can also be used to evaluate key characteristics of active pharmaceutical ingredients (APIs) or drug products. By using labeled peptide compound 492, Figure 2 The biodistribution of compound 491 is shown, revealing the specificity of these agents, which are rarely or not detectable in other organ systems. Figure 15 I also support this point. Figure 15Compound 492 was shown to be detected only in endometriosis tissue and not in healthy uterine tissue. Figure 3 The vaginal administration and detection at the lesion site of compound 494 are illustrated. This has been successfully demonstrated with other labeled peptides and other routes of administration, including but not limited to SQ, IV, and PO administration as viable options, all of which have different pharmacokinetic characteristics. Figure 4 In vitro experiments confirmed Figure 3 Visual observations showed that the peptide could penetrate vaginal tissue, and that vaginal administration of the peptide required approximately 3 hours to penetrate human vaginal tissue. This is consistent with imaging performed using a publicly available imaging agent via an animal model, confirming the accuracy of the imaging agent.

[0312] The agents and peptides described in this article show promise in monitoring disease progression or assessing the effectiveness of therapeutic treatments. Figure 3 and Figure 4 The ability to administer these agents via multiple routes, as described herein, provides flexibility in the delivery of these imaging, diagnostic, and / or therapeutic agents. For example... Figure 6 The data shows time points between 20 minutes and 72 hours, with imaging observed up to 48 hours after blood removal in subjects with endometriosis, and no imaging observed in subjects without endometriosis after blood removal. Figure 7 In this context, peptides are used to track disease progression and / or lesions treated with medication, thereby providing a quantitative assessment of therapeutic efficacy and disease burden over time. Figure 7 The imaging, diagnostic, and efficacy effects of the agent are shown in animals administered two SQ doses of compound 492 at several weeks intervals. The final necropsy report of the ex vivo lesion analysis is consistent with the in vivo visualization of the agent administration. Follow-up of lesion regression in the same mice after administration of two SQ doses at several weeks intervals indicates that daily dosing is not necessary for efficacy.

[0313] Example 2: Identification of effective dosage and dosing regimen like Figures 8 to 10 As illustrated in the figure, the purpose of this embodiment is to identify an approximate effective dose and determine the optimal dosing regimen. Figures 8 to 21 Compounds 491 or 492 were used as examples, but the results were also replicated and confirmed using many published compounds.

[0314] Figure 8 The lesion weights obtained in mice with endometriosis after daily administration of compound 491 for 3 weeks (left panel) or 6 weeks (right panel) are shown. The therapeutic agent improved with longer treatment duration, especially when administered at lower doses. This evidence provides valuable guidance for optimizing treatment regimens to achieve maximum therapeutic outcomes, particularly when using lower dose levels.

[0315] Figure 9 The pharmacokinetic exposure of compound 491 at 1 mg / kg in mice, as analyzed in serum, is shown. When compared with... Figures 6 to 8 Compared to the data, the pharmacodynamic effects and lesion-specific persistence of compound 491 extend beyond its pharmacokinetic presence in serum, indicating its potential for sustained imaging, diagnosis, and treatment.

[0316] Figure 10 This finding was supported by cell proliferation assays. A single dose of SEQ ID NO: 491 exerted a pharmacodynamic effect lasting up to five days post-treatment, which was more durable than its serum pharmacokinetics. The pharmacodynamic effect of compound 491 on 12Z human endometriosis cells was determined using a proliferation assay. 96-well plates were treated with extracellular matrix for 1 hour, and then 12Z human endometriosis cells were seeded into the wells at a concentration of 5000 cells / 50 μL assay medium. After overnight incubation at 37°C, 5% CO2, the medium was aspirated, and fresh Prigrow III medium supplemented with 10% FBS and 1% PS containing different concentrations of the peptide of interest was added to each well. Cells were treated with the peptide for only one day, after which fresh medium was added to each well daily for the next four days. To determine the effect of the peptide, the medium was mixed with PrestoBlue, which turned pink within 4–6 hours. Fluorescence in each well was measured at 540 nm and 590 nm using a Biotek HTX microplate reader. IC50 50 The graphs were calculated using Graph Pad Prism 9.0, providing a quantitative measurement of the peptide's effect on cell proliferation. One dose of compound 491 showed a durable pharmacodynamic effect (shown 5 days after treatment). This suggests that increased dosing frequency may potentially enhance the results, but is not essential for efficacy.

[0317] Example 3: In vitro characterization of the agent The aim of these studies is to demonstrate that diagnostic peptides remain cellularly permeable and capable of visualization even when conjugated to the imaging portion, without being quenched by conjugation, intracellular localization, or binding of the peptide to its β-linkin target. Furthermore, the imaging portion does not interfere with the diagnostic peptide's ability to localize in the cytoplasm, nor does it disrupt its cytoplasmic β-linkin binding, translation from the cytoplasm to the inner membrane, or its β-linkin membrane binding. Cy7-labeled compound 491 (compound 492) binds to the target β-linkin, successfully translocates to the target bound to the inner membrane, and can be imaged with the portion conjugated to the peptide without signal disruption from its conjugation to the peptide or target binding, exhibiting rapid intracellular uptake and longer retention in diseased tissues than in serum. These in vitro datasets support in vivo imaging and pharmacodynamic studies illustrated in other figures.

[0318] like Figure 11 As shown in Figure 12, compound 492 binds directly to its target β-linkin within the cell; it is cell-permeable and binds to the target in the cell's cytoplasm. This binding does not occur in the cell nucleus and does not show disruption of membrane binding.

[0319] To determine whether compound 491 is cell-permeable, the cellular localization of compound 492, its binding to its mate (β-linkin), and whether it can be visualized using a fluorophore, human endometriosis 12z cells were incubated with compound 492 for 1 h. Membrane fraction, cytoplasmic fraction, and nuclear fraction were extracted. BCA analysis was performed to determine protein concentration. Equal amounts of protein from all fractions, along with compound 492, were run on a non-denaturing PAGE gel. These results showed that both compound 492 and the β-linkin / compound 492-binding complex were visualized and separated on native gels, and that compound 492 was present in the membrane and cytoplasmic fractions but not in the nuclear fraction, confirming the mechanism of action.

[0320] To determine at what time point compound 491 binds to β-linkin in the cytoplasm, human endometriosis 12z cells were administered once with 0.5 μM compound 492 at different time points: 1 h, 3 h, 5 h, 7 h, 1 day, 2 days, and 3 days. After administration, the cells were washed with PBS buffer and cytoplasmic fractions were extracted. Natural gels were run and imaged. The results in Figure 14 suggest that compound 492 binds to β-linkin within 1 hour of incubation, with the increased signal intensity lasting for 24 h and exhibiting a stable signal over several days. These findings are based on previous experiments: (1) FP experiments, in which binding occurred after 1 hour and persisted for at least 24 hours (data not shown), and (2) imaging experiments in rodent model lesions, in which binding persisted in the lesions for at least 48 hours (data not shown). Figure 6Rodents without endometriosis showed no signs of imaging agent after blood removal. Figure 6 Additionally, this is consistent with the proliferation results, which showed that only one dose of compound 491 provided a sustained pharmacodynamic effect of reduced cell proliferation.

[0321] The results in Figure 12 show that compound 492 binds to β-linkin for 1 hour. Incubation and signal stability last for at least 3 days. These findings are based on our previous experiments: (1) the FP experiment, in which binding occurs after 1 hour and persists for at least 24 hours, and (2) imaging experiments in rodent lesions, in which binding remains in the lesions beyond blood clearance. This is consistent with animal imaging results in mice, which show that a dose of compound 492 is specifically retained in the lesions.

[0322] To visualize the effect of compound 491 on β-linkin in the cytoplasmic fraction, human endometriosis (12z cells) were administered 0.5 μM of compound 492, and the effects were analyzed at time points: 1 h, 3 h, 5 h, 7 h, 1 day, 2 days, and 3 days after treatment. Following administration, cells were washed with PBS buffer, and the cytoplasmic fraction was extracted. The native gel was run and imaged using Cy7. The gel was then transferred for Western blotting using a nitrocellulose membrane and detected with a β-linkin antibody. Chemiluminescence imaging was used to visualize the Western blotting. Figure 13 The results suggest that after compound 492 binds to the cytoplasm, β-linkin begins to decrease.

[0323] To visualize the effect of compound 491 on β-linkin in the membrane fraction, human endometriosis 12z cells were administered 0.5 μM of compound 492 once, and the effects were analyzed at different time points: 1 h, 3 h, 5 h, 7 h, 1 day, and 2 days post-treatment. Following administration, cells were washed with PBS buffer, and the non-cytoplasmic / non-nuclear fraction was extracted. The native gel was run and imaged using Cy7. The gel was then transferred for Western blotting using a nitrocellulose membrane and detected with a β-linkin antibody. Chemiluminescence imaging was used to visualize the Western blotting. Figure 13 The results suggest that β-linkin remains stable in the membrane fraction.

[0324] These observations suggest that peptides can be inherently flexible, as their interaction with one of the (TCF4) binding sites can influence the allosteric conformation of other (BCL9) binding sites, even if only one binding site physically interacts with compound 491. This flexibility may facilitate the relocation and / or supportive binding of β-linkin from the cytoplasm to the membrane, allowing for its Wnt4 rescue activity and confirming its mechanism of action.

[0325] Example 4: Species Homology This paper also shows that peptides can have high homology across species (in this embodiment, compound 491 has over 95% homology across multiple animal species) and are therefore applicable to all animal and mammalian species for imaging, diagnosis, and therapeutic interventions. Based on the data presented in Table 3, the binding characteristics of compound 491 to β-linkin show fundamental similarity across multiple mammalian species. Therefore, compound 492 and other imaging agents utilizing compound 491 can be effectively used in animal studies to visualize the progression or elimination of endometriosis using therapeutic agents or other compounds. This offers significant advantages compared to using animal cohorts and is useful for evaluating therapeutic efficacy for many reasons, including but not limited to the ability to view efficacy in real time without sacrificing animals and to observe the efficacy of therapeutic agents in the same animals, which is particularly useful in heterogeneous diseases such as endometriosis.

[0326] Binding data across all mammals are shown in Table 3. As shown in Table 3, the binding of compound 491 to β-linkin is almost identical among mice, rats, dogs, and humans.

[0327]

[0328] Example 5: Allosteric conformation of β-linkin target and loop flexibility generate Wnt4 activity for both efficacy and safety. The purpose of this embodiment is to demonstrate the allosteric conformational changes of β-linkin, where binding at one site induces a structural transformation throughout the molecule, allowing it to translocate from the cytoplasm to its interaction with the cell membrane. β-linkin is known to inherently possess several structures that allow it to reside in either the membrane or the cytoplasm. It was previously assumed that the TCF4 and BCL9 binding sites could not be simultaneously affected by binding to small molecules because these two binding sites are located on opposite sides of a large protein. However, Table 4 describes how the binding of agents and / or peptides to β-linkin can have an effect across two sites, similarly indicating the inherent full-protein flexibility of β-linkin due to its conformational adaptation. This flexibility allows cytoplasmic β-linkin to adopt a conformation that promotes membrane localization, similar to the effect of Wnt4 agonists. Therefore, this demonstrates the ability to bind both cytoplasmic and membrane-bound forms of β-linkin due to its flexibility. Relocation and / or support for β-linkin's membrane-binding ability allows for the efficacy of the agent, while allowing β-linkin to remain in the membrane allows for both the safety and mechanism of action of the peptide. This activity is shown in… Figure 15 In this study, after reaching equilibrium, the cellular localization of peptides was mostly at the membrane, suggesting Wnt4 activity of the peptides. Figure 16 Quantified Figure 15 The observations in the study.

[0329] The binding of the agent to one site in β-linkin can promote allosteric conformational changes, affecting both the BCL9 and TCF4 binding sites, as shown in Table 4.

[0330]

[0331] Example 6: Mid-target effect and downstream target This example demonstrates the on-target effects and mechanism of action of the agent (particularly compound 491), and its influence on the β-linkin signaling pathway, leading to its potential for imaging, diagnosis, and / or treatment of diseases driven by β-linkin dysregulation. Endometriosis regression was observed in mice after 3 or 6 weeks of treatment with compound 491. Figure 8 Therefore, mid-target changes were examined in the excised endometriotic lesions. Figures 17-21 The effects of compound 491 administration on endometriotic lesions extracted after treatment in animals were shown. Reduced cell epithelial proliferation was observed. Figure 17 Increased apoptosis of cell epithelium ( Figure 18 Increased recruitment of activated macrophages Figure 19 ), and reduced RNA of downstream target genes of β-linkin ( Figure 20 ) and protein reduction in downstream target genes of β-linkin ( Figure 21 These figures support the mechanism of action and on-target activity of these peptides, and these experimental groups have been replicated with many published compounds.

[0332] Figure 22 It was also shown that labeled peptides targeting the downstream target protein MMP2 can be used to image diseased tissue (in this example, endometriosis) in a mouse model of endometriosis.

[0333] Example 7: Peptide pKa value and pH difference driving peptide specificity in endometriosis cells.

[0334] The specificity of the peptide for diseased cells has been documented. This is supported by the understanding that unhealthy cells exhibit a different intracellular pH (pHi) compared to healthy cells, thereby allowing increased permeability in pathological cells and decreased permeability in healthy cells. We tested endometriosis cells and healthy cells via an intracellular pH indicator dye (Invitrogen™, catalog number P35372) and found that the pH of healthy cells was 7.2, while that of endometriosis cells was 4.5. To assess whether compound 491 undergoes changes in membrane permeability with pH, ​​PAMPA was performed across pH 3 to pH 10 according to published literature. We confirmed that the permeability of compound 491 differs significantly from high to low permeability according to pH. As two examples, the pKa distributions of compounds 491 and 430 show pKa values ​​of 6.7 and 5.2, respectively; both values ​​fall between the pH values ​​of endometriosis cells (4.5) and healthy cells (7.2). In summary, these data suggest that these compounds can be observed to drive the selectivity of endometriosis cells by exhibiting dramatic changes in cell permeability based on protonation state in response to different pH environments.

[0335] Example 8: The peptides disclosed herein do not exhibit off-target activity.

[0336] The Panlabs SAFETYScan assay covers 491 compounds. Panlabs SAFETYScan is a safety panel with 78 targets used for safety assessment in drug discovery to predict adverse clinical events.

[0337] Compound 491 did not exhibit any off-target activity, including against transporters used for arrhythmia assessment, hERG, CaV, and NaV, even at the highest concentrations tested.

[0338] Example 9: The peptides disclosed herein do not affect the HPG axis, clinical chemistry, or hematology.

[0339] To visualize potential off-target effects on the reproductive organ system, the uterus and ovaries, as well as the peritoneal wall, were visualized by H&E following treatment with compound 491. In these organs (uterus and ovaries); Figure 23 No toxic or necrotizing off-target effects were observed. Furthermore, compound 491 did not alter the uterine cycle / HPG axis or uterine weight at autopsy (data not shown). Following treatment with compound 491, standard veterinary clinical chemistry parameters (Table 5) and hematological results (data not shown) were within normal ranges. Further toxicological studies confirmed that no adverse reactions were observed at the maximum feasible dose (>2000 × expected clinical dose exposure).

[0340]

[0341] In summary, the preliminary data lay a solid foundation demonstrating that the peptides from our library can be used as effective diagnostic agents because these peptides (1) bind directly to β-linkin even when conjugated to the imaging portion, (2) are cell-permeable even when conjugated to the imaging portion, (3) have a detectable signal that is not diminished by conjugation to the peptide, (4) exhibit a sustained half-life in the lesion after blood clearance, (5) are not absorbed by healthy cells, (6) accurately detect the lesion during in vivo imaging compared to ex vivo autopsy reports, (7) can quantify disease burden and therapeutic efficacy, (8) show pH-selective targeting of diseased cells based on pKa properties, (9) show no toxicity or off-target activity, and (10) are non-hormonal. Therefore, our central hypothesis is that novel non-hormonal peptides specific to endometriosis cells conjugated to the iron imaging portion will be used as safe and targeted MRI imaging agents. This imaging agent is expected to reliably, specifically, and sensitively detect endometriosis using a non-invasive method.

[0342] Example 10: Pharmacokinetic (PK) analysis confirmed the blood clearance of the peptide.

[0343] PK studies were conducted in CD1 healthy mice to perform spectral analysis of the absorption, distribution, and excretion of compound 491 in plasma via LC / MS (Table 6). Compound 491 was detected up to 24 h post-administration, at which point it was cleared from the bloodstream, similar to imaging results. Subjects with endometriosis specifically retained the compound in their lesions, and it remained visible after plasma clearance, whereas subjects without endometriosis showed no detectable amount of the compound after clearance from the bloodstream. Figure 1 and Figure 6 In summary, these data confirm that the labeled compound 491 has imaging potential for visualizing endometriosis lesions beyond its plasma presence.

[0344]

[0345] Example 11: Evaluation of the pharmacokinetics, therapeutic activity, and target binding of diagnostic compounds We tested several compounds in the library that specifically bind to β-linkin and investigated their relationship with therapeutic pharmacodynamics and pharmacokinetics in plasma and lesions. The aim of these experiments was to investigate whether compounds need to possess high therapeutic activity to demonstrate diagnostic efficacy.

[0346] Reporter assays and target binding were performed according to the protocol in Table 4. Protein binding was measured in five human plasma aliquots (final 10 μM) and incubated at 37°C for 4 hours using a rapid equilibration dialysis apparatus (Thermo Scientific™). Samples from both sides of the dialysis apparatus were prepared for LC-MS / MS analysis, with propranolol hydrochloride used as a positive control. The percentage of protein binding was calculated as follows: Protein binding (%) = (Area p - Area b / Area p) × 100. Plasma stability was analyzed by pre-incubating human plasma (990 μL) at 37°C and 75 rpm for 5 min. 10 μL of the compound was added. 50 μL aliquots were collected in duplicate into 1.5 mL tubes containing 150 µL warfarin (200 ng / mL) at 0, 0.5, 2, 3.5, 4, 4.5, and 5 min. The tubes were incubated at 37°C and 75 rpm for up to 120 min. The samples were then centrifuged at 17000×g for 10 min at 4 °C. 100 μL of the supernatant was transferred to a 96-well plate containing 100 μL of water, vortexed, and then centrifuged at 3750 rpm for 10 min. Pharmacokinetic analysis was performed by administration of the compound at 1 mg / kg to CD1 mice, with blood samples collected at the following time points: before administration and 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post-administration. After centrifugation, the collected plasma was analyzed by LC-MS / MS to determine the t1 / 2 and peptide concentration (in ng / ml) based on a standard curve. The pharmacodynamic effects of each peptide were analyzed by intraperitoneal administration of a low dose of 0.325 mg / kg or 1 mg / kg / day for 3 weeks. The results are shown in Table 8.

[0347] A key property is the binding affinity of the peptide to its target. All compounds exhibited some degree of plasma stability; however, crucially, they were specifically retained in lesions for a longer period than they exist in the bloodstream, allowing for disease imaging after plasma clearance. Compounds with longer half-lives showed undetectable plasma levels at 24 hours but significant retention within lesions, indicating potent diagnostic compounds. Changes in plasma degradation and their inverse relationship with protein binding suggest that, unbound by theory, intracellular proteins or target binding protect compounds from degradation. Unbound by theory, compounds may be protected from plasma proteases by intracellular localization or binding to intracellular targets after intracellular translocation, suggesting that target binding may contribute to reducing degradation in lesions. Retention within lesions exceeding plasma levels, regardless of therapeutic potency, confirms their potential as diagnostic candidates. Unbound by theory, rapid clearance from the bloodstream may contribute to reduced therapeutic efficacy; however, it still allows for effective diagnostic performance. Therefore, compounds with or without strong therapeutic potency remain strong diagnostic candidates due to their clearance from the bloodstream and specific uptake by disease cells.

[0348] Example 12: Conjugation of diagnostic peptides with iron-based components Peptides were conjugated with functionalized iron oxide nanoparticles to enable MRI detection. For this embodiment, iron oxide nanoparticles <100 nm were used because these small particles have good cell-targeting potential, and below 100 nm, the immune system is unlikely to recognize them. Additionally, smaller nanoparticles have been observed to exhibit an enhanced permeability effect (EPR) as unhealthy cells are surrounded by vascular endothelial cells with enhanced permeability via leaky connections.

[0349] The examples described herein depict peptide conjugation using methods including functionalized iron oxide nanoparticles or [iron]DOTA, enabling their visualization via MRI imaging. Figure 24AIn this study, the iron nanoparticles, functionalized iron oxide nanoparticles, and conjugation kit were obtained from Ocean Nanotech (San Diego, CA). Hydrodynamic dimensions, PDI, and zeta potential were confirmed using the Malvern Zetasizer (Malvern Panalytical, Malvern, Worcestershire, United Kingdom). To enable the conjugation of amine-functionalized iron oxide nanoparticles with peptides containing cysteine ​​residues at the N-terminus, the nanoparticles were reacted with sulfonyl-SMCC (succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylic acid ester) to promote the formation of a stable amide bond. The free thiol group present on the peptide reacts with the intermediate functionalized iron oxide nanoparticles to generate a peptide-iron oxide conjugate. See also: Figure 24B To achieve the conjugation of iron oxide nanoparticles with peptides via carboxyl-functionalized nanoparticles, the carboxyl groups on the nanoparticles were chemically activated using carbodiimide chemistry involving EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) to form a stable amide bond with an amine group at the N-terminus of the peptide. For [iron]DOTA-conjugated peptides, the DOTA moiety was first linked to the peptide via solid-phase peptide synthesis (SPPS), targeting the N-terminus, C-terminus, or specific side chains to ensure the functionality and stability of the final complex. Next, iron ions were introduced into the DOTA peptide conjugate to promote efficient chelation of iron within the DOTA cage. The resulting iron-conjugated peptides were purified using high-performance liquid chromatography (HPLC) to remove any unreacted components and byproducts, followed by thorough characterization of the complex.

[0350] Example 13: Another example of imaging.

[0351] Peptides were synthesized using fluorophores to achieve imaging as disclosed herein (Table 7):

[0352]

[0353] 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 to which this disclosure pertains. Publications cited herein and the materials they cite are specifically incorporated by reference.

[0354] Those skilled in the art will recognize or be able to determine numerous equivalents of the specific embodiments of the present disclosure described herein using experiments no more than those employed in the conventional manner. Such equivalents are intended to be covered by the appended claims.

Claims

1. An imaging agent comprising a peptide or peptide mimic conjugated to an imaging portion via a linker, wherein: (a1) The peptide or peptide mimic comprises an amino acid sequence according to Formula I: R-X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 - X 13 -X 14 -X 15 -X 16 (Formula I), in: R is NH2, acetylated, stearic acid, palmitic acid, myristic acid, lauric acid, C1-C8 hydrocarbon, C1-C8 fatty acid, or absent; X1 is M, G, β-alanine, leucine, valine, or absent; X2 is W, N-methyl-W, R, Y, F, citrulline, or K. X3 is P, W, N-methyl-W, N-ethyl-W, N-methyl-A, N-ethyl-A, L, Pip, Aib, Y, or F; X4 is E, Q, N, or D; X5 is S, α-methyl-S, K, D, Orn, T, or E; X6 is I, Chg, H, or L; X7 is either L or I; X8 is D, N, E, or Q; X9 is D, E, K, Q, or Orn; X 10 It is H or methyl-H; X 11 It is V, α-methyl-V, Chg, L, I, or valine; X 12 It is Q, Aib, S, R, or N; X 13 It is R, K, citrulline, Orn, D, or E; X 14 It is V, I, L or valine; X 15 It is W, Y, or F; and X 16 It is R, G, or does not exist; or (a2) The peptide or peptide mimic comprises an amino acid sequence according to Formula II. X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 - X 13 -X 14 -X 15 -X 16 (Formula II) in: X1 is M or does not exist; X2 is S, I, G, T, A, L, or does not exist; X3 is R, K, or does not exist; X4 is a positively charged amino acid, citrulline, Orn, D, E, 8-aminooctanoic acid, or an aminocarboxylic acid with between 4 and 12 carbon atoms; X5 is M, ortholeucine, Orn, D, E, K, H, R, K, 8-aminooctanoic acid, an aminocarboxylic acid with between 4 and 12 carbon atoms, or is absent; X6 is W, Y, F, or N-methyl-A; X7 is F, I, L, Chg, Cha, or Tie; X8 is L, I, or A; X9 is L, I, or A; X 10 It is C, S, A, Abu, C(me) or S(Bzl); X 11 It is F, H, A, K, E, Chg, Cng, or Orn; X 12 It is W, Y, A, or F; and X 13 It is G, GABA, or does not exist; X 14 It is V, I, L, or valine, or it is absent; X 15 It is W, Y, F or does not exist; and X 16 It is R, G, or does not exist; and (b) The connector is absent or the connector is conjugated to the imaging portion and the peptide or peptide mimic.

2. The imaging agent according to claim 1, wherein the imaging component is a fluorescent molecule, a magnetic resonance imaging (MRI) contrast agent, a radionuclide, a microbubble, a computed tomography (CT) contrast agent, or a photoacoustic contrast agent.

3. The imaging agent according to claim 2, wherein the imaging component is a fluorophore, fluorescent dye, dye, pigment, fluorescent transition metal, or fluorescent protein.

4. The imaging agent according to claim 2, wherein the imaging portion is a paramagnetic ion complex or superparamagnetic magnetite particles.

5. The imaging agent according to claim 2, wherein the imaging component is a gadolinium-based contrast agent, a manganese-based contrast agent, an iron oxide contrast agent, an iron-platinum contrast agent, or an iron complex.

6. The imaging agent according to claim 2, wherein the imaging portion comprises at least one selected from 18 F, 64 Cu、 68 Ga and 99m Radionuclides in the Tc group.

7. The imaging agent according to claim 2, wherein the imaging portion comprises superparamagnetic iron oxide nanoparticles (SPION) or DOTA-Fe(III) complex.

8. The imaging agent according to claim 2, wherein the imaging portion is gold nanoparticles or an iodide compound.

9. The imaging agent according to claim 1, wherein the linker comprises one or more of the following: amino acids, –NH(CH2) n C(O)O–、–(OCH2CH2) m –、–O–、–NH–、C1-C6 alkylene、C1-C6 haloalkylene、C1-C6 alkoxy、C1-C6 heteroalkylene、C2-C6 alkenyl、C2-C6 alkyneyl、alkylamino、alkylthio、alkylcarbonyl、alkoxycarbonyl、alkylsulfonyl、C3-C 10 Cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-C 10 aryl or 5- to 10-membered heteroaryl or combinations thereof, wherein each of the following is a C1-C6 alkylene group, C1-C6 haloalkylene group, C1-C6 alkoxy group, C1-C6 heteroalkylene group, C2-C6 alkenyl group, C2-C6 alkyneyl group, alkylamino group, alkylthio group, alkylcarbonyl group, alkoxycarbonyl group, alkylsulfonyl group, C3-C 10 Cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-C 10 Aryl and 5- to 10-membered heteroaryl groups are optionally and independently bounded by 1, 2, or 3 R groups. 9 Replace; of which: n is an integer from 1 to 10 independently; m is an integer independently from 1 to 10; and R 9 Independently, it is a halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 heteroalkyl, –C(O)H, –C(O)OH, –CN, C3-C 10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-C 10 Aryl, 5- to 10-membered heteroaryl, -C(O)(C1-C4 alkyl), -C(O)O(C1-C4 alkyl), –C(O)NH2, –C(O)NH(C1-C4 alkyl), –C(O)N(C1-C4 alkyl)2, -NH2, -NH(C1-C4 alkyl), -N(C1-C4 alkyl)2, -NH(C2-C4 alkylene)-OH, -NH(C2-C4 alkylene)-O-(C1-C4 alkyl), -OH, -O(C 1-C4 alkyl), -O(C1-C4 haloalkyl), -O(C2-C4 alkylene)-NH2, -O(C2-C4 alkylene)-NH-(C1-C4 alkyl), -O(C2-C4 alkylene)-N-(C1-C4 alkyl)2, -O(C1-C4 alkylene)-C(O)OH, -O(C1-C4 alkylene)-C(O)O-(C1-C4 alkyl), -O(C2-C4 alkenyl), -O(C1-C4 alkylene)-(C6-C 10 aryl), -O (C1-C4 alkylene)- (5- to 10-membered heteroaryl), –O (C6-C 10 aryl), -SH, S(O)2OH, -S(O)2 (C1-C4 alkyl), -S(O)2NH2, -S(O)2NH (C1-C4 alkyl) or –S(O)2N (C1-C4 alkyl)2; or two R 9 Together with the atoms to which they are attached, they form C3-C 10 Cycloalkyl or 3- to 10-membered heterocyclic alkyl rings.

10. The imaging agent according to claim 9, wherein the linker comprises cysteine, -NH(CH2) n C(O)-、-C(O)(CH2) n NH-, -(OCH2CH2) m -、-O-、-NH-、C1-C6 alkylene、C1-C6 alkoxy、C1-C6 heteroalkylene、C3-C 10 Cycloalkylene or 3- to 10-membered heteroalkylene or combinations thereof, wherein each C1-C6 alkylene, C1-C6 alkoxy, C1-C6 heteroalkylene, C3-C 10 Cycloalkylene and 3- to 10-membered heterocycloalkylene are optionally independently bound by 1, 2, or 3 R's. 9 Replace, where n, m and R 9 As defined in claim 36.

11. The imaging agent of claim 1, wherein the connector is in contact with the peptide or peptide mimicry at X1 to X... 16 At least one of the amino acids is up-conjugated.

12. The imaging agent of claim 1, wherein the connector is conjugated to the N-terminus of the peptide or peptide mimic.

13. The imaging agent of claim 12, wherein the peptide or peptide mimic further comprises -NH2 at its C-terminus.

14. The imaging agent of claim 1, wherein the connector is conjugated to the C-terminus of the peptide or peptide mimic.

15. The imaging agent of claim 14, wherein the peptide or peptide mimic further comprises a C1-C8 alkyl-C(O)- at the N-terminus.

16. The imaging agent of claim 14, wherein the peptide or peptide mimic further comprises CH3(CH2) at its N-terminus. p C(O)-, where p is an integer from 0 to 7.

17. The imaging agent of claim 14, wherein the peptide or peptide mimic further comprises CH3(CH2)6C(O)- at its N-terminus.

18. The imaging agent of claim 1, wherein the peptide or peptide mimic comprises the amino acid sequence SEQ ID NO:496 or SEQ ID NO:

497.

19. The imaging agent according to claim 18, wherein the imaging portion is iron oxide nanoparticles.

20. The imaging agent of claim 18, wherein the imaging portion is iron chelated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

21. The imaging agent of claim 1, wherein the peptide or peptide mimic is cyclic or bicyclic.

22. The imaging agent of claim 21, wherein the peptide or peptide mimic is cyclized by Cys-Cys disulfide bonds, cyclized by amide bonds, cyclized using hydrocarbon pinning, or cyclized using click chemistry.

23. The imaging agent of claim 22, wherein the amide bond is a head-to-tail amide bond between the N-terminus and the C-terminus, wherein the amide bond is a head-to-side-chain amide bond between the N-terminus and the internal COOH, wherein the amide bond is a side-to-tail amide bond between the internal NH2 and the C-terminus, wherein the amide bond is a side-to-side-chain amide bond between the internal NH2 and the internal COOH, or any combination thereof.

24. A method for detecting endometriosis lesions in a subject, comprising: (a) Administering to the subject a formulation comprising the imaging agent according to any one of claims 1 to 23; and (b) Imaging the subject to determine the presence of the imaging agent.

25. The method of claim 24, further comprising: (c) Administering the subject a second β-linkin-binding peptide or a peptide mimic; and (d) Wait at least 1 week, and then repeat steps (a) and (b).

26. The method of claim 24, wherein the imaging in (b) further comprises creating an image and displaying the image.

27. The method of claim 26, wherein the image is a video, a still image, a live video, or any combination thereof.

28. The method of claim 27, wherein the subject or the image is visualized or displayed by the imaging agent during surgery performed on the subject.

29. The method of claim 28, wherein the imaging agent is administered during or before surgery for the diagnosis, staging, and / or resection of endometriosis.

30. The method of claim 24, wherein the preparation is administered vaginally or intravenously.

31. The method of claim 24, wherein the imaging in (b) includes in vivo fluorescence imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), ultrasound imaging, computed tomography (CT), or photoacoustic imaging.

32. The method of claim 24, wherein the subject is a human, a cultured cell line, or an ex vivo tissue extracted from an animal.

33. The method of claim 24, wherein the imaging includes whole-body imaging.

34. The method of claim 24, wherein the second β-linkin-binding peptide or peptide mimic comprises an amino acid sequence according to Formula I: R-X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 - X 13 -X 14 -X 15 -X 16 (Equation I), where: R is NH2, acetylated, stearic acid, palmitic acid, myristic acid, lauric acid, C1-C8 hydrocarbon, C1-C8 fatty acid, or absent; X1 is M, G, β-alanine, leucine, valine, or absent; X2 is W, N-methyl-W, R, Y, F, citrulline, or K. X3 is P, W, N-methyl-W, N-ethyl-W, N-methyl-A, N-ethyl-A, L, Pip, Aib, Y, or F; X4 is E, Q, N, or D; X5 is S, α-methyl-S, K, D, Orn, T, or E; X6 is I, Chg, H, or L; X7 is either L or I; X8 is D, N, E, or Q; X9 is D, E, K, Q, or Orn; X 10 It is H or methyl-H; X 11 It is V, α-methyl-V, Chg, L, I, or valine; X 12 It is Q, Aib, S, R, or N; X 13 It is R, K, citrulline, Orn, D, or E; X 14 It is V, I, L or valine; X 15 It is W, Y, or F; and X 16 It is R, G, or does not exist.

35. The method of claim 34, wherein the second β-linkin-binding peptide or peptide mimic has the amino acid sequence Oct-W-NMeA-Q-Orn*-ILDE*H-Chg-QRVWG-NH2 (SEQ ID NO: 491).

36. The method of claim 24, wherein the second β-linkin-binding peptide or peptide mimic comprises the amino acid sequence according to Formula II: X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 - X 13 -X 14 -X 15 -X 16 (Equation II), where: X1 is M or does not exist; X2 is S, I, G, T, A, L, or does not exist; X3 is R, K, or does not exist; X4 is a positively charged amino acid, citrulline, Orn, D, E, 8-aminooctanoic acid, or an aminocarboxylic acid with between 4 and 12 carbon atoms; X5 is M, ortholeucine, Orn, D, E, K, H, R, K, 8-aminooctanoic acid, an aminocarboxylic acid with between 4 and 12 carbon atoms, or is absent; X6 is W, Y, F, or N-methyl-A; X7 is F, I, L, Chg, Cha, or Tie; X8 is L, I, or A; X9 is L, I, or A; X 10 It is C, S, A, Abu, C(me) or S(Bzl); X 11 It is F, H, A, K, E, Chg, Cng, or Orn; X 12 It is W, Y, A, or F; X 13 It is G, GABA, or does not exist; X 14 It is V, I, L, or valine, or it is absent; X 15 It is W, Y, F or does not exist; and X 16 It is R, G, or does not exist.

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