Compositions and methods for imaging and treating endometriosis

Abstract

This specification provides peptides that bind to β-catenin and compositions comprising such peptides. Agents utilizing peptides that directly bind to β-catenin can be used for imaging, diagnosis, and treatment of diseases caused by β-catenin dysfunction. This specification also provides methods for using such peptides and compositions in imaging, diagnosis, and treatment of tissue-invasive diseases, including endometriosis.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 504,185, filed on 24 May 2023, which is incorporated herein by reference in whole.

[0002] Statements concerning the rights of the government This invention was made with government support under grant number HD100289 granted by the National Institutes of Health. The government has certain rights to this invention.

[0003] Sequence List This application, filed in ST.26 format, created on 24 May 2024, and titled "170502-2010 Sequence Listing," contains a sequence listing of 566,033 bytes. The contents of the sequence listing are incorporated herein by reference in their entirety. [Background technology]

[0004] Endometriosis (EMS) affects approximately 10% of women and young women, impacting over 7.4 million women in the United States alone and over 176 million women worldwide. EMS is the leading cause of disability and infertility among women of reproductive age, with 40% of infertile women suffering from EMS. The average age at diagnosis is 28. Debilitating symptoms include painful menstruation, chronic pain, pain during intercourse, and infertility. In addition to human suffering, the productivity losses and direct healthcare costs associated with this condition in the United States exceed $90 billion annually. EMS is caused by retrograde menstruation, in which viable endometrial tissue flows backward through the fallopian tubes into the abdominal cavity. There, the endometrial tissue attaches to multiple external sites (e.g., fallopian tubes, ovarian fossa, peritoneal wall, ligaments, and intestines) and responds to hormones (Sampson, JA, Am J Pathol 3, 93-110 143, 1927). The gold standard for diagnosing endometriosis is visual examination using laparoscopy, preferably with histological confirmation. Because there is no superior non-invasive test for endometriosis, the diagnosis of this disease is often significantly delayed. Therefore, non-invasive methods for detecting endometriosis are needed.

[0005] Currently, the only way to diagnose endometriosis is through laparoscopic surgery, and the average delay from symptom onset to diagnosis is a very long 4 to 12 years. Current efforts to use scanning techniques are at best experimental. Ultrasound is limited by its field of view and depends on the operator's skill level, and experimental MRI of endometriosis shows great variability in protocol (T1T2) and utilizes gadolinium, which is particularly toxic to women of reproductive age. Because the results are highly inconsistent and the sensitivity is low to detect certain widespread subtypes of endometriosis, these methods are not suitable as an alternative to surgery. [Overview of the Initiative]

[0006] To begin addressing this significant unmet need in the diagnosis of endometriosis, this specification discloses non-invasive diagnostic agents targeting endometriosis. A non-invasive diagnostic agent available on a broad market, including for superficial endometriosis which can cause widespread and painful symptoms, can provide early detection. For patients, this can eliminate years of pain, anxiety, and confusion currently experienced before a proper diagnosis, and can lead to changes in healthcare costs. Early intervention can also lead to better outcomes for women with endometriosis, such as preserving fertility.

[0007] The development of a definitive diagnostic approach that specifically targets endometriotic lesions is expected to enhance both the reliability and reproducibility of results, while minimizing dependence on variables such as instrumentation and operator expertise. The targeted diagnostic strategy of this disclosure can overcome the limitations currently faced by laparoscopic evaluation and contrast agents, providing a more reliable and reproducible means of diagnosing endometriosis.

[0008] The agents of this disclosure can be used in whole-body scans with readily available conventional MRI machines, including models with low Tesla values. This includes compatibility with mobile and open MRI systems, enhancing accessibility and comfort, particularly for individuals with claustrophobia.

[0009] Because contrast agents like gadolinium reduce accuracy, the use of more powerful Tessa machines or PET / MRI scans is required. These machines are limited to selecting the imaging center and can at best only visualize limited subtypes of endometriosis. The potential of our technology as a targeting probe is that it can significantly improve lesion detection without requiring the highest resolution equipment. Most available MRI machines are conventional models with lower magnetic field strengths, which are less expensive but ineffective with gadolinium contrast agents. The agents and methods of this disclosure have the potential to make the diagnosis of endometriosis more accessible to all populations, including in underserved locations, particularly in situations where only lower-intensity MRI machines are available. Furthermore, the solutions of this disclosure would be immediately marketable.

[0010] The diagnostic agents of this disclosure can be delivered to all women, regardless of age, ethnicity, or economic status, who are suspected of having endometriosis or who are at high risk of having such disease.

[0011] When used in conjunction with whole-body MRI scans, the imaging agent can diagnose endometriotic lesions of all subtypes and stages in any organ system. The goal is to provide both patients and healthcare providers with better insights into disease progression and the prognosis of data-driven care. For example, endometriosis of the bladder may present as painful UTIs, and endometriosis of the appendix may present as extremely painful appendicitis. Importantly, this MRI approach also enables the detection and characterization of malignancies, providing patients with crucial information to ensure they receive appropriate care.

[0012] The target imaging agent of this disclosure contains a peptide conjugated to an imaging component, such as an iron-based component. Since iron / iron oxide has been proven safe, this solution is expected to not only outperform but also avoid the serious side effects of gadolinium.

[0013] The key characteristics of an effective diagnostic agent are retention in the target lesion and rapid clearance from the bloodstream, which creates a window of imaging opportunity from blood clearance to degradation of the imaging agent within the lesion. Based on our research, our pharmacokinetic (PK) data in both the lesion and bloodstream indicate an imaging timeframe of at least one day. This duration is beneficial because it provides scheduling flexibility, allowing scans to be performed even if appointments are delayed. Other experimental techniques based on contrast technology have shorter imaging windows, which may be impractical or difficult for clinical use.

[0014] Currently, tracking the success of clinical trials for endometriosis treatment is limited to highly subjective and nonspecific pain. Non-surgical diagnostic methods are revolutionary and will enable data-driven assessments of the impact of treatment on disease progression.

[0015] The agents disclosed herein can serve not only as efficacy biomarkers but also help measure the appropriate duration of treatment necessary to eliminate the disease. This approach could revolutionize the development of endometriosis treatment by eliminating the need for second-look laparoscopy. Also disclosed are β-catenin conjugates that can be used to treat endometriosis. These diagnostic and therapeutic agents, used together, can eliminate endometriosis as a long-term disease.

[0016] Details of one or more embodiments of this disclosure are given in the accompanying drawings and the following description. Other features, purposes, and advantages of this disclosure will become apparent from the description and drawings, as well as the claims. [Brief explanation of the drawing]

[0017] [Figure 1]Figure 1 shows the identification of subjects with endometriosis via labeled compounds 492-494 as an imaging diagnostic tool. Visualization of the lesions is consistent between in vivo and ex vivo imaging. This is for illustrative purposes only, and similar tests have been performed with other compounds, including compound 495. [Figure 2] Figure 2 shows the identification of the in vivo distribution of compounds 491 and 492 via the Cy7-labeled version of the peptide (compound 492). Compound 491 is highly specific to diseased tissues and is present in little to no quantity, or undetectable, in other organs including the heart, lungs, uterus, ovaries, GI organs, and bone. This is illustrative and has been done with other compounds, including compounds 493-495. [Figure 3] Figure 3 shows the intravaginal administration of compound 494, which supports the idea that intravaginal administration is a viable option for distributing the drug to the lesion. This is also done with other administration routes, including but not limited to SQ, IV, and PO administration, as well as with other labeled peptides. [Figure 4] Figure 4 shows that the peptide can penetrate vaginal tissue and that intravaginal administration of the peptide takes approximately 3 hours to pass through human vaginal tissue. This is consistent with imaging performed in animal models using a labeled agent (compound 491 is shown here for illustrative purposes). [Figure 5] Figure 5 shows that the elimination pathway can be determined using labeled agents / peptides, and for this figure, compound 492 is represented as feces. [Figure 6] Figure 6 shows the time course of spectral imaging of C57BI / 6 albino mice treated with compound 494. This demonstrates that labeling can be used on live mice to identify endometriotic lesions after blood clearance (approximately 24 hours later; here indicating SQ delivery), and that scanning at multiple time points can show the retention time of the drug in endometriotic lesions, in this case, 24 to at least 48 hours after administration. This is illustrative and has been done with other compounds, including compounds 492-495, using different administration routes with different serum pharmacokinetic profiles. [Figure 7] Figure 7 shows the tracking of lesion reduction in the same mice after two SQ administrations several weeks apart, demonstrating that daily administration is not necessary for efficacy. This is illustrative and has been done with other compounds, including compound 492 (shown here). [Figure 8] Figure 8 shows that efficacy improves with longer treatment times, especially when administered at lower doses. [Figure 9] Figure 9 shows pharmacokinetic drug exposures analyzed in serum. Compared to Figures 6 and 7, the pharmacokinetic effect lasts longer than that analyzed in serum. [Figure 10] Figure 10 shows a cell proliferation assay confirming that a single dose of compound 491 has a long-lasting pharmacodynamic effect within the lesion, and that this effect persists longer than that of serum pharmacokinetics (shown 5 days after treatment). Figure 10 also shows that while more frequent administration may be beneficial, it is not necessary for efficacy. [Figure 11] Figure 11 shows that compound 492 directly binds to the target β-catenin within the cell, and that compound 492 is cell-permeable, binding to the target in the cytoplasm of the cell rather than the nucleus, allowing imaging without signal quenching due to intracellular localization of the peptide or imaging agent. [Figure 12A] Figures 12A and 12B show that compound 492 binds to β-catenin after 1 hour of incubation, that compound 492 is present in the cytoplasm, and that the signal remains stable for at least 3 days. [Figure 12B] Figures 12A and 12B show that compound 492 binds to β-catenin after 1 hour of incubation, that compound 492 is present in the cytoplasm, and that the signal remains stable for at least 3 days. [Figure 13]Figure 13 shows the downstream effects of the binding described in Figure 12. Figure 13 shows that after the binding of compound 492 to β-catenin in the cytoplasm, endogenous cytoplasmic β-catenin protein begins to decrease. In the nuclear fraction, the same trend was observed, even though the peptide did not directly interact with the nucleus, but instead sequestered excess cytoplasmic β-catenin, preventing its translocation to the nucleus and redirecting it towards the membrane. [Figure 14A] Figures 14A to 14C show that endogenous β-catenin protein remains stable in the non-nuclear / non-cytoplasmic (membrane) fraction and that the binding of the peptide to cytoplasmic β-catenin does not inhibit the binding of β-catenin to the membrane. Figures 14A to 14C show the amount of β-catenin after incubation with 0.5 μM compound 492 in the cytoplasmic fraction (Figure 14A), membrane fraction (Figure 14B), or nuclear fraction (Figure 14C). [Figure 14B] Figures 14A to 14C show that endogenous β-catenin protein remains stable in the non-nuclear / non-cytoplasmic (membrane) fraction and that the binding of the peptide to cytoplasmic β-catenin does not inhibit the binding of β-catenin to the membrane. Figures 14A to 14C show the amount of β-catenin after incubation with 0.5 μM compound 492 in the cytoplasmic fraction (Figure 14A), membrane fraction (Figure 14B), or nuclear fraction (Figure 14C). [Figure 14C] Figures 14A to 14C show that endogenous β-catenin protein remains stable in the non-nuclear / non-cytoplasmic (membrane) fraction and that the binding of the peptide to cytoplasmic β-catenin does not inhibit the binding of β-catenin to the membrane. Figures 14A to 14C show the amount of β-catenin after incubation with 0.5 μM compound 492 in the cytoplasmic fraction (Figure 14A), membrane fraction (Figure 14B), or nuclear fraction (Figure 14C). [Figure 15] Figure 15 shows that the imaging peptide is not detected in normal uterine tissue of animals with endometriosis but is found in lesions, thus supporting its disease specificity. This figure also shows the cellular localization of the peptide, which is mostly membrane-bound after reaching equilibrium, thereby demonstrating the peptide's Wnt4 activity. [Figure 16]Figure 16 quantifies the intracellular localization of the disease lesions observed in Figure 15, thereby supporting the mechanism of action observed in Figure 15. [Figure 17] Figure 17 shows that cell proliferation in epithelial cells of endometriotic lesions is reduced after administration of compound 491 to animals, which supports the mechanism of action and on-target activity. [Figure 18] Figure 18 shows that administration of compound 491 to animals increased cell apoptosis in epithelial cells of endometriotic lesions, which supports the mechanism of action and on-target activity. [Figure 19] Figure 19 shows that activated macrophages are recruited to the lesion site in animals after administration of compound 491, supporting the mechanism of action and on-target activity. [Figure 20] Figure 20 shows that lesions extracted from animals treated with compound 491 exhibit a reduction in RNA of the target gene downstream of β-catenin, thus supporting the mechanism of action and on-target activity. [Figure 21] Figure 21 shows that lesions extracted from animals treated with compound 491 exhibit a decrease in the protein of the β-catenin target gene, thus supporting the mechanism of action and on-target activity. [Figure 22] Figure 22 shows imaging of endometriotic lesions in animals demonstrating the successful targeting and binding of a labeled peptide to the target protein MMP2 downstream of β-catenin. [Figure 23] Figure 23 shows that β-catenin peptide (referred to here as compound 491 for illustrative purposes) does not alter the tissue structure of the uterus or ovary. Top: As expected, normal proliferation, glands, stroma, and myometrium were observed in a cross-section of the uterus. Magnification = 100x. Bottom: The ovary was normal, showing follicular formation at all stages from primordial follicle to corpus luteum. Magnification = 400x. Sections were stained with H&E. [Figure 24A]Figures 24A and 24B support the successful synthesis of the MRI agent using zeta potential analysis before and after conjugation of compound 496 with functionalized iron oxide. [Figure 24B] Figures 24A and 24B support the successful synthesis of the MRI agent using zeta potential analysis before and after conjugation of compound 496 with functionalized iron oxide. [Modes for carrying out the invention]

[0018] Unless otherwise defined, all technical terms, notations, and other technical and scientific or specialized terms used herein are intended to have the same meaning as those generally understood by those skilled in the art in which the claimed subject matter belongs. Where applicable, terms that have a generally understood meaning are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from those generally understood in the art.

[0019] Throughout this application, various embodiments may be presented in range format. It should be understood that the range format is merely for convenience and brevity and should not be interpreted as a definitive limitation to the scope of this disclosure. Therefore, range descriptions should be considered as specifically disclosing all possible subranges, as well as the individual numbers within those ranges. For example, a range description such as 1-6 should be considered as having specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and similarly, individual numbers within ranges such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0020] definition As used herein and in the claims, the singular forms "a," "and," and "the" include multiple references unless the context explicitly indicates otherwise. For example, the term "a sample" includes multiple samples, as well as mixtures thereof.

[0021] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement, including determining the presence or absence of an element (e.g., detection). Such terms may include quantitative, qualitative, or both quantitative and qualitative determinations. Alternatively, evaluation may be relative or absolute. “Detecting the presence of ~” includes determining the quantity of something that is present, as well as determining whether or not it is present.

[0022] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” may be a biological entity containing expressed genetic material. A biological entity may be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject may be a tissue, cell, or offspring of a biological entity obtained in vivo or cultured in vitro. A subject may be a mammal. A mammal may be a human. A subject may be diagnosed or suspected to be at high risk of disease. The disease may be endometriosis. In some cases, a subject may not necessarily be diagnosed or suspected to be at high risk of disease.

[0023] The term "in vivo" is used to describe events that occur within the living body of a subject.

[0024] The term "ex vivo" is used to describe events that occur outside the living body of the subject. An "ex vivo" assay is not performed on the subject; rather, it is performed on a sample separate from the subject. An example of an "ex vivo" assay performed on a sample is an "in vitro" assay.

[0025] The term "in vitro" is used to describe events that occur in a container holding an experimental reagent, separating the reagent from the living organism from which the substance is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are utilized. In vitro assays can also encompass cell-free assays in which intact cells are not utilized.

[0026] As used herein, a number preceded by the term "approximately" refers to a number plus or minus 10% of that number. A range preceded by the term "approximately" refers to a range of minus 10% of the minimum value and plus 10% of the maximum value.

[0027] As used herein, the terms “treatment” or “treating” are used in reference to a medical or other intervention regimen for the purpose of obtaining a beneficial or desired outcome in a recipient. Beneficial or desired outcomes include, but are not limited to, therapeutic benefits and / or preventive benefits. Therapeutic benefits may also refer to the eradication or alleviation of the symptom or underlying disease being treated. Therapeutic benefits may also be achieved by eradicating or relieving one or more physiological symptoms associated with an underlying disorder so that improvement is observed in the subject, even though the subject may still be affected by the underlying disorder. Preventive benefits include delaying, preventing, or eliminating the onset of a disease or illness; delaying or eliminating the onset of symptoms of a disease or illness; slowing, stopping, or improving the progression of a disease or illness; or any combination thereof. With respect to preventive benefits, subjects at risk of progression of a particular disease, or who report one or more physiological symptoms of a disease, may receive treatment, even if they have not been diagnosed with the disease.

[0028] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include encoded and unencoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone.

[0029] As used herein, “peptide mimetic” means a peptide mimetic that includes some changes in normal peptide chemistry. Peptide mimetic typically enhances some properties of the original peptide, such as increasing stability, increasing efficacy, enhancing delivery, or increasing half-life. Methods for preparing peptide mimetic based on known polypeptide sequences are described, for example, in U.S. Patents 5,631,280, 5,612,895, and 5,579,250. The use of peptide mimetic may include incorporating non-amino acid residues having non-amide bonds at a given position. One embodiment of the present disclosure is a peptide mimetic in which a compound has an amino acid component replaced by a bond, peptide backbone, or appropriate mimetic. Some non-native amino acids that may be suitable amino acid mimetic models include β-alanine, L-α-aminobutyric acid, L-γ-aminobutyric acid, L-α-aminoisobutyric acid, L-ε-aminocaproic acid, 7-aminoheptanoic acid, L-aspartic acid, L-glutamic acid, N-ε-Boc-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-α-Boc-N-δCBZ-L-ornithine, N-δ-Boc-N-α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.

[0030] Many abbreviations used in the sequences of terms in this specification are defined in Table 1.

[0031] [Table 1]

[0032] The section headings used in this specification are for organizational purposes only and should not be construed as limiting the subjects described herein.

[0033] approach Beta-catenin-binding peptides that can be used for the treatment of endometriosis are disclosed in WO2020 / 106995, which is incorporated by reference in whole for instruction on these peptides, as well as their preparation methods and use. As disclosed herein, these peptides can also be used for diagnostic purposes due to their specificity for endometriosis.

[0034] In normal cells, β-catenin binds to E-cadherin at the cell membrane, resulting in high concentrations of β-catenin at the membrane and low concentrations in the cytoplasm and nucleus.73–76 Conversely, in the pathogenesis of endometriosis, β-catenin separates from the membrane and accumulates in the cytoplasm due to either receptor dysregulation or somatic mutation. Cytoplasmic β-catenin is thought to bind to the estrogen receptor (ESR1) and translocate to the nucleus. The nuclear transcription factor activity of β-catenin upregulates matrix metalloproteinases (MMPs), chloride 3 channels (ClC-3), and other proteins, thereby initiating transformation, invasion, migration, and fibrosis of endometriotic cells and potentially leading to lesions. Epithelial-mesenchymal transition (EMT), involving TGF-β and Wnt81, is essential for the establishment and proliferation of endometriotic lesions because it disrupts the cytoskeleton and intercellular tissue of epithelial cells, producing highly migratory mesenchymal cells. Furthermore, human endometriotic tissue exhibits high expression levels of β-catenin-activated CIC-3, matrix metalloproteinase-9 (MMP9), and SOX proteins. MMP9 supports angiogenesis, enabling endometrial cells to proliferate outside the uterus. Notably, high expression levels of CIC-3 correlate with endometriotic symptoms, including infertility, dysmenorrhea, pelvic pain, dyspareunia, and lesion diameter.

[0035] All the downstream events underlying the pathogenesis of endometriosis are mediated by β-catenin (e.g., mutant Wnt4 [germline], mutant β-catenin [somatic], and overproduction of estrogen (E2) [hormonal]). GWAS studies have revealed a strong association between WNT4 mutations and endometriosis. This is expected, considering the increased likelihood of endometriosis in individuals with a family history of the disease. Furthermore, WNT4 gene expression is significantly higher in orthotopic endometrium than in diseased orthotopic endometrium. Importantly, the wild-type WNT4 protein prevents β-catenin translocation and dysregulation into the nucleus by binding to cytoplasmic β-catenin and transporting it to the membrane. Mutant or downregulated Wnt4 proteins enable the accumulation of β-catenin in the cytoplasm, its translocation to the nucleus, and subsequently the activation of transcription factors that activate IL6, MMP2, MMP7, MMP9, CIC-3, and other P-catenin activating proteins. Our unique peptide candidate specifically targets cytoplasmic β-catenin and inhibits excessive translocation of cytoplasmic β-catenin to the nucleus by redirecting it toward the membrane, similar to Wnt4 activity.

[0036] Therefore, cytoplasmic β-catenin-specific markers represent an attractive approach for targeting endometriosis. Although β-catenin had been considered an untargetable drug due to its protein-protein interactions, we identified suitable cell-permeable cyclic peptide candidates with acceptable ADME profiles for pharmaceutical use using a combination of in vitro, in vivo, and in silico tools. Cytoplasmic accumulation of β-catenin has been observed throughout all stages of endometriosis, including atypical endometriosis, typical endometriosis, and cancerous lesions arising from endometriosis, making it a primary target for diagnosis and treatment.

[0037] Endometriosis can range from isolated small lesions to larger areas of invasive endometrial tissue affecting pelvic organs, including the ovaries, fallopian tubes, intestines, and bladder. The American Society for Reproductive Medicine's classification system categorizes endometriosis into four stages based on clinical findings regarding lesion characteristics (size, location, and surface lesions or DIE), as well as the degree of intraoperative pelvic adhesions. Genetic testing of lesions has confirmed all classifications and organ locations that have mutations in somatic β-catenin or genes that regulate cytoplasmic β-catenin. Furthermore, endometriotic mesenchymal stem cells promote fibrosis in ovarian endometriomas through the Wnt / β-catenin pathway, by producing paracrine TGF-β1 and Wnt1. Multiple types of endometriotic lesions (e.g., atypical, DIE, rectovaginal, and ovarian endometrioma subtypes) may have oncogenic mutations in genes known to upregulate β-catenin, including ARID1A, PIK3CA, KRAS, and PPP2R1A. In short, it is shown that a diagnostic agent targeting cytoplasmic β-catenin could address all stages and subtypes of endometriosis.

[0038] Importantly, the identification of malignant tumors has been successful through MRI findings. This approach, when combined with targeted diagnostics, can identify cancerous lesions, including those that are endometriotic. Early detection of malignant transformation is crucial for proper management. Endometriosis-associated ovarian cancer (EAOC) has been successfully detected on MRI by (1) identification of mural nodule enhancement, (2) tumor size growth (specifically, tumors larger than 9 cm serve as an independent predictor of ovarian cancer), and (3) the absence of low signal intensity on T2, which has been shown to be caused by secretions from malignant tumors that dilute bleeding.

[0039] The agents and methods of this disclosure utilize an innovative approach that uses pH-sensitive peptides as pharmaceuticals for the diagnosis of targeted endometriosis. Specificity to endometriotic tissue is represented by a novel mechanism driven by the pKa of the weakly acidic peptide, thereby enabling selective permeability in diseased tissue. Furthermore, to the best of our knowledge, our group was the first (and only) to identify a three-point pH difference between endometriotic cells and normal cells.

[0040] Our initial toxicity findings did not show any observable toxicity associated with the peptide in our library. We attribute this to our unique approach, which specifically targets cytoplasmic β-catenin in endometriotic tissue via Wnt4-like activity. While β-catenin attachment to the membrane is maintained for cellular health, and disruption of β-catenin's membrane binding can lead to proto-oncogene-like effects, Wnt4 activity is necessary for uterine decidualization, maintenance of the GI duct, and prevention of anemia. Furthermore, our approach is non-hormonal.

[0041] The peptides described herein are potent and have been shown to exhibit a high safety profile even at exposure levels with effects exceeding 2000 times. In addition, peptide degradation products (unlike small molecules) pose little to no risk of toxicity. Therefore, peptides conjugated to MRI-compatible iron are expected to be very safe, as the iron component is not associated with the toxicity typically associated with gadolinium.

[0042] The use of targeted molecular agents for diagnosing endometriosis significantly improves accuracy by exclusively binding to diseased cells, surpassing conventional methods that rely on the absorption of nonspecific contrast agents. The method of this disclosure includes a highly specific peptide designed for pinpoint accuracy in detecting endometriotic lesions down to the sub-millimeter level. This approach is expected to improve reproducibility, reliability, sensitivity, and specificity across all lesion subtypes and organs using standard MRI equipment, thereby increasing availability and reducing scanning costs.

[0043] β-catenin-binding peptide This specification discloses β-catenin conjugates that may be useful in a variety of imaging, diagnostic, and / or therapeutic settings, including, for example, detecting, treating, inhibiting, preventing, monitoring, staging, or mitigating endometriosis (EMS) and its associated symptoms.

[0044] In some embodiments, these agents may be useful for imaging, diagnosing, and / or setting up treatments for colorectal cancer, hepatocellular carcinoma, endometrial cancer, ovarian cancer, medulloblastoma, glioblastoma, melanoma, and other diseases associated with dysregulated Wnt / β-catenin pathways, including Alzheimer's disease.

[0045] In some embodiments, these drugs can be used for the diagnosis, imaging, and treatment of endometriosis, uterine fibroids, adenomyosis, ovarian cysts, polycystic ovary syndrome (PCOS), tubal endometriosis, and other gynecological conditions including cervical polyps, endometrial polyps, and vaginal polyps, and / or their symptoms.

[0046] In some embodiments, the β-catenin binding agent includes a peptide. β-catenin binding peptides are described in WO2020 / 106995, which is incorporated by reference in whole for instruction on these peptides as well as their preparation and use.

[0047] As disclosed herein, the β-catenin-binding peptides of this disclosure can be used to selectively target endometriotic lesions with a drug. In some embodiments, the peptide targets diseased tissue having a pathogenesis related to β-catenin dysregulation. In some embodiments, the drug and peptide can identify diseased tissue. In some embodiments, the drug and peptide preferentially target diseased tissue over normal tissue. In some embodiments, the peptide is undetectable in normal tissue, such as uterine tissue. While we do not wish to be bound by theory, the peptide is selective for β-catenin, has a cell-permeable structure, and is directed towards the cytoplasm where there is an excess of available targets in diseased tissue (cytoplasmic β-catenin).

[0048] The peptides of this disclosure may directly visualize and / or address the pathogenesis of EMS, improve disease progression, and prevent or delay recurrence of EMS. In some cases, β-catenin-binding peptides specifically bind to EMS lesions. In some cases, β-catenin-binding agents can reduce existing EMS lesions. In some cases, β-catenin-binding peptides can inhibit dysregulation of the Wnt pathway, which is thought to be one of the underlying causes of EMS. β-catenin-binding peptides may limit the pathogenesis of EMS by preventing the infiltration of endometrial cells. β-catenin-binding peptides may limit the pathogenesis of EMS by inducing apoptosis. β-catenin-binding peptides may induce macrophages that help clear lesions. Alternatively, or in addition, β-catenin-binding peptides may suppress epithelial-mesenchymal transition (EMT). This suppression can limit the proliferation of endometrial lesions. Alternatively, or in addition to the above, β-catenin-binding peptides may reduce paracrine production of TGF-β1 and Wnt1, thereby reducing fibrosis in ovarian endometriomas. In some cases, β-catenin-binding peptides reduce disease burden and inflammation caused by the disease. In some cases, by downregulating cytoplasmic β-catenin and inhibiting the β-catenin complex with TCF / LEF transcription factors, the expression of procytokine IL-6 may be reduced, thereby reducing the expression of target genes including but not limited to IL-6.

[0049] In some cases, β-catenin-binding peptides bind to cytoplasmic β-catenin and return to the membrane via Wnt4-like activity. In some cases, Wnt4-like activity / rescue mechanisms can help maintain bone and colon regeneration while downregulating integrity. In some cases, Wnt4-active peptide agents may be helpful in recurrent implantation failure. In some cases, the Wnt4 rescue mechanism can lead to cytoplasmic β-catenin-specific classical Wnt antagonism downstream by redirecting β-catenin's nuclear localization to the membrane, thereby inhibiting or regulating abnormal transcription factor activity while maintaining important intercellular junctions. These represent an intriguing approach to imaging, diagnosing, and treating diseases caused by β-catenin dysregulation, including but not limited to endometriosis, adenomyosis, fibroids, colon cancer, skin cancer, endometrial cancer, and ovarian cancer.

[0050] Furthermore, β-catenin-binding peptides may reduce the expression of downstream proteins, including but not limited to matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 7 (MMP7), SOX (SRY-related high-mobility group box transcription factor), interleukin-6 (IL6), fibronectin 1 (Fn1), or chloride C3 channel (CIC-3). In some embodiments, this can result in a reduction of angiogenesis. Inhibition or reduction of angiogenesis can inhibit the ability of endometrial cells to proliferate outside the uterus.

[0051] The peptides described herein may be optimized for their affinity to their desired target, absorption or uptake into cells, stability, protease resistance, and other factors. The affinity of a peptide to its intended target can be modified using a variety of methods. For example, affinity can be improved using amino acid substitutions. Furthermore, changes in peptide size, changes in cyclization position, increased hydrophobicity, and modifications of amino acids or the incorporation of artificial amino acids into the peptide structure may affect the affinity of the peptide. Exemplary modifications include the incorporation of N-methyl and α-methyl amino acids.

[0052] The embodiments provided include peptides, peptide mimetics, and / or β-catenin binders having the same or similar structures and properties as the molecules described herein. Such peptide mimetics comprise small protein-like chains designed to mimic peptides. These include peptide mimetics involving peptide modifications or the design of similar systems that mimic peptides such as peptoids and β-peptides.

[0053] Therefore, in this specification, the formula X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 A peptide or peptide mimetic comprising an amino acid sequence having the formula is disclosed, where X1 is M or null, X2 is S, I, G, T, A, L, or null, X3 is R, K, or null, X4 is a positively charged amino acid, citrulline, Orn, D, E, 8-aminooctanoic acid, or an aminocarboxylic acid having 4 to 12 carbon atoms, X5 is M, norleucine, Orn, D, E, K, H, R, K, 8-aminooctanoic acid, an aminocarboxylic acid having 4 to 12 carbon atoms, or null, X6 is W, Y, F, or N-methyl A, X7 is F, I, L, Chg, Cha, or Tle, X8 is L, I, or A, X9 is L, I, or A, X 10is 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, X 13 is G, GABA, or null, X 14 is V, I, L, norvaline, or null, X 15 is W, Y, F, or null, X 16 is R, G, or null.

[0054] In this specification, a peptide or peptidomimetic comprising an amino acid sequence having the formula X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 is provided, wherein R is NH2, acetylation, stearic acid, palmitic acid, myristic acid, lauric acid, C1-C8 hydrocarbon, C1-C8 fatty acid, or null, X1 is M, G, β-alanine, norleucine, norvaline, or null, 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 L or I, X8 is D, N, E, or Q, X9 is D, E, K, Q, or Orn, X 10 is H or methyl-H, X 11 is V, α-methyl V, Chg, LI, 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, X 15 is W, Y, or F, X 16is R, G, or null.

[0055] In some embodiments, the peptide or peptide mimetic has the amino acid sequence Ac-CW-{NMeA}-QK * ILDD * -H-{Chg}-QRVWG-NH2 (SEQ ID NO: 496) or (Ahx)-NH-W-NMeA-QK * ILDD * It contains H-Chg-QRVWG-NH2 (Sequence ID 497).

[0056] Accordingly, the Specified Publication discloses peptides and peptide mimetic agents comprising any one amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 499, or peptides and peptide mimetic agents comprising amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 499.

[0057] In some embodiments, the imaging component is iron oxide nanoparticles. Therefore, in some embodiments, the imaging agent is of formula:

[0058] [ka] It has.

[0059] In some embodiments, the peptides or peptide mimes of this disclosure can be covalently conjugated to superparamagnetic iron oxide nanoparticles (SPIONs) (e.g., pegged SPIONs) via a linker using the method described in Liu et al. Nanoscale 2012 4:2306, which is incorporated by reference in whole for teaching this method.

[0060] In some embodiments, the imaging component is iron chelated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Therefore, in some embodiments, the imaging agent is of the formula {DOTA-[iron]}-(Ahx)-NH-W-NMeA-QK * ILDD * It contains H-Chg-QRVWG-NH2 (compound 499, sequence number 499).

[0061] In some embodiments, the peptide has pKa values ​​ranging from about 4.5 to about 7.2, including 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.

[0062] In some embodiments, the peptide directly binds to β-catenin. In some embodiments, 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 enables 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 β-catenin membrane binding. In some embodiments, the peptide prevents β-catenin from acting as a transcription factor to oncogenes, MMP2, MMP7, SOX, IL6, Fn1, or chloride C3 channel (CIC-3). In some embodiments, the peptide prevents transformation, invasion, migration, fibrosis, or any combination thereof of endometriosis (EMS) cells. In some embodiments, the peptide activates cell apoptosis and macrophages in diseased tissue. In some embodiments, systemic necrosis does not occur. In some embodiments, the peptide does not affect the HPX axis (i.e., the hormonal cycle). 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 interferes with oncogenic transcription factor activity. In some embodiments, the peptide has an EC of about 50 μM, about 30 μM, about 10 μM, about 5 μM, about 1 μM, 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 less than about 5 pM. 50It downregulates Wnt pathway activity. In some embodiments, the peptide is not naturally occurring. In some embodiments, the peptide is a cyclized peptide. In some embodiments, the peptide is a bicyclic peptide. In some embodiments, the peptide is cyclized by a Cys-Cys disulfide bond. In some embodiments, the peptide is cyclized by an amide bond. In some embodiments, the amide bond is a head-to-side chain between the N-terminus and the C-terminus. In some embodiments, the amide bond is a head-to-side chain between the N-terminus and the internal COOH. In some embodiments, the amide bond is a side chain-to-tail between the internal NH2 and the C-terminus. In some embodiments, the amide bond is a side chain-to-side chain between the internal NH2 and the internal COOH. In some embodiments, the peptide is cyclized using hydrocarbon stapling. In some embodiments, the peptide is cyclized using click chemistry.For several purposes, the peptides are 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 It has 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, The amino acid residues are 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. In some embodiments, the peptide contains one or more non-natural amino acids. In some embodiments, one or more non-natural amino acids are N-methylamino acids.

[0063] Examples of peptide sequences include compounds 1-499 (sequence numbers 1-499) listed in Table 2.

[0064] [Table 2-1]

[0065] [Table 2-2]

[0066] [Table 2-3]

[0067] [Table 2-4]

[0068] Table 2-5

[0069] Table 2-6

[0070] Table 2-7

[0071] Table 2-8

[0072] Table 2-9

[0073] Table 2-10

[0074] Table 2-11

[0075] Table 2-12

[0076] Table 2-13

[0077] Table 2-14

[0078] Proteins downstream of β-catenin can also be targeted for treatment and imaging for endometriosis and other gynecological diseases. Labeled peptides targeting MMP2 have been shown to visualize lesions (see, for example, Figure 13). These agents can also be used for other diseases affected by β-catenin dysregulation.

[0079] Peptide synthesis The peptides described herein can be produced by synthetic methods. For example, solid-phase peptide synthesis (SPPS) allows for the rapid assembly of peptide chains by sequential reaction of amino acid derivatives on an insoluble porous support. The solid support often contains small polymer resin beads functionalized with reactive groups (such as amine or hydroxyl groups) that link to the nascent peptide chain. Since the peptide remains covalently bound to the support throughout the synthesis, excess reagents and byproducts can be removed by washing and filtration. Each amino acid bound to the N-terminus of the peptide chain can be protected on its N-terminus and side chain using an appropriate protecting group such as Boc (acid-labile) or Fmoc (base-labile), depending on the side chain and the protection strategy used (see below).

[0080] A typical SPPS procedure is usually one of several iterative cycles of N-terminal deprotection and coupling reactions. The resin may be washed between each step. First, an amino acid is often coupled to the resin. Next, the amine is usually deprotected and then coupled with the free acid of a second amino acid. This cycle can be repeated until the desired sequence is synthesized. The SPPS cycle may further include a capping step, which can block the unreacted amino acid terminals from reacting. At the end of the synthesis, the crude peptide can be cleaved from the solid support. This step often involves removing all protecting groups simultaneously using a strong acid such as trifluoroacetic acid or a nucleophile. To remove organically soluble byproducts, the crude peptide can be precipitated from a nonpolar solvent such as diethyl ether. The crude peptide can be purified using reversed-phase HPLC. Byproducts can be removed using a continuous chromatography process such as the MCSGP process to maximize yield without sacrificing purity levels.

[0081] The peptides described herein may be cyclic peptides. Cyclic peptides can offer several advantages in certain situations. For example, cyclic peptides are often more resistant to proteases than their acyclic counterparts and are therefore metabolized more slowly. On the other hand, cyclic peptides have a longer-acting depot effect than their corresponding linear counterparts. Cyclic peptides can be used to mimic the structure of biologically active peptides (e.g., peptide hormones) and can bind to in vivo drug targets.

[0082] The peptides described herein may have some flexibility in their ring structure. This flexibility allows for different conformations (confirmation) and enables the peptide to bind to targets that accept different conformational structures. The peptide can penetrate cells and exert its effects on intracellular protein targets (cell-permeable peptides). The large surface area of ​​cell-permeable peptides is superior to that of 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, where binding can occur when the structure of β-catenin changes from a cytoplasmic (stable) structure to an unstable structure that allows for membrane binding. The ability of flexible peptides to bind to cytoplasmic β-catenin allows them to exhibit activity and efficacy, while the ability of the same peptide bound to β-catenin to migrate toward the cell membrane ensures safety during use. Therefore, flexible peptides are advantageous in diagnostic and imaging applications because they are flexible and allow for both safety and efficacy, and can function as Wnt4 agonists. Peptides possess conformations that can mimic molecular structures typically found at protein-protein interaction interfaces.

[0083] The peptides described herein may be cyclized using disulfide crosslinks. Peptide disulfide crosslinks can bond two thiol (SH) groups from the side chains of cysteine ​​or cysteine ​​analogs. Undesirable bonding can be prevented by using appropriate protecting group chemistry, and either specific intramolecular or intermolecular oxidation can be achieved. Generally, disulfide crosslinks can be formed intermolecularly (two peptide molecules are linked via a disulfide crosslink), or intramolecularly (cyclization within a single peptide molecule), resulting in either a homodimer (two identical peptides) or a heterodimer (two different peptides).

[0084] Cyclic peptides can also be synthesized by linking the amino (N) terminus of a peptide to the carboxyl (C) terminus via an amide bond. The amino side chains of Lys and Orn, as well as the carboxyl side chains of Asp and Glu, may also be used to construct cyclic peptides via amide bonds. Depending on the functional group of the peptide, cyclic peptide synthesis often employs one of four different methods: head-tail between the N-terminus and the C-terminus, head-side chain between the N-terminus and the internal COOH (e.g., the β-COOH group of Asp or the γ-COOH group of Glu), side-tail between the internal NH2 and the C-terminus (e.g., the ε-NH2 group of Lys), and side-side chain between the internal NH2 and the internal COOH (e.g., the ε-NH2 group of Lys having either the β-COOH group of Asp or the γ-COOH group of Glu).

[0085] The peptides described herein may be cyclized using staple peptide synthesis. Peptide stapling is typically achieved by incorporating α,α-disubstituted non-native amino acids having terminal olefin tethers of varying lengths. Subsequent olefin metathesis creates carbon-carbon bond tethers between amino acid side chains to cyclize the peptide. Alternatively, staple peptides may be produced using Fmoc solid-phase synthesis chemistry, which is generally known in the art.

[0086] The peptides described herein may be further cyclized using click chemistry. Using various combinations of protected amino acids modified with alkyne groups, clickable functional groups can be incorporated into the peptide during synthesis and subsequently click-reacted with azidic acid. The resulting peptides are separated from the resin to yield triazole-containing peptides. These functional groups can be further introduced by post-synthesis modifications to produce structurally constrained peptides.

[0087] The peptides described herein may be modified to increase their stability. N-methyl modification has generally been shown to 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 is incorporated herein by reference for such disclosure). In some embodiments described herein, peptides are modified with one or more N-methyl analogs to native amino acids. The incorporation of one or more N-methyl amino acids can increase proteolytic resistance by 10 to 10,000 times compared to the resistance of peptides consisting of native residues. In some embodiments, the proteolytic resistance of peptides containing one or more N-methylamino acids is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 400, 500, 600, 700, 800, 90, 10,000 times greater than that of peptides consisting of native residues.

[0088] Imaging agent and method This specification discloses imaging agents comprising one or more of the β-catenin-binding peptides of this disclosure conjugated to an imaging component. Also disclosed is a method for detecting diseased tissue in a subject, comprising administering the imaging agent of this disclosure to the subject and imaging the subject or an object for the presence of the imaging component within the subject's living body.

[0089] In some embodiments, the diseased tissue is an endometriotic lesion. In some embodiments, the diseased tissue is a fibroid. In some embodiments, the diseased tissue is a tumor of colon cancer, ovarian cancer, endometrial cancer, and skin cancer.

[0090] This specification describes methods in all living cells and species. In some embodiments, the subject is human. In some embodiments, the subject is animal. In some embodiments, the subject is cultured cells. In some embodiments, the subject is ex vivo disease tissue taken from animals. In some embodiments, the subject is ex vivo disease tissue taken from humans.

[0091] In some embodiments, the imaging component is inherently cell-permeable. In other embodiments, the imaging component is not originally cell-permeable, but becomes permeable when conjugated with a cell-permeable peptide and functions effectively as cargo for the cell-permeable peptide.

[0092] The peptides and peptide mimes of this disclosure may be labeled with imaging components to function as in vivo imaging probes. In some embodiments, the label is at the N-terminus. In some embodiments, the label is at the C-terminus. In some embodiments, the label is attached to an amino acid side chain. In some embodiments, the label substitutes an atom or molecule in the amino acid sequence (i.e., radiolabeling). In some embodiments, the label is followed by modification. In some embodiments, the modification is lipid, acetylation, and / or amidation.

[0093] The advantage of using the imaging agents described herein is that the peptides bind to targets in the diseased tissue, thus aiding in the signaling of diseased tissue against the background noise of normal tissue. This means that the agents can help visualize very small lesions (less than 1 mm in mice, as described in the examples) and / or lesions located deep within the body, where the detection limits in scanning techniques without imaging agents are much higher or nonspecific. This is important for detecting metastatic diseases such as endometriosis or cancer, where the size and extent of diseased tissue in the patient are unknown. Ensuring the identification of all lesions or tumors is crucial for pathology, staging, and recurrence.

[0094] These imaging agents can revolutionize medical imaging, disease identification, and staging. In some embodiments, the technology may be used for initial diagnosis, providing high-resolution, comprehensive imaging to facilitate accurate disease detection. Other embodiments may use the technology as a tool for surgical preparation, helping to plan procedures in detail by providing thorough visualization of the diseased site. Furthermore, the diagnosis can serve as a benchmark for tracking the progression of an ongoing disease, helping to monitor the patient's health over time and adjust treatment protocols accordingly. There are embodiments in which the technology can evaluate treatment effectiveness during and after the procedure, providing valuable feedback on treatment effectiveness. In some embodiments, repeated scans may be performed, which is particularly useful in chronic or long-term diseases requiring continuous monitoring. In some embodiments, the imaging agent is a companion diagnostic tool to therapeutic agents. In some embodiments, the diagnostic tool may be combined with therapeutic techniques (i.e., theranostics), integrating diagnosis and treatment into a single system for a more holistic and individualized approach to patient care. In some embodiments, the diagnostic compound may also elicit therapeutic efficacy or effects. In some embodiments, peptide imaging agents may also be used to evaluate the properties of active pharmaceutical ingredients (APIs) or drug products, including but not limited to bioavailability, pharmacokinetics (PK), and exclusion pathways.

[0095] Some widely used imaging components include organic dyes for optical imaging, radionuclides for PET and SPECT imaging, and Gd3+ chelating agents for MR imaging. Functional groups of peptides available for conjugation include, but are not limited to, ε-amino groups on lysine side chains, guanidinium groups on arginine side chains, carboxyl groups on aspartic acid or glutamic acid, cysteine ​​thiols, and phenols on tyrosine. The most common conjugation reactions are the coupling of carboxyl and amine mediated by carbodiimide / N-hydroxysuccinimidyl (EDC / NHS), maleimide conjugation to thiol groups, and diazonium modification of phenols on tyrosine.

[0096] Radiolabeled peptides can also be used as peptide-based imaging agents. Radionuclide-based PET / SPECT imaging can detect imaging agents at concentrations ranging from micromolar to picomolar. This allows for minimizing the use of peptides to mitigate any harmful biological effects. To deploy peptide-based probes for PET / SPECT imaging, the radionuclide should be labeled with the target peptide. Some radionuclides used to label peptides are effective in SPECT imaging. 99m Tc, 123 I and 111In are used in PET imaging. 18 F, 64 Cu, and 68These radionuclides generally attach to peptides via chelating agents or radiolabeled groups after synthesis. Some widely used chelating agents 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-glutaric acid-4,7-acetic acid), diethylenetriaminepentaacetic acid (DTPA), mercaptoacetylglycylglycylglycine (MAG3), diaminedithiol (DADT), and 2-hydrazidonicotinic acid (hydrazidonicotinic acid). Examples include acid (HYNIC), N-succinimidyl-4-18F-fluorobenzoic acid (18FSFB), N-succinimidyl-5-iodo-3-pyridinecarboxylic acid (SIB), and N-succinimidyl-3-iodobenzoic acid (SIPC).

[0097] In this specification, the peptide compositions described in this patent may be labeled with contrast agents for CT scans, including but not limited to iron-based, iodine-based, or gold-based contrast agents, or other types of imaging components. These agents containing high atomic number elements such as iodine or gold exhibit excellent X-ray attenuation properties, thereby enabling improved visualization during CT scans.

[0098] The design of fluorophore-labeled peptides is similar to that of radiolabeled peptides, except that the fluorophore is used as a substitute for a radionuclide. Various dyes are commercially available (e.g., cyanine dyes from GE Healthcare and Alexa Fluor dyes from Invitrogen). Fluorescence imaging offers the advantages of high-resolution, non-invasive, and safe detection, but its light transmittance and tissue autofluorescence limit the use of fluorophores in vivo. The introduction of hybrid derivatives incorporating both fluorescent tags and radiolabeling opens new avenues in the field of image-guided surgery.

[0099] In some embodiments, the fluorescent agent is selected from the group consisting of fluorophores, fluorescent dyes, dyes, pigments, fluorescent transition metals, and fluorescent proteins. 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, JA133, AlexaFluor488, AlexaFluor546, AlexaFluor633, AlexaFluor555, AlexaFluor647, DAPI, TMR, R6G, GFP, enhanced GFP, CFP, ECFP, YFP, Citrin, Venus, YPet, CyPet, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine, and Europium.

[0100] Such peptides may include, but are not limited to, SEQ ID NOs: 491 to 499. Compound 495 variant contains a mini-PEG (polyethylene glycol) linker inserted between Cy7-labeled cysteine ​​and the subsequent amino acid. This modification may be beneficial for solubility, stability, or bioavailability.

[0101] The peptides described herein may also be optimized into dimeric or multimeric forms to alter their ability to target receptors. Dimeric and tetrameric forms of similar peptides may exhibit higher binding affinity compared to their monomeric counterparts. Different types of linkers may be introduced between the peptide and the imaging component to optimize these multimeric peptide complexes. These linkers, differing in length, flexibility, and hydrophilicity, can significantly influence the overall binding affinity of the peptide. Using this strategy, heterodimers may be constructed using one or more peptides described herein, which may allow for targeting multiple excess targets and potentially lead to enhanced imaging results.

[0102] In certain embodiments, the fluorescent agent has excitation and emission wavelengths in a range of red and near-infrared spectra. In certain embodiments, the fluorescent agent has excitation and emission wavelengths in the range of 400–1300 nm, or 440–1100 nm, or 550–800 nm, or 600–900 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). Types of probes having excitation and emission wavelengths in other spectra, such as the visible spectrum and ultraviolet spectrum, may also be used in certain embodiments. In particular, fluorophores such as fluorescent dyes or pigments of specific carbocyanins or polymethines can be used as fluorescent agents, for example, U.S. Patent No. 6,747,159 (2004) by Caputo et al., U.S. Patent No. 6,448,008 (2002) by Caputo et al., U.S. Patent No. 6,136,612 (2000) by Della Ciana et al., U.S. Patent No. 4,981,977 (1991) by Southwick et al., U.S. Patent No. 5,268,486 (1993) by Waggoner et al., U.S. Patent No. 5,569,587 (1996) by Waggoner et al., U.S. Patent No. 5,569,766 (1996) by Waggoner et al. U.S. Patent No. 5,486,616 (1996) by al. Waggoner, U.S. Patent No. 5,627,027 (1997) by Brush, et al. U.S. Patent No. 5,808,044 (1998) by Reddington, et al. U.S. Patent No. 5,877,310 (1999) by Shen, et al. U.S. Patent No. 6,002,003 (1999) by Leung et al. U.S. Patent No. 6,004,536 (1999) by Waggoner, et al. U.S. Patent No. 6,008,373 (1999) by Minden, et al. U.S. Patent No. 6,043,025 (2000) by Minden, et al. U.S. Patent No. 6,127,134 (2000) by Waggoner, et al. U.S. Patent No. 6,130,094 (2000), Waggoner, et al.U.S. Patent No. 6,133,445 (2000) by Licha, et al., U.S. Patent No. 7,445,767 (2008) by Licha, et al., U.S. Patent No. 6,534,041 (2003) by Licha et al., U.S. Patent No. 7,547,721 (2009) by Miwa et al., U.S. Patent No. 7,488,468 (2009) by Miwa et al., U.S. Patent No. 7,473,415 (2003) by Kawakami et al., as well as International Publication No. 96 / 17628, European Patent No. 0796111 B1, European Patent No. 1181940 B1, European Patent No. 0988060 B1, International Publication No. 98 / 47538, International Publication No. 00 / 16810, European Patent No. 113822 Examples include Patent No. B1, International Publication No. 01 / 43781, European Patent Application No. 1237583A1, International Publication No. 03 / 074091, European Patent No. 1480683B1, International Publication No. 06 / 072580, European Patent Application No. 1833513A1, European Patent Application No. 1679082A1, International Publication No. 97 / 40104, International Publication No. 99 / 51702, International Publication No. 01 / 21624, and European Patent Application No. 1065250A1, and Tetrahedron Letters 41,9185-88(2000).

[0103] Examples of fluorescent dyes include, for example, Cy5.5, Cy5, Cy7.5, and Cy7 (GE® Healthcare), AlexaFluor660, AlexaFluor680, AlexaFluor790, and AlexaFluor750 (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 Examples include 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), as well as Kodak®X-SIGHT®650, Kodak®X-SIGHT®691, and Kodak®X-SIGHT®751 (Carestream® Health).

[0104] In some embodiments, the imaging component is an MRI agent. In some embodiments, the MRI agent may be Gd(salt), Ir(salt), functionalized iron oxide, iron oxide, paramagnetic chemical exchange saturation transfer (CEST) agent, 19F active substance, manganese, melanin, or a substance that shortens or extends T1 or T2, or a combination thereof. In certain embodiments, the Gd MRI agent may be compounds such as DOTA-Gd, DTPA-Gd, Gd in a polymer chelate, and Gd fixed with a negative charge on the layer. In certain embodiments, as described, the iron MRI agent may be compounds such as DOTA-Ir, DTPA-Ir, functionalized iron oxide, functionalized iron oxide in a polymer chelate, and iron fixed with a negative charge on the layer.

[0105] In certain embodiments, the iron oxide MRI agent may be a compound such as small paramagnetic iron oxide (SPIO) or very small SPIO, or superparamagnetic iron oxide nanoparticles (SPION), with or without a dextran or other stable layer. Superparamagnetic iron oxide nanoparticles (SPION) themselves are not inherently cell-permeable. However, their uptake into cells can be improved by surface functionalization with appropriate ligands or peptides that promote internal translocation into target cells. This can be achieved by various mechanisms such as receptor-mediated endocytosis, macropinocytosis, or direct membrane translocation.

[0106] In some embodiments, imaging agents can be enhanced to be taken up by cells by functionalizing them with cell-permeable peptides (CPPs). CPPs are a class of short-chain peptides that can efficiently cross the cell membrane and facilitate the internal movement of cargo molecules such as nanoparticles, proteins, or nucleic acids. By attaching CPPs to SPION or other imaging agents, the cell permeability of nanoparticles is improved, enabling their incorporation into target cells for a variety of biomedical applications, including but not limited to imaging diagnostics for MRI. In certain embodiments, the paramagnetic CEST MRI agent may be a compound such as a lanthanide complex.

[0107] In some embodiments, the MRI agent may be bound to the layer via bonding such as maleimide chains, NHS esters, click chemistry, or other covalent or non-covalent approaches, or combinations thereof. In some embodiments, the MRI agent may be loaded without the addition of any exogenous agents, i.e., the layer and the MRI agent alone.

[0108] In some embodiments, the imaging component is a drug for use in PET. 18 F, 64 Cu, 11 C, 13 N, 15 O, 68 Ga, 89 Zr, 82 Rb, 14 C, 26 AI, 124 I, 76 Br, and 52F This may include, but is not limited to, e. In some embodiments, the imaging component is a drug for use in SPECT. 99m Tc, 67 Ga, 192 Ir, 111 In, 123 I, 201 Tl, 131 I, 125 I, 133 Xe, and 57 The imaging components may include, but are not limited to, Co. In some embodiments, the imaging components are alpha-emitting radionuclides, which may include, but are not limited to, At-211, Bi-212, Bi-213, Ra-223, Ac-225, Th-227, and Po-210. In some embodiments, the imaging components are beta-emitting radionuclides, which may include, but are not limited to, Cu-67, Y-90, Ag-111, I-131, Pm-149, Sm-153, Ho-166, Lu-177, Re-186, Re-188, Sr-89, P-32, and Tl-204.

[0109] In some embodiments, 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, diagnostic, or therapeutic agents to endometriotic 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 have an average diameter of 1 to 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), a sulfur hexafluoride-filled microbubble contrast agent with a phospholipid shell. SonoVue® enhances the visualization of vascular and tissue perfusion in a variety of applications, including echocardiography and localized liver lesion characterization.

[0110] The microbubble shells may be functionalized with peptide mimetic molecules disclosed herein. This process typically involves conjugating peptides to lipids, proteins, or polymers that form the microbubble shells.

[0111] Microbubbles themselves are generally too large to directly enter cells. However, by combining microbubbles with ultrasonic energy, it becomes possible to enhance cell membrane permeability and facilitate the transport of molecules into cells via sonoporation. By applying ultrasonic energy to a target area, the microbubbles vibrate or collapse, generating a mechanical force that temporarily increases the permeability of the nearby cell membrane. This process, called sonoporation, allows attached molecules (e.g., drugs, genes) to enter the cell. Ultrasonic imaging is used to monitor the progress of the procedure. Microbubbles enhance ultrasonic image contrast, enabling real-time visualization and evaluation of the target area.

[0112] 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.

[0113] In some embodiments, the contrast agent is administered during the scan. In some embodiments, the image is administered less than 15 minutes, less than 30 minutes, less than 60 minutes, less than 90 minutes, 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 before the scan. In some embodiments, the scan may help identify diseased tissue. In some embodiments, the scan may be read as contrast. In some embodiments, the signal persists in the lesion for a period exceeding the drug's PK in the system.

[0114] 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 application site. In some embodiments, the signal can be used to track the ADME profile (i.e., bioavailability or root of elimination) of the drug.

[0115] Linker In some embodiments, the peptide may be conjugated with a linker. The linker component can be used to produce the drug of the present invention by covalently conjugating one or more fluorophores, quenchers, biological modifiers, and non-fluorescent reporters to an enzymatically cleavable oligopeptide or any biological modifier. It should be understood that, where a linker is present, there are no restrictions on the specific structure, size, or content of the linker. The linker may contain a variety of functional groups, such as maleimides, dithiopyridyls, thiols, azides, alkenes, or alkynes, which allow for the assembly of molecules of diverse structures.

[0116] The linker may be a homofunctional or heterofunctional linker. For example, an amine (NH2) functionalized component may be reacted with a bifunctional crosslinking agent designed to react with an amino group. Particularly useful conjugation reagents that can promote linker formation or promote covalent bonding between, for example, a fluorophore and an enzymatically cleavable oligopeptide may include N-hydroxysuccinimide (NHS) esters and / or maleimides. NHS esters can react with, for example, the amine group of a peptide or fluorophore. Maleimides can react with the sulfhydryl group of another molecule. Other particularly useful linker components include bifunctional crosslinking agents such as N-succinimidyl 3-(2-pyridyldithio)propionic acid (SPDP), long-chain SPDP, maleimidobenzoic acid-N-hydroxysuccinimidate (MBS), succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylic acid (SMCC), succinimidyliodoacetic acid (SIA), and 6-aminohexanoic acid (Ahx).

[0117] In certain embodiments, the linker, if present, may be a derivative of a diamine. Diamine components or derivatives can be optionally derivatized with carboxylic acids to provide linker arms of varying lengths and chemical properties for chemically linking molecules. Non-limiting examples of diamines include ethylenediamine (EDA), propylenediamine, spermidine, spermine, hexanediamine, and diamine-amino acids such as homolysine, lysine, ornithine, diaminobutyric acid, and diaminopropionic acid. In other embodiments, components of the imaging agent may be chemically linked to dicarboxylic acids, such as succinic acid, glutaric acid, suberic acid, or adipic acid. In one embodiment, the linker is aminoethylmaleimide.

[0118] In certain embodiments, the linker may be formed from an azide component that can react with a substituted alkyne in a Huisgen[3+2] type cycloaddition of the azide and acetylene. In certain embodiments, the azide or alkyne linker can be bonded to a polyethylene glycol (PEG) component, for example, an enzymatically cleavable oligopeptide. Other intended linkers include propargylglycine, pentanoyl, pentic acid, propargyl acid, and / or propargylamine components.

[0119] In some embodiments, polyethylene glycol (PEG) modification may be used as a linker. PEGylation can enhance peptide solubility and improve handling and formulation. Furthermore, PEGylation can lead to increased stability by extending the peptide half-life by protecting it from enzymatic degradation. Cellular uptake can be improved by increased endocytosis and avoidance of efflux transporters, while overall toxicity can be reduced. PEGylation can also enhance the water solubility of peptides while simultaneously reducing their renal clearance. The pharmacokinetics of a drug can be regulated based on PEG chain length, allowing for adjustment of release rate, biodistribution, and efflux kinetics. In some embodiments, direct conjugation linkers may be used in which imaging molecules are directly immobilized on the peptide. Another embodiment involves the use of peptide linkers composed of short amino acid sequences that provide biodegradability and flexibility, designed to have specific properties. In other embodiments, carbohydrate linkers may be utilized, which exhibit biocompatibility and ease of synthesis and can be modified to modulate their properties. Another embodiment may utilize ester and amide linkers, where the amide linker provides significant stability and degradation resistance in vivo, and the ester linker is engineered to cleave under specific conditions. Further embodiments include disulfide linkers designed to cleave by glutathione, a reducing agent found in cells, enabling the selective release of imaging molecules in specific biological regions. Another embodiment is a heterobifunctional linker having two distinct functional groups to allow the linkage of two different types of molecules. Enzyme-cleavable linkers designed to cleave in response to specific enzymes may also be used, providing a mechanism for controlling the release of imaging molecules. These linkers optimize the desired in vivo distribution and pharmacokinetics, as well as enable the intended use of the imaging agent. These embodiments offer innovative strategies in the design and application of peptide imaging agents, improving their efficacy and adaptability in a wide variety of imaging, clinical, and diagnostic applications.

[0120] In certain embodiments, a fluorophore, quencher, or other reporter is directly bound to the imaging agent using a reactive NHS ester group on the fluorophore, quencher, or reporter that reacts with an amine group on an enzymatically cleavable oligopeptide. In certain other embodiments, a carboxylic acid group on the fluorophore, quencher, or other reporter may be activated in situ with activators known in the art, such as 2-(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU), 1-ethyl-3-(3'-dimethylaminopropyl)-carbodimide hydrochloride (EDC), N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-disuccinimidyl carbonate (DSC). In other embodiments, a reporter containing a sulfhydryl or thiol group may be chemically bound to the agent via a bifunctional crosslinking agent having a second component that can react with the sulfhydryl (thiol) group. Examples of such crosslinking agents include, as mentioned above, SPDP, long-chain SPDP, SIA, MBS, SMCC, and others well known in the art.

[0121] Useful linker components include both natural and unnatural amino acids, oligopeptides, such as linear or cyclic oligopeptides, and nucleic acids.

[0122] The linker may be a peptide or peptide component and optionally includes a proteolytic or non-proteolytic cleavage site, such as an ester bond, which can be cleaved by a change in pH at the site of interest.

[0123] Image-guided surgery This specification discloses methods for using the imaging agents of the present disclosure for image-guided surgery. In some embodiments, various techniques are utilized for image-guided surgery to enable improved diagnosis, accuracy, and precision during surgical procedures. In some embodiments, a surgeon may use the contrast agents of the present disclosure to diagnose the staging of endometriosis and cancer. In some embodiments, a surgeon may use the contrast agents of the present disclosure to detect which organ system an endometriotic lesion or cancerous tumor is located on. In some embodiments, a surgeon may use the imaging agents of the present disclosure to identify and / or stage a disease. In some embodiments, a surgeon may use the imaging agents of the present disclosure to aid in the resection of a lesion or tumor.

[0124] In some embodiments, a surgeon may use the imaging agents of the present disclosure to aid in the excision of a lesion or tumor. In some embodiments, this includes fluorescence-guided surgery (FGS). FGS uses a fluorescent probe or dye, which accumulates in the tumor after being ingested or injected into the patient. When these probes or dyes are exposed to light of a specific wavelength, they emit fluorescence, which can be detected by a specialized camera system and visualized by the surgeon, thus aiding in the visualization of the tumor. The imaging system used to visualize fluorescence in FGS typically includes a light source for exciting the fluorescent compound, a camera for imaging the emitted fluorescence, and a display monitor for the surgeon to view the image. For example, the "da Vinci Surgical System" has been modified to include a near-infrared fluorescence imaging system for real-time image-guided surgery. To visualize fluorescence during photodynamic therapy (PDT), handheld devices such as the SPY Elite System, Artemis and Artemis Pro (Quest Medical Imaging), as well as Fluobeam (Fluoptics), Hamamatsu PDE-Neo, and Visee PDT Imaging System (Visee Medical) are used. In some embodiments, a handheld probe (Leica FL560 (Leica Microsystems)) may be used as part of a surgical microscope system to visualize fluorescence in areas that may be difficult to reach with a microscope.

[0125] In some embodiments, the imaging agent of the present disclosure can also be visualized by a device such as the LightPath Imaging System (Lightpoint Medical), a handheld device, which can detect carbon-11 ( 11 C), nitrogen-13( 13 N), oxygen-15( 15 O), Copper-64( 64 Cu), Gallium-68( 68 Ga), Fluorine-18 (18 FIt is designed to detect Cherenkov emission emitted from specific types of radiation tracers, including ).

[0126] This specification describes a method for identifying, visualizing, diagnosing, and surgically excising diseased tissue. This method involves applying an imaging agent to the patient, thereby facilitating the visualization of the diseased tissue, either visually or with machine assistance. In some embodiments, images of the area of ​​luminescent lesion tissue are obtained before the surgical procedure. In some embodiments, this process is performed in real time. In some embodiments, the captured images are displayed to the surgeon. In some embodiments, the images are a single still image or a series of still images. In some embodiments, the images are a video. The images are then presented to the surgeon during surgery using a reading device, allowing the surgeon to visually identify the luminescent diseased tissue in the patient. The surgeon may directly observe the identified luminescent tissue or use a dedicated visualization machine for assistance. Based on the identified luminescent lesion tissue, the surgeon can perform a surgical diagnosis, stage the disease, and / or proceed with the excision of the lesion tissue accordingly.

[0127] In some embodiments, the drug is visualized by magnetic resonance imaging (MRI). In some embodiments, computed tomography (CT) may be used. In further embodiments, imaging methods such as X-ray and single-photon emission computed tomography (SPECT) may be used. In other embodiments, ultrasound imaging may be used. In some embodiments, the peptide may be used in optical imaging, which uses light to examine the molecular composition of tissue, thereby assisting in surgery where the tumor or target is near the surface. In some embodiments, positron emission tomography (PET) is used to guide certain surgical procedures. In some embodiments, PET is used in combination with CT.

[0128] In some embodiments, the drug is visualized using an MRI machine with a lower magnetic field strength, which is less expensive but ineffective with gadolinium contrast agents.

[0129] In some embodiments, drugs are visualized using 0.064T portable MRI machines. These machines are designed for point-of-care applications, making MRI technology more accessible and affordable. They can be used at the patient's bedside and are particularly useful in situations where conventional MRI would be limited. While they lack detail due to their low magnetic field strength, they offer significant value in certain clinical situations, such as diagnosing stroke in emergencies.

[0130] In some embodiments, the drug is visualized using a low-field MRI machine (0.2T–0.4T). These machines are suitable for larger patients or those with claustrophobia due to their open design. These machines are less expensive but produce images lacking detail. They are typically used for limb imaging, although some are capable of whole-body scans.

[0131] In some embodiments, the drug is visualized using a 1.5T MRI machine. These machines are standard in clinical imaging and offer a balance between image quality, speed, and cost. They are versatile and suitable for a wide range of diagnostic purposes, including imaging of the brain, spine, and musculoskeletal system.

[0132] In some embodiments, the drug is visualized using a 3T MRI machine. These machines provide high image quality and detail, making them useful for advanced imaging needs such as detailed brain and musculoskeletal system examinations and high-resolution imaging. Although they are more expensive, they can improve patient throughput with faster imaging times.

[0133] In some embodiments, drugs are visualized using ultra-high field MRI (7T or higher). These machines are primarily used in research, and these scanners provide excellent detail for medical research. While beneficial for imaging small structures and functional MRI, they are generally not used in standard clinical practice due to their high cost and technical requirements.

[0134] In some embodiments, the drug is visualized using an open-bore MRI machine. These machines are designed to alleviate claustrophobia or accommodate larger patients, and they have lower magnetic field strengths (approximately 0.5T), resulting in images with less detail than those of a closed-bore machine, but providing a more comfortable experience for certain patients.

[0135] In some embodiments, the drug is visualized using a front-open MRI machine. These machines are similar to open MRIs but are designed to improve comfort and usability, particularly in examinations where specific positioning is required. They offer an alternative for patients with claustrophobia or mobility issues.

[0136] MRI machines are generally selected based on imaging needs, patient considerations, and cost-effectiveness, and image quality can be improved by using contrast agents. 1.5T and 3T machines are the most commonly available for clinical use outside of specialized imaging centers due to their versatility and balance of image quality, speed, and cost.

[0137] In some embodiments, to diagnose endometriotic lesions of all subtypes and stages in any organ system, the drug is visualized in a whole-body scan, i.e., a scan that does not only include the pelvis. This can provide both patients and healthcare professionals with better insights into disease progression and the prognosis of data-driven care. For example, endometriosis of the bladder may present as a painful UTI, and endometriosis of the appendix may present as extremely painful appendicitis. Importantly, this MRI approach can also enable the detection and characterization of malignancies, providing patients with crucial information so that they can receive appropriate care. For example, but not limited to, whole-body scans can be used to detect endometriotic lesions in the appendix, brain, spine, thyroid, lungs, liver, gallbladder, pancreas, spleen, kidneys, adrenal glands, bladder, ovaries, uterus, intestines, or any combination thereof.

[0138] In one embodiment, this disclosure discloses the use of an imaging agent during endometriosis surgery. In a particular embodiment, the drug is introduced into the abdominal cavity concurrently with the surgical procedure. In an alternative embodiment, the drug is administered prior to the surgical intervention, at 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, or more than 4 weeks before the surgery.

[0139] Furthermore, in further embodiments, the drug may be administered via alternative routes, including but not limited to intravenous (IV), subcutaneous (SC), vaginal, rectal, oral, and intramuscular (IM) routes. By using this method, surgeons gain the ability to visualize endometriotic lesions with superior accuracy, thereby facilitating their identification in terms of size, location, and disease staging. This visualization can be achieved either visually or through the use of handheld tools or imaging devices. With this enhanced visual information, surgeons can then proceed with the careful excision of endometriotic lesions while minimizing the risk of inadvertently removing normal tissue. This technique represents a significant advance in endometriosis surgery, enabling improvements in surgical outcomes and patient care.

[0140] This can be applied to surgery for diseases involving β-catenin dysregulation, 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 used for the diagnosis, imaging, and treatment of gynecological diseases including endometriosis, uterine fibroids, adenomyosis, ovarian cysts, polycystic ovary syndrome (PCOS), vas deferens endometriosis, and cervical polyps, endometrial polyps, and vaginal polyps, and / or their symptoms.

[0141] Pharmaceutical composition This specification discloses peptides and peptide mimetic agents comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 1 to SEQ ID NOs: 500 or any one of SEQ ID NOs: 1 to SEQ ID NOs: 499, on a pharmaceutically acceptable carrier.

[0142] In some embodiments, the agents and / or peptides described herein are formulated into pharmaceutical compositions. The pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive components that facilitate the treatment of the active agent and / or peptide into a pharmaceutically useful preparation. The appropriate formulation varies depending on the chosen route of administration. Summaries of the pharmaceutical compositions described herein can be found, for example, in 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, NY, 1980, and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), which are incorporated herein by reference for such disclosures.

[0143] In some embodiments, the agents and / or peptides described herein are administered alone or in combination with pharmaceutically acceptable carriers, excipients, or diluents in pharmaceutical compositions. Administration of the agents and / or peptides and compositions described herein may be achieved by any method that enables delivery of the agents and / or peptides to the site of action. Such methods include, but are not limited to, delivery via intestinal routes (including oral, gastric, or duodenal nutrient duct, anal suppositories, and rectal enemas), parenteral routes (including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intracosal, intravascular, intravenous, intravitreous, epidural, and subcutaneous injections or infusions), inhalation, percutaneous, transmucosal, sublingual, buccal, and topical (including epithelial, skin, enema, eye drops, ear drops, intranasal, vaginal, and intrauterine) administration, but the most appropriate route may vary, for example, depending on the recipient's disease and impairment. As just one example, the drugs and / or peptides described herein may be administered locally to the area requiring treatment, for example, by local injection during surgery, topical application such as creams or ointments, injection, catheter, implantation, or insertion device. Administration may also be by direct injection at the site of the lesioned tissue or organ.

[0144] In some embodiments, the pharmaceutical composition is in solution form. In some embodiments, the formulation utilizes a buffer such as sodium acetate or histidine buffer. In some embodiments, NMP, DMSO, or polypropylene glycol are used as cosolvents. In some embodiments, an anti-aggregation agent such as PS80 is used. In some embodiments, NaOleate, 2-phenoxyethanol, niacinamide, HPBCD, and trehalose are used as excipients. In some embodiments, the formulation is an aqueous suspension. In some embodiments, Na-CMC, PS80, PBS, and trehalose are used as excipients.

[0145] 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, this consists of a mixture of cotton:soybean:olive, corn and cotton, sesame and cotton oil. In some embodiments, an emulsifier is used (including, but not limited to, PS80, PS20, Kolliphor EL, Lecethin, or several combinations). In some embodiments, NMP, ethanol, propylene glycol, and DMSO may be used as solvents.

[0146] In some embodiments, pharmaceutical compositions suitable for oral administration are presented as dispersion units such as capsules, cachetes, or tablets, as powders or granules, as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-based emulsions in aqueous or water-based emulsions in oil-based liquids, each containing a predetermined amount of the active ingredient. In some embodiments, the active ingredient is provided as a bolus, lick, or paste.

[0147] Pharmaceutical compositions for oral use include tablets, compressible capsules made of gelatin, and sealed soft capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets may optionally be made by compression or molding, together with one or more adjuncts. Compressed tablets may optionally be prepared by compressing the active ingredient in a free-flowing form, such as a powder or granules, with a binder, an inert diluent, or a lubricant, surfactant, or dispersant using a suitable machine. Molded tablets may be made by molding a mixture of powdered pharmaceuticals and / or peptides moistened with an inert liquid diluent using a suitable machine. In some embodiments, tablets are coated or scored and formulated to result in delayed or controlled release of the active ingredient therein. All formulations for oral administration may be in dosages suitable for such administration. Compressible capsules may contain the active ingredient in combination with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active agent and / or peptide may be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In some embodiments, stabilizers are added. Sugar-coated tablet cores are provided with a suitable coating. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the coating of the tablets or sugar-coated tablets for identification or to characterize different combinations of doses of the active agent and / or peptide.

[0148] In some embodiments, the pharmaceutical composition is formulated for parenteral administration by injection, such as bolus injection or continuous infusion. Formulations for injection may be provided in unit dosage forms, for example, in ampoules or multi-dose containers, along with additional preservatives. The composition may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain compounding agents such as suspending agents, stabilizers, and / or dispersants. The composition may be provided in unit or multi-dose containers, such as sealed ampoules and vials, and may be stored in powder form, or in a lyophilized state requiring only the addition of a sterile liquid carrier, such as saline or distilled water free of pyrogenic substances, immediately before use. Instantaneous injections and suspensions may be prepared from the sterile powders, granules, and tablets of the types described above.

[0149] Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous (oil-based) sterile injection solutions of active agents and / or peptides, which may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the desired recipient, and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickeners. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. The aqueous injection suspension may contain a substance that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain a suitable stabilizer or agent that increases the solubility of the agent and / or peptide to enable the preparation of higher concentration solutions.

[0150] The pharmaceutical composition may be formulated as a Devot preparation. Such long-acting preparations may be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Therefore, for example, the drug and / or peptide may be formulated in a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, for example, as a sparingly soluble salt.

[0151] For buccal or sublingual administration, the composition may take the form of tablets, lozenges, pastels, or gels formulated in the conventional manner. Such compositions may contain sucrose and the active ingredient in the main component, which may be flavored, such as acacia or tragacanth.

[0152] The pharmaceutical composition may also be formulated into rectal or vaginal compositions such as suppositories or retaining enemas, for example, by including conventional suppository bases such as cocoa butter, polyethylene glycol, or other glycerides. Upon insertion, the suppository base liquefies or becomes water-miscible at body temperature, allowing the components to remain in contact with the mucous membrane for a sufficiently long period to have imaging, diagnostic, and / or therapeutic effects. The weight percentage of the suppository base varies depending on the size of the body openings of humans and / or animals, the drug composition required to have imaging, diagnostic, and / or therapeutic effects, and its physicochemical properties that allow it to remain solid below room temperature. In some embodiments, the suppository contains about 50% to over 99% by weight of the suppository base, or about 75% to over 99% by weight of the suppository base. In some embodiments, the suppository contains about 75% to about 98% by weight of polyethylene glycol. In some embodiments, the suppository contains 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 (U.S. Patent Application No. 2009 / 0311290, which is incorporated herein by reference for such disclosure).

[0153] In some cases, the pharmaceutical composition may contain an absorption enhancer such as sodium caprate. Some of these cases include compositions and dosage forms used rectally or vaginally, as described herein.

[0154] Pharmaceutical compositions may be administered topically, rectally, i.e., by non-systemic administration. This includes applying the agents and / or peptides of the Disclosure to the external epidermis or cheek cavity, and injecting such agents and / or peptides dropwise into the rectum or vagina, such that the agents and / or peptides do not enter the bloodstream as much as possible. In contrast, systemic administration refers to oral, intravenous, intraperitoneal, and intramuscular administration.

[0155] Pharmaceutical compositions suitable for topical administration include solutions, lotions, vibrators, creams, ointments, gels, foams, transdermal patches, powders, solids, sponges, tapes, vapors, pastes, tinctures, microparticles, microcapsules, nanoparticles, liposomes, or emulsions, including liquid or semi-liquid formulations suitable for penetration through the skin, such as those suitable for delivery to the vagina or rectum. The active ingredient may be present in the formulation at a concentration of 0.001 w / w% to 10 w / w%, for example, 1% to 2% by weight, in the case of topical administration.

[0156] Pharmaceutical compositions for administration by inhalation are conveniently delivered by injectors, nebulizers, pressurized packs, or other convenient means of delivery such as aerosol sprays. Pressurized packs may contain a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. In the case of pressurized aerosols, the dose unit may be determined by providing a valve for delivering a measured amount. Alternatively, for administration by inhalation or aeration, the pharmaceutical formulation may take the form of a dry powder composition, e.g., a powder mixture of the drug and / or peptide with a suitable powder base such as lactose or starch. The powder composition may be provided in unit dosage forms such as capsules, cartridges, gelatin, or blister packs, in which the powder can be administered using an inhaler or injector.

[0157] In some embodiments, the agents disclosed herein are formulated to achieve delivery of the drug and / or peptide to specific areas of the gastrointestinal tract. For example, the agents and / or peptides disclosed herein are formulated for oral delivery by means of bioadhesive polymers, pH-sensitive coatings, time-dependent biodegradable polymers, microflora activation systems, etc., to achieve delivery of the drug and / or peptide to specific areas of the gastrointestinal tract.

[0158] In some embodiments, the agents and / or peptides disclosed herein are formulated to achieve delivery of the agent and / or peptide to a specific area of ​​the urogenital or anorectal mucosa. For example, the agents and / or peptides disclosed herein are formulated for intravaginal delivery by bioadhesive polymers, pH-sensitive coatings, time-dependent biodegradable polymers, microflora-activating systems, etc., to achieve delivery of the agent and / or peptide to a specific area 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 agent and / or peptide from the dosage form into which the agent and / or peptide is incorporated, over a period of time according to a desired profile. Controlled release profiles include, for example, sustained release, long-term release, pulsed release, and delayed release profiles. In contrast to immediate-release compositions, controlled-release compositions allow for delivery of the agent to a subject over a period of time according to a predetermined profile. Such release rates provide prolonged imaging and / or therapeutically effective levels of the drug, resulting in a longer-lasting pharmacological response while minimizing side effects compared to conventional rapid-release formulations. These prolonged responses offer numerous unique advantages not achieved with the corresponding short-acting, immediate-release formulations.

[0159] Approaches for delivering intact therapeutic, imaging, and / or diagnostic agents to specific areas of the genitourinary system (e.g., vagina) or gastrointestinal tract (e.g., colon) include: (i) Polymer coating: Intact molecules can be delivered to the colon without being absorbed in the upper part of the intestine by coating drug molecules with appropriate polymers that degrade only in the colon. In addition, polymer coating can provide a protective or controlled-release profile for vaginal formulations. (ii) pH-sensitive polymer coating: Delivery systems targeting the intestine, colon, and vagina can be based on coating tablets or pellets filled into conventional hard gelatin capsules. The most commonly used pH-dependent coating polymer is methacrylic acid copolymer (commonly known as Eudragit® S), more specifically Eudragit® L and Eudragit® S. Eudragit® L100 and S100 are copolymers of methacrylic acid and methyl methacrylate. (iii) coating with biodegradable polymers, (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) bioadhesion systems, (x) coating with microparticles, and (xi) osmotically controlled drug delivery.

[0160] Another approach to drug delivery or controlled-release systems targeting the vagina and colon involves embedding a drug in a polymer matrix and capturing that drug for release in the vagina or colon. These matrices may be pH-sensitive or biodegradable. Matrix-based systems, such as multi-matrix (MMX) based delayed-release tablets, ensure drug release in the vagina or colon.

[0161] The compositions and methods described herein may include the delivery of therapeutic, imaging, and / or diagnostic agents by various dosage forms and devices, including those described above and below. Many of the dosage forms described below have several advantages. These may include local delivery of therapeutic, imaging, and / or diagnostic agents and / or peptides. Local delivery can reduce side effects that may be associated with systemic delivery and off-target effects. In some cases, the dosage forms and devices described herein can improve patient compliance by, for example, providing long-term or continuous delivery of the agents and / or peptides described herein. In some cases, these dosage forms and devices can further increase tolerability and safety.

[0162] The peptides and compositions described herein may be incorporated into an intravaginal ring for delivery. Delivery of therapeutic, imaging, and / or diagnostic substances via an intravaginal ring (IVR) enables local delivery and increases the safety and tolerability of therapeutic, imaging, and / or diagnostic substances. IVR can further offer several advantages, including bypass gastrointestinal absorption and first pass through the liver / renal system, reduced effective dose, continuous delivery and / or controlled release profiles, extended intervals between doses, reduced side effects, reduced serum drug concentrations, patient self-administration, and improved patient satisfaction. IVR can successfully deliver drugs, including hydrophilic and high molecular weight agents, to organs affected by EMS, including deep EMS (DIE). The intravaginal ring delivery system is distributed by tissue absorption rather than serum absorption and can reach all organs affected by EMS in the peritoneal cavity.

[0163] A vaginal ring typically consists 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 a desired period of time, and then removed by the user. The ring may be solid or hollow containing therapeutic, imaging, and / or diagnostic components, or it may be a porous material from which the drug is released. The ring may optionally include a third outer velocity-controlling elastomer layer that does not contain the drug. Optionally, the third ring may contain a second drug for a dual-release ring. The drug may be incorporated into polyethylene glycol throughout the silicone elastomer ring to function as a reservoir for the drug to be delivered. In some cases, the IVR may include silicone, compressed tablets, or lyophilized gels.

[0164] Pessaries, cups, strips, tablets, and suppositories are other examples of drug delivery systems that may be used in this disclosure. These systems have been used for the delivery of vaginal contraceptives and are widely described in the literature.

[0165] Another example of a delivery system is a vaginal sponge and foam. The desired drug may be incorporated into a silicone matrix, which is coated onto a cylindrical drug-free polyurethane vaginal sponge, as described in the literature.

[0166] In some embodiments, the IVR comprises peptides or compositions described herein, formulated as suppositories, solutions, lotions, stimulant combinations, creams, ointments, gels, foams, transdermal patches, powders, solids, sponges, tapes, pastes, tinctures, emulsions, microparticles, microcapsules, nanoparticles, liposomes, or capsules containing microparticles, microcapsules, nanoparticles, or liposomes. The IVR has been further modified to include various delivery vehicles, such as silicone inserts, compressed tablets, or lyophilized gels, to optimize the release profile of hydrophilic inhibitors or high molecular weight drugs, e.g., 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 may also include one or more absorption enhancers, such as sodium caprate.

[0167] In some embodiments, the IVRs described herein are formulated to contain about 0.01 mg to about 5000 mg of a drug and / or peptide. In some embodiments, the IVR may 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 a drug and / or peptide.

[0168] In some embodiments, the IVRs described herein are formulated to deliver approximately 0.01 mg to approximately 1000 mg of drugs and / or peptides per day. In some embodiments, the IVRs are formulated to deliver approximately 0.01 mg, approximately 0.05 mg, approximately 0.1 mg, approximately 0.5 mg, approximately 1 mg, approximately 5 mg, approximately 10 mg, approximately 20 mg, approximately 40 mg, approximately 60 mg, approximately 80 mg, approximately 100 mg, approximately 150 mg, approximately 200 mg, approximately 400 mg, approximately 600 mg, approximately 800 mg, or approximately 1000 mg of drugs and / or peptides per day.

[0169] The peptides and compositions described herein may be incorporated into a tampon device for delivery. The tampon device typically includes a vaginal tampon having a proximal and distal end. The distal end is a cup-shaped, porous foam portion that fits around the cervix and contains the peptides or compositions described herein for delivery. The device may further include a non-absorbent shaft having a distal opening and extending into the tampon through the porous foam cup to guide blood flow to the absorbent material. Optionally, a retrieval string or tape connected to the tampon device is also included. Absorbent vaginal tampons may contain or be coated with the peptides or compositions described herein and may be used as medicinal tampons for delivery.

[0170] The peptides and compositions described herein may be incorporated into a solid for topical delivery. The solid may take the form of a pessary or a vaginal or anal suppository. The solid dosage form may melt when it reaches body temperature. Alternatively, the solid may retain its structure and release the incorporated composition described herein. The solid may be a pessary designed to provide a support within the vagina. A vaginal sponge may be embedded with the composition described herein for intravaginal delivery.

[0171] The peptides and compositions described herein may be incorporated into topical formulations for delivery. Topical agents are applied to body surfaces such as skin or mucous membranes. In some examples, body surfaces include, but are not limited to, epithelial tissue, mucous membranes, peritoneum, epithelium, and endometrium. The agents and / or peptides are absorbed by the body surface to achieve a topical or systemic effect. Topical agents are optionally formulated in the class of topical solutions, lotions, stimulant mixtures, creams, ointments, gels, foams, transdermal patches, powders, solids, sponges, tapes, vapors, pastes, or tinctures. Topical solutions may typically be administered as low-viscosity washes, sprays, or infusions having water or alcohol as the base. Lotions can be thicker and more emollient than solutions. These are usually oils mixed in water and may have less alcohol than solutions. Stimulant mixtures are mixtures that separate into two or three parts over time. The mixture may be oil mixed with an aqueous solution and must be shaken to form a suspension before use. Cream is an emulsion in which oil and water are in approximately equal proportions. Ointment is a homogeneous, viscous, semi-solid formulation, most commonly a high-viscosity, oily, concentrated oil (80% oil - 20% water). Ointment may contain a hydrocarbon base, an absorption base, a water-soluble base, an emulsifying base, or a vegetable oil, or any combination thereof. Ointment is formulated in a base that may contain, but is not limited to, a hydrocarbon base such as solid paraffin, soft paraffin, microcristan wax, or ceresin; a water-absorbing base such as lanolin or beeswax; a water-soluble base such as macrogol 200, 300, or 400; an emulsifying base such as emulsifying wax or cetrimide; or a vegetable oil such as olive oil, coconut oil, sesame oil, almond oil, or peanut oil.

[0172] A gel is a semi-solid emulsion. Non-limiting examples of useful emulsifiers include acrylic polymers (thickeners of the carbomer brand manufactured by Voveon, Inc., e.g., carbomer 934P), polyoxyethylene-10-stearyl ether, polyoxyethylene-20-stearyl ether, cetostearyl alcohol, cetyl alcohol, cholesterol, diglycol stearate, glyceryl monostearate, glyceryl stearate, polyglyceryl-3-oleate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lanolin, polyoxyethylene lauryl ether, methylcellulose, polyoxyethylene stearate, polysorbate, propylene glycol monostearate, sorbitan esters, stearic acid, or mixtures of two or more of these.

[0173] The amount of emulsifier in the topical formulation may be in the range of about 1 to about 40% by weight, based on the total weight of the topical formulation, and in some embodiments, in the range of about 5 to about 30% by weight.

[0174] The gel formulations described herein may contain one or more gelling agents. Non-limiting examples of useful gelling agents include carboxylic acid polymers, including acrylic acid polymers crosslinked with crosslinking agents such as sucrose allyl ether (e.g., carbomer brand thickeners), cetostearyl alcohol, hydroxymethylcellulose, polyoxyethylene-polyoxypropylene copolymer, sodium carboxymethylcellulose, polyvinylpyrrolidone, or mixtures of two or more thereof.

[0175] The amount of gelling agent in the topical gel formulation may be in the range of about 0.1 to about 10% by weight, based on the total weight of the topical formulation, and in some embodiments, about 0.1 to about 1% by weight.

[0176] The gel formulations described herein may further contain one or more alkalizing agents, such as sodium hydroxide, in an amount of less than about 2% by weight, as gelation activators.

[0177] The formulation may contain one or more further excipients known in the art, such as water and a thickener, such as colloidal silicon dioxide.

[0178] Thermoreversible gels are liquid formulations that become a gel when inserted rectally or vaginally. Thermoreversible gels allow for easier administration and positioning than conventional suppositories or pessaries and can prevent leakage of the dosage form. Thermoreversible gels are formulated as polymer solutions consisting of thermoreversible polymers (e.g., poloxamers combined with mucosal adhesive polymers that allow the gel to adhere to mucous membranes). In situ's thermoreversible liquid gel formulations, also called thermoreversible "liquid suppositories," are liquid at low temperatures (below 10°C) and become a gel at body temperature.

[0179] The active agent may be incorporated into surgical tape for occlusive dressings. The drug may be applied as an ointment or gel to reach the mucous membrane by vaporization. Pastes combine oil, water, and powder. Tinctures typically contain a high percentage of alcohol for application to the skin.

[0180] The peptides and compositions described herein may be incorporated into transdermal patch delivery. Transdermal patches provide controlled drug release either through a porous membrane covering a drug reservoir or by melting a thin layer of drug embedded in a patch adhesive at body temperature.

[0181] The compositions described herein may be incorporated into a film for delivery. The film is a thin, small polymer formulation that can be easily inserted into the vaginal cavity without the use of an applicator and without causing discomfort. Vaginal films are easier to apply than other types of vaginal formulations such as pessaries, foams, and gels (Rohan LC et al., AAPS Journal 2009;11:78-87, which is incorporated herein by reference for such disclosure).

[0182] The peptides and compositions described herein may be incorporated into intrauterine devices (small, often T-shaped devices inserted into the uterus for delivery). IUDs typically contain copper, progestogens, or levonorgestrel. The compositions described herein are incorporated into IUD devices and released slowly over time.

[0183] The peptides and compositions described herein may be formulated for systemic delivery. Parenteral injections may be formulated for bolus injection or continuous infusion. Pharmaceutical compositions may be in a form suitable for parenteral injection, such as a sterile suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulations such as suspending agents, stabilizers, and / or dispersants. Pharmaceutical formulations for parenteral administration comprise an aqueous solution of the peptides described herein in a water-soluble form. Suspensions of the peptides described herein may be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. The suspensions may also contain suitable stabilizers or agents that increase the solubility of such peptides described herein and / or reduce aggregation of the peptides, in order to enable the preparation of highly concentrated solutions. Alternatively, the peptides described herein may be lyophilized or in powder form for reconstitution with a suitable vehicle, such as sterile water free of pyrogens, before use. In some embodiments, the purified peptides are administered intravenously.

[0184] The peptides and compositions described herein may be formulated for oral delivery. Pharmaceutical compositions that can be used orally include, in addition to tablets and push-fit capsules made of gelatin, soft, sealed capsules made of gelatin, and contain plasticizers such as glycerol or sorbitol. Tablets may optionally be made by compression or molding together with one or more accessory components. Compressed tablets may optionally be prepared by mixing with a binder, an inert diluent, or a lubricant, surfactant, or dispersant and compressing the active ingredient in a free-flowing form such as powder or granules with a suitable machine. Molded tablets may be made by molding a mixture of powdered drugs and / or peptides moistened with an inert liquid diluent with a suitable machine. In some embodiments, the tablets are formulated to be coated or scored to provide delayed or controlled release of the active ingredient therein. Formulations for oral administration may be in dosages suitable for such administration. Push-fit capsules may contain the active ingredient in combination with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active drug and / or peptide may be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In some embodiments, a stabilizer is added. The sugar-coated tablet core is provided with a suitable coating. For this purpose, a concentrated sugar solution may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solution, and a suitable organic solvent or solvent mixture. Dyes or pigments may be added to the coating of the tablets or sugar-coated tablets for identification or to characterize different combinations of dosages of the active drug and / or peptide.

[0185] Additional pharmaceutical approaches to the targeted delivery of therapeutic agents to specific regions of the gastrointestinal tract are known. Chourasia M K, 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 Sci Tech. 2015 August;16(4):731-741. Each of these references is hereby incorporated by reference herein for such disclosure.

[0186] It should be understood that, in addition to the components specifically mentioned above, the agents and / or peptides and compositions described herein may include other agents customary in the art related to the type of formulation in question. For example, agents suitable for oral administration may include flavoring agents.

[0187] The peptides and compositions described herein may contain further excipients to improve their imaging, diagnostic, and therapeutic capabilities. The compositions may also contain absorption enhancers or penetration enhancers to increase absorption across the epidermal or mucosal surface and membrane permeability. Enhancers formulated in the compositions described herein include, but are not limited to, sulfoxides such as dimethyl sulfoxide (DMSO), pyrrolidones such as laurocapram (1-dodecyl azacycloheptan-2-one) and n-methyl-2-pyrrolidone, terpenes and terpenoids, essential oils, oxazolidinones such as 4-decycloxazolidin-2-one, urea, cyclopentadecalactone, and sodium N-[8-(2-hydroxybenzoyl)amino] Examples may include caprylates (SNACs), 8-(N-2-hydroxy-5-chlorobenzoyl)-aminocaprylic acid (5-CNACs), medium-chain fatty acids, salts and derivatives, sodium caprate, sodium caprylate, protease inhibitors and ω-3 fatty acids, liquid mixed micelle sprays, lipid polymer micelles, alkyl glycosides, chitosan, 2-N,N-dimethylaminopropionate dodecyl (DDAIP), cell membrane lipid components, nanoparticles, liposomes, ligands, and lipophilic modifications.

[0188] The peptides and compositions described herein may be formulated as solutions, lotions, vibrating mixtures, creams, ointments, gels, foams, mucosal adhesion compositions, emulsions, liposomes, coatings, cores, matrices, and lyophilized products.

[0189] Method of administration In some embodiments, the pharmacokinetic and / or peptide compositions described herein are used to prepare agents for imaging, diagnosing, and / or treating diseases, illnesses, or symptoms in mammals that would benefit from the administration of β-catenin pharmacokinetic and / or peptides. A method for treating any of the diseases or illnesses described herein in a mammal requiring such treatment comprises administering a pharmaceutical composition comprising at least one pharmacokinetic and / or peptide described herein to the mammal in an imaging and / or therapeutically effective amount.

[0190] Diseases, illnesses, or conditions for which treatment with the compositions described herein may be beneficial include, but are not limited to, endometriosis, endometrial lesions, endometriomas, superficial endometriotic implants, deep endometriosis, chronic pain, central sensitization, myofascial pain, adnexal tumors, infertility, dysmenorrhea, genetic predisposition, non-menstrual pelvic-abdominal pain, dyspareunia, bowel symptoms (diarrhea, muscle cramps, constipation), painful defecation (dysfecation), ovarian masses or ovarian tumors, painful bladder symptoms, and urinary dysfunction.

[0191] In certain embodiments, compositions containing the agents and / or peptides described herein are administered for prophylactic and / or therapeutic purposes. For specific therapeutic, imaging, and diagnostic applications, the compositions are administered to patients already suffering from a disease or illness in an amount sufficient to cure or at least partially block at least one symptom of the disease or illness. The effective amount for this application will vary depending on the severity and course of the disease or illness, previous treatments, the patient's health status, weight, and response to the drug, as well as the judgment of the treating physician. The effective amount for imaging and therapy will be determined at will, but is not limited to, by methods including clinical trials of dose escalation and / or dose range exploration.

[0192] For prophylactic use, compositions containing the agents and / or peptides described herein are administered to patients who are susceptible to or at risk of developing a particular disease, disorder, or illness. Such amounts are defined as “a prophylactically effective amount or dose.” For this use, the exact amount will also vary depending on the patient’s health condition, weight, etc. When used in patients, the effective amount for this use may vary depending on the severity and course of the disease, disorder, or illness, previous treatments, the patient’s health condition and response to the drug, and the judgment of the treating physician. In one embodiment, prophylactic treatment involves administering a pharmaceutical composition containing the agents and / or peptides described herein to a mammal that has previously experienced at least one symptom of the disease being treated and is currently in remission, in order to prevent recurrence of the disease or symptoms of the illness.

[0193] In certain embodiments where the patient's condition does not improve, drugs and / or peptides are administered chronically, i.e., over a long period including the patient's lifetime, at the discretion of the physician, to alleviate, or otherwise manage or limit the symptoms of the patient's disease or illness.

[0194] In certain embodiments where the patient's condition is improving, the dose of the administered drug is temporarily reduced or temporarily stopped for a period of time (i.e., “drug-free days”). In certain embodiments, the length of the drug-free period is between two days and one year, and includes, in just a few examples, periods of two, three, four, five, six, seven, ten, twelve, fifteen, twenty, twenty, twenty-eight, or longer than twenty-eight days. The dose reduction during the drug-free period is, in just a few examples, between 10% and 100%, and includes, in just a few examples, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

[0195] Once the patient's condition improves, a maintenance dose is administered as needed. Subsequently, in certain embodiments, the dose and / or frequency of administration is reduced, depending on the symptoms, to a level at which the improved disease, impairment, or illness is maintained. However, in certain embodiments, the patient requires intermittent treatment over a long period if symptoms recur.

[0196] The amount of a given drug corresponding to such a quantity varies depending on factors such as the specific inhibitor, the state and severity of the disease, and the unique characteristics of the subject or host requiring treatment (e.g., weight, sex), but nevertheless, it is determined according to the specific circumstances surrounding the case, including, for example, the specific drug being administered, the route of administration, the disease being treated, and the subject or host being treated.

[0197] Generally, doses used for the treatment of adult humans typically range from about 0.01 mg to about 5000 mg per day. In some embodiments, doses used for the treatment of adult humans range from about 0.01 mg to about 1000 mg per day. In some embodiments, the desired dose is provided as a single dose or as divided doses administered simultaneously or at appropriate intervals, preferably as subdoses of two, three, four times, or more times per day. In other embodiments, the compositions described herein are formulated for sustained release over periods of several hours, several days, or several months. In some embodiments, the composition is formulated for delivery over a period of 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.

[0198] In some embodiments, the appropriate daily dose for the agents and / or peptides described herein is approximately 0.01 to approximately 50 mg / kg of body weight. In some embodiments, the daily dose or amount of the active ingredient in the dosage form may be less or more than the range shown herein, based on many variables relating to the individual treatment regimen. In various embodiments, the daily dose and unit dose may be modified depending on many variables, including, but not limited to, the activity of the agent and / or peptide used, the disease or illness being treated, the mode of administration, the requirements of the individual subject, the severity of the disease or illness being treated, and the physician's judgment.

[0199] The toxicity and efficacy of such imaging and treatment regimens are not limited to, but include LD 50 ,I C 50 , EC 50 , and ED 50 This is determined by standard pharmaceutical procedures in cell culture or experimental animals, including the determination of the LD50. The dose-to-toxicity ratio is the therapeutic index, which is the LD50. 50 and ED 50 It is expressed as a ratio between . In certain embodiments, data obtained from cell culture assays and animal studies are used to formulate a range of therapeutically effective daily doses and / or therapeutically effective unit doses for use in mammals, including humans. In some embodiments, the daily dose of the drugs and / or peptides described herein is the ED with minimal toxicity. 50 It is within the range of blood concentrations including [specific component]. In certain embodiments, the range of daily doses and / or unit doses vary within this range depending on the dosage form used and the route of administration utilized.

[0200] In any of the embodiments described herein, there are further embodiments in which an 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 by injection to a mammal and / or (e) administered topically to a mammal and / or (f) administered non-systemically or topically to a mammal.

[0201] In any of the embodiments described above, there are further embodiments that include a single dose of an effective amount of the drug and / or peptide, including (i) the drug and / or peptide is administered once daily, or (ii) the drug and / or peptide is administered to a mammal multiple times over a day. In further embodiments, there are embodiments in which the drug and / or peptide is administered continuously over a period of time.

[0202] Further embodiments include multiple doses of an effective amount of the drug and / or peptide, including any of the above embodiments in which (i) the drug and / or peptide is administered continuously or intermittently as a single dose, (ii) the interval between multiple doses is every 6 hours, (iii) the drug and / or peptide is administered to a mammal every 8 hours, (iv) the drug and / or peptide is administered to a mammal every 12 hours, and (v) the drug and / or peptide is administered to a mammal every 24 hours.

[0203] In any of the embodiments described above, there are further embodiments that include a drug and / or peptide incorporated into a delivery vehicle to provide sustained delivery of the drug. In some embodiments, delivery is sustained over 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 embodiments, the drug and / or is incorporated into a transdermal patch, tape, suppository, vaginal suppository, vaginal tampon, vaginal ring, vaginal strip, vaginal capsule, vaginal tablet, vaginal pessary, vaginal cup, vaginal sponge, or intrauterine device.

[0204] In further or alternative embodiments, the method includes a drug-free period during which the administration of the drug and / or peptide is temporarily interrupted or the amount of the drug and / or peptide being administered is temporarily reduced, and the administration of the drug and / or peptide is resumed at the end of the drug-free period. In one embodiment, the length of the drug-free period varies from 2 days to 1 year.

[0205] In certain cases, it is appropriate to administer at least one of the drugs and / or peptides described herein in combination with one or more other therapeutic agents. Exemplary further therapeutic agents include, but are not limited to, hormonal therapies, including fertility agents, combination fertility agents, selective progesterone receptor antagonists, selective progesterone receptor agonists, gonadotropin-releasing hormone receptor antagonists, gonadotropin-releasing hormone receptor agonists, antiretroviral agents, anti-cancer agents, anti-inflammatory agents, nonsteroidal anti-inflammatory drugs, or any combination thereof.

[0206] 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 alone 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 to the patient is increased by administering one of the agents and / or peptides described herein together with another agent (including treatment regimens) that also has a therapeutic benefit.

[0207] In a particular embodiment, the agent and / or peptide described herein is co-administered with a second therapeutic agent. In some cases, the agent and / or peptide described herein and the second therapeutic agent modulate different aspects of the disease, disorder, or illness being treated, thereby providing an overall greater effect than either therapeutic agent administered alone. In any case, regardless of the disease, disorder, or illness being treated, the overall benefit to the patient may be additive of the two therapeutic agents, or the patient may receive a synergistic benefit.

[0208] In certain embodiments, various therapeutically effective amounts of the agents and / or peptides disclosed herein may be used in formulating pharmaceutical compositions and / or in treatment regimens in which the agents and / or peptides disclosed herein are administered in combination with one or more further agents, such as further therapeutically effective drugs or adjuvants. The effective amounts of the agents and other agents used in the combination treatment regimens are optionally determined with respect to the active ingredients themselves by means similar to those specified herein. Furthermore, the preventive / treatment methods described herein include the use of metronomic dosing, i.e., providing more frequent and lower doses to minimize toxic side effects. In some embodiments, the combination treatment regimens include treatment regimens in which the administration of the agents and / or peptides described herein is initiated before, during, or after treatment with a second agent described herein and continues until any point during treatment with the second agent or until after the completion of treatment with the second agent. This also includes treatments in which the agents and / or peptides described herein, as well as a second agent used in combination, are administered simultaneously or at different times, and / or at shorter or longer intervals, during the treatment period. Concomitant treatments also include periodic treatments that are initiated and discontinued at various points in time to aid in the clinical management of the patient.

[0209] It should be understood that dosing regimens for imaging, diagnosing, treating, preventing, or alleviating diseases in which relief is sought may be modified to suit various factors (e.g., the disease, disorder, or illness the subject is suffering from, the subject's age, weight, sex, diet, and medical condition). Therefore, in some examples, the dosing regimen used will differ, and in some embodiments, it will deviate from the dosing regimens specified herein.

[0210] With regard to the combination therapies described herein, the dosage of the co-administered drugs and / or peptides will vary depending on the type of co-agent used, the specific drug used, the disease or illness being treated, etc. In further embodiments, when co-administered with one or more other therapeutic agents, the drugs and / or peptides provided herein will be administered simultaneously with or sequentially with one or more other therapeutic agents.

[0211] In combination therapy, multiple therapeutic agents (one of which is one of the drugs and / or peptides described herein) are administered in any order or simultaneously. When administered simultaneously, the multiple therapeutic agents are provided, to name just a few, in a single unified form or in multiple forms (e.g., as a single pill or as two separate pills).

[0212] The agents and / or peptides described herein, as well as combination therapies, are administered before, during, or after the onset of a disease or illness, and the timing of administration of compositions containing the agents and / or peptides varies. Therefore, in one embodiment, the agents and / or peptides described herein are used as prophylactic agents and are continuously administered to subjects prone to developing a disease or illness to prevent its onset. In another embodiment, the agents and / or peptides and compositions are administered to subjects during the onset of symptoms or as soon as possible after the onset. In certain embodiments, the agents and / or peptides described herein are administered as soon as possible after the onset of a disease or illness is detected or suspected, for a period necessary to treat the disease. In some embodiments, the period necessary for treatment varies, and the treatment period is adjusted to the specific needs of each subject. For example, in certain embodiments, the agents and / or peptides described herein, or formulations containing the agents and / or peptides, are administered for at least two weeks, approximately one month, to approximately five years.

[0213] The peptides and compositions described herein may be administered concurrently with other therapeutic agents. In the embodiments described herein, the cyclic peptide compositions may be administered with drugs for treating osteoporosis, including but not limited to alendronate, ibandronate, risedronate, zoledronic acid, denosumab, calcitonin, estrogen, raloxifene, bazedoxifene, teriparatide, abaloparatide, or any combination thereof.

[0214] Embodiment Embodiment 1. An imaging agent comprising a peptide or peptide mimetic conjugated to an imaging component by a linker, (a) A peptide or peptide mimetic may have the 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 The formula includes an amino acid sequence, in which, R is NH2, acetylated, stearic acid, palmitic acid, myristic acid, lauric acid, C1-C8 hydrocarbon, C1-C8 fatty acid, or null. X1 is M, G, β-alanine, norleucine, norvaline, or null. 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-methylS, K, D, Orn, T, or E. X6 is I, Chg, H, or L. X7 is L or I, X8 is D, N, E, or Q. X9 is D, E, K, Q, or Orn. X 10 is H or methyl-H, X 11 is V, α-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, X 15 is W, Y, or F, X 16 is R, G, or null, (b) The linker is null or the linker is an imaging agent that conjugates an imaging component and a peptide or peptidomimetic.

[0215] Embodiment 2. An imaging agent comprising a peptide or peptidomimetic conjugated to an imaging component by a linker, (a) The peptide or peptidomimetic has the formula: X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 and comprises an amino acid sequence according to, wherein, X1 is M or null, X2 is S, I, G, T, A, L, or null, X3 is R, K, or null, X4 is a positively charged amino acid, citrulline, Orn, D, E, 8-aminooctanoic acid, or an aminocarboxylic acid having 4 to 12 carbons, X5 is M, norleucine, Orn, D, E, K, H, R, K, 8-aminooctanoic acid, an aminocarboxylic acid having 4 to 12 carbons, or null, 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 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, X 13 is G, GABA, or null, X 14 is V, I, L, norvaline, or null, X 15 is W, Y, F, or null, X 16 is R, G, or null, (b) The linker is null or the linker is an imaging agent that conjugates with an imaging component and a peptide or peptidomimetic.

[0216] Embodiment 3. The imaging agent according to Embodiment 1 or 2, wherein the imaging component is a fluorescent molecule, a contrast agent for magnetic resonance imaging (MRI), a radionuclide, microbubbles, a contrast agent for computed tomography (CT), or a contrast agent for photoacoustic imaging.

[0217] Embodiment 4. The imaging agent according to Embodiment 1 or 2, wherein the imaging component is a fluorophore, a fluorescent dye, a dye, a pigment, a fluorescent transition metal, or a fluorescent protein.

[0218] Embodiment 5. The imaging agent according to Embodiment 1 or 2, wherein the imaging component is a paramagnetic ion complex or a superparamagnetic magnetite particle.

[0219] Embodiment 6. The imaging agent according to Embodiment 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.

[0220] Embodiment 7. The imaging components are: 18 F, 64 Cu, 68 Ga, 99m The imaging agent according to Embodiment 1 or 2, which is a radionuclide comprising at least one selected from the group consisting of Tc.

[0221] Embodiment 8 The imaging agent according to Embodiment 1 or 2, wherein the imaging component comprises superparamagnetic iron oxide nanoparticles (SPION) or a DOTA-Fe(III) composite.

[0222] Embodiment 9. The imaging agent according to Embodiment 1 or 2, wherein the imaging component is gold nanoparticles or an iodine compound.

[0223] Embodiment 10. The linker is 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 alkenylene, C2-C6 alkylynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C3-C 10 Cycloalkylene, 3-10 member heterocycloalkylene, C6-C 10 The compound comprises one or more aryl or 5-10 member heteroarylenes, where each C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 alkoxy, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkylynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C3-C 10 Cycloalkylene, 3-10 member heterocycloalkylene, C6-C 10 Aryl and 5-10 membered heteroarylenes are optionally given 1, 2, or 3 R 9 It is independently replaced by, n is an independent integer between 1 and 10. m is an independent integer between 1 and 10. R 9These are independently halogens, C1-C4 alkyls, C2-C4 alkenyls, C2-C4 alkynyls, C1-C4 haloalkyls, C1-C4 alkoxys, C1-C4 haloalkoxys, C1-C4 heteroalkyls, -C(O)H, -C(O)OH, -CN, and C3-C 10 Cycloalkyl, 3-10 member heterocycloalkyl, C6-C 10 Aryl, 5-10 member heteroaryl, -C(O)(C1-C4alkyl), -C(O)O(C1-C4alkyl), -C(O)NH2, -C(O)NH(C1-C4alkyl), -C(O)N(C1-C4alkyl)2, -NH2, -NH(C1-C4alkyl), -N(C1-C4alkyl)2, -NH(C2-C4 alkylene)-OH, -NH(C2-C4 alkylene)-O-(C1-C4alkyl), -OH, -O(C1 -C4alkyl), -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)-(5-10 member heteroaryl), -O(C6-C 10 Ariel), - SH, S(O)2OH, -S(O)2(C1-C4alkyl), -S(O)2NH2, -S(O)2NH(C1-C4alkyl), or -S(O)2N(C1-C4alkyl)2, or two R 9 Along with the atoms to which they are attached, C3-C 10 An imaging agent according to any one of Embodiments 1 to 9, which forms a cycloalkyl or 3- to 10-membered heterocycloalkyl ring.

[0224] Embodiment 11. The linker is 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 The compounds include cycloalkylenes, 3-10 member heterocycloalkylenes, or combinations thereof, each comprising C1-C6 alkylene, C1-C6 alkoxy, C1-C6 heteroalkylene, and C3-C 10 Cycloalkylenes and 3- to 10-membered heterocycloalkylenes may optionally have 1, 2, or 3 R groups. 9 They are independently substituted by n, m, and R 9 The imaging agent according to Embodiment 10, as defined in Embodiment 36.

[0225] Embodiment 12. The linker is X1~X 16 The imaging agent according to any one of embodiments 1 to 11, wherein at least one of the amino acids is conjugated with a peptide or peptide mimetic.

[0226] Embodiment 13. The imaging agent according to any one of Embodiments 1 to 11, wherein the linker is conjugated to the N-terminus of a peptide or peptide mimetic.

[0227] Embodiment 14. The imaging agent according to Embodiment 13, wherein the peptide or peptide mimetic further comprises -NH2 at the C-terminus.

[0228] Embodiment 15. The imaging agent according to any one of Embodiments 1 to 12, wherein the linker is conjugated to the C-terminus of a peptide or peptide mimetic.

[0229] Embodiment 16. The imaging agent according to Embodiment 15, wherein the peptide or peptide mimetic further comprises a C1-C8 alkyl-C(O)- at the N-terminus.

[0230] Embodiment 17. The peptide or peptide mimetic has a CH3(CH2) group at its N-terminus. p The imaging agent according to Embodiment 15, further comprising C(O)-, where p is an integer from 0 to 7.

[0231] Embodiment 18. The imaging agent according to Embodiment 15, wherein the peptide or peptide mimetic further comprises CH3(CH2)6C(O)- at the N-terminus.

[0232] Embodiment 19. The imaging agent according to any one of Embodiments 1 to 12, wherein the peptide or peptide mimetic comprises the amino acid sequence of SEQ ID NO: 496 or SEQ ID NO: 497.

[0233] Embodiment 20. The imaging agent according to any one of Embodiments 1 to 21, wherein the imaging component is iron oxide nanoparticles.

[0234] Embodiment 21. The imaging agent according to any one of Embodiments 1 to 21, wherein the imaging component is iron chelated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0235] Embodiment 22. An imaging agent according to any one of Embodiments 1 to 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.

[0236] Embodiment 23. An imaging agent according to any one of Embodiments 1 to 21, wherein the imaging component is a fluorophore, a fluorescent dye, a dye, a pigment, a fluorescent transition metal, or a fluorescent protein.

[0237] Embodiment 24. The imaging agent according to any one of Embodiments 1 to 21, wherein the imaging component is superparamagnetic iron oxide nanoparticles (SPION) or a gadolinium composite.

[0238] Embodiment 25. The imaging components are: 18 F, 64 Cu, 68 Ga, or 99m An imaging agent according to any one of Embodiments 1 to 21, wherein the radionuclide is Tc.

[0239] Embodiment 26. The imaging agent according to any one of Embodiments 1 to 21, wherein the imaging component is gold nanoparticles or an iodine compound.

[0240] Embodiment 27. The imaging agent according to any one of Embodiments 1 to 26, wherein the peptide or peptide mimetic is cyclic or bicyclic.

[0241] Embodiment 28. The imaging agent according to Embodiment 27, wherein the peptide or peptide mimetic is cyclized by a Cys-Cys disulfide bond.

[0242] Embodiment 29. The imaging agent according to Embodiment 27, wherein the peptide or peptide mimetic is cyclized by an amide bond.

[0243] Embodiment 30. The imaging agent according to Embodiment 29, wherein the amide bond is a head-tail between the N-terminus and the C-terminus.

[0244] Embodiment 31. The imaging agent according to Embodiment 29, wherein the amide bond is a head-side chain between the N-terminus and the internal COOH.

[0245] Embodiment 32. The imaging agent according to Embodiment 29, wherein the amide bond is a side chain-tail between the internal NTh and the C-terminus.

[0246] Embodiment 33. The imaging agent according to Embodiment 27, wherein the amide bond is a side chain-side chain between the internal NTh and the internal COOH.

[0247] Embodiment 34. The imaging agent according to Embodiment 27, wherein the peptide or peptide mimetic is cyclized using hydrocarbon stapling.

[0248] Embodiment 35. The imaging agent according to Embodiment 27, wherein the peptide or peptide mimetic is cyclized using click chemistry.

[0249] Embodiment 36. The imaging agent according to any one of Embodiments 1 to 35, wherein the peptide consists of fewer than 81 amino acid residues.

[0250] Embodiment 37. The imaging agent according to any one of Embodiments 1 to 36, wherein the peptide comprises one or more non-natural amino acids.

[0251] Embodiment 38. The imaging agent according to Embodiment 36, wherein one or more non-natural amino acids are N-methyl amino acids.

[0252] Embodiment 39. A method for detecting endometriotic lesions in a subject, (a) Administering a preparation containing an imaging agent as described in any one of Embodiments 1 to 38 or 60 to 85 to a subject, (b) Imaging the subject to determine the presence of imaging components Methods that include...

[0253] Embodiment 40. (c) Administering a therapeutically effective amount of a second β-catenin-binding peptide or peptide mimetic to the subject, (d) Wait for at least one week, then repeat steps (a) and (b) The method according to embodiment 39, further including the method described in embodiment 39.

[0254] Embodiment 41. The method according to Embodiment 39 or 40, wherein the imaging in (b) further includes creating an image and displaying the image.

[0255] Embodiment 42. The method according to Embodiment 41, wherein the image is a video, a still image, a real-time video, or any combination thereof.

[0256] Embodiment 43. The method according to Embodiment 42, wherein the subject or image is visualized or displayed by an imaging agent during a surgical procedure performed on the subject.

[0257] Embodiment 44. The method according to Embodiment 43, wherein the imaging agent is administered during or before surgery for the diagnosis, staging, and / or excision of endometriosis.

[0258] Embodiment 45. The method according to any one of Embodiments 36 to 44, wherein the formulation is administered vaginally or intravenously.

[0259] Embodiment 46. The imaging in (b) is the method according to any one of Embodiments 36 to 45, including 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.

[0260] Embodiment 47. The method according to any one of Embodiments 36 to 46, wherein the subject is a human.

[0261] Embodiment 48. The method according to Embodiment 47, wherein imaging includes imaging the whole body.

[0262] Embodiment 49. The method according to any one of Embodiments 36 to 48, wherein the test subject is a cultured cell line.

[0263] Embodiment 51. The method according to any one of Embodiments 36 to 48, wherein the test subject is ex vivo tissue extracted from an animal.

[0264] Embodiment 52. The second β-catenin-binding peptide or peptide mimetic is given by 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 The formula includes an amino acid sequence, in which, R is NH2, acetylated, stearic acid, palmitic acid, myristic acid, lauric acid, C1-C8 hydrocarbon, C1-C8 fatty acid, or null. X1 is M, G, β-alanine, norleucine, norvaline, or null. 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-methylS, K, D, Orn, T, or E. X6 is I, Chg, H, or L. X7 is L or I, X8 is D, N, E, or Q. X9 is D, E, K, Q, or Orn. X 10 is H or methyl-H, X 11 It is V, α-methyl V, Chg, L, I, or norvaline. X 12 is Q, Aib, S, R, or N, X 13 These are R, K, citrulline, Orn, D, or E. X 14 These are V, I, L, or norvaline. X 15 is W, Y, or F, X 16 The method according to any one of embodiments 36 to 51, wherein is R, G, or null.

[0265] Embodiment 53. The second β-catenin-binding peptide or peptide mimetic has the amino acid sequence Oct-W-NMeA-Q-Orn * -ILDE * The method according to Embodiment 52, comprising H-Chg-QRVWG-NH2 (Sequence ID 491).

[0266] Embodiment 54. The second β-catenin-binding peptide or peptide mimetic is given by formula: X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13-X 14 -X 15 -X 16 The formula includes an amino acid sequence, in which, X1 is M or null, X2 is S, I, G, T, A, L, or null. X3 is R, K, or null. X4 is a positively charged amino acid, citrulline, Orn, D, E, 8-aminooctanoic acid, or an aminocarboxylic acid having 4 to 12 carbon atoms. X5 is M, norleucine, Orn, D, E, K, H, R, K, 8-aminooctanoic acid, an aminocarboxylic acid having 4 to 12 carbon atoms, or null. 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 is C, S, A, Abu, C(me), or S(Bzl), X 11 These are F, H, A, K, E, Chg, Cng, or Orn. X 12 is W, Y, A, or F, X 13 is G, GABA, or null. X 14 is V, I, L, norvaline, or null. X 15 is W, Y, F, or null, X 16 The method according to any one of embodiments 36 to 51, wherein is R, G, or null.

[0267] Embodiment 55. The method according to any one of Embodiments 39 to 54, wherein the peptide binds to cytoplasmic β-catenin and inhibits the translocation of β-catenin to the cell nucleus.

[0268] Embodiment 56. The method according to any one of Embodiments 39 to 54, wherein the peptide binds to cytoplasmic β-catenin to maintain or increase membrane-bound β-catenin.

[0269] Embodiment 57. The method according to any one of Embodiments 39 to 54, wherein the peptide binds to cytoplasmic β-catenin to prevent β-catenin from acting as a transcription factor to oncogenes, matrix metalloproteinase 9 (MMP9), or chloride C3 channel (ClC-3).

[0270] Embodiment 58. The method according to any one of Embodiments 39 to 54, wherein the peptide binds to cytoplasmic β-catenin to prevent transformation, invasion, migration, fibrillation, or any combination thereof of EMS cells.

[0271] Embodiment 59. The method according to any one of Embodiments 39 to 54, wherein the peptide binds to cytoplasmic β-catenin, preventing β-catenin from binding to the estrogen receptor (ESR1).

[0272] Embodiment 60. The method according to any one of Embodiments 39 to 54, wherein the peptide binds to cytoplasmic β-catenin, and the membrane activity of β-catenin is not reduced.

[0273] Embodiment 61. The method according to any one of Embodiments 39 to 54, wherein the peptide is bound to cytoplasmic β-catenin, and the β-catenin-E-cadherin bond is not reduced.

[0274] Embodiment 62. The method according to any one of Embodiments 39 to 54, wherein the peptide inhibits the activity of oncogenic transcription factors.

[0275] Embodiment 63. The method according to any one of Embodiments 39 to 54, wherein the amount of nuclear β-catenin in the cells of the subject is reduced by at least 5%.

[0276] Embodiment 64. The method according to Embodiment 62, wherein the reduction in nuclear β-catenin is in the cells of a control subject that was not administered a therapeutically effective amount of the pharmaceutical composition.

[0277] Embodiment 65. The method according to Embodiment 62, wherein the reduction in nuclear β-catenin is in the cells of the subject obtained before the subject developed the disease.

[0278] Embodiment 66. The method according to Embodiment 62, wherein the reduction in nuclear β-catenin is for cells from subjects obtained at different time points.

[0279] Embodiment 67. The method according to any one of Embodiments 39 to 66, wherein the therapeutically effective dose is approximately 0.01 mg to approximately 1000 mg.

[0280] Embodiment 68. Formula III: Labeled-linker-peptide (Formula III) An imaging agent, (a) The label is an imaging component, (b) The peptide is given by 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) The formula includes an amino acid sequence, in which, X1 is M, G, β-alanine, norleucine, norvaline, or null. 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-methylS, K, D, Orn, T, or E. X6 is I, Chg, H, or L. X7 is L or I, X8 is D, N, E, or Q. X9 is D, E, K, Q, or Orn. X 10 is H or methyl-H, X 11 It is V, α-methyl V, Chg, L, I, or norvaline. X 12 is Q, Aib, S, R, or N, X 13 These are R, K, citrulline, Orn, D, or E. X 14 These are V, I, L, or norvaline. X 15 is W, Y, or F, X 16 is R, G, or null, (c) The linker is either null or conjugates with the label and peptide, and is an imaging agent.

[0281] Embodiment 69. The imaging agent according to Embodiment 68, wherein the label is a fluorescent molecule, a contrast agent for magnetic resonance imaging (MRI), a radionuclide, a microbubble, a contrast agent for computed tomography (CT), or a contrast agent for photoacoustic imaging.

[0282] Embodiment 70. The imaging agent according to Embodiment 68, wherein the label is a fluorophore, a fluorescent dye, a dye, a pigment, a fluorescent transition metal, or a fluorescent protein.

[0283] Embodiment 71. The imaging agent according to Embodiment 68, wherein the label is a paramagnetic ion complex or superparamagnetic magnetite particles.

[0284] Embodiment 72. The imaging agent according to Embodiment 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.

[0285] Embodiment 73. The sign is, 18 F, 64 Cu,68 Ga, 99m The imaging agent according to Embodiment 68, which is a radionuclide comprising at least one selected from the group consisting of Tc.

[0286] Embodiment 74. The imaging agent according to Embodiment 68, wherein the label comprises superparamagnetic iron oxide nanoparticles (SPION) or a DOTA-Fe(III) composite.

[0287] Embodiment 75. The imaging agent according to Embodiment 68, wherein the label is gold nanoparticles or an iodine compound.

[0288] Embodiment 76. The linker is 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 alkenylene, C2-C6 alkylynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C3-C 10 Cycloalkylene, 3-10 member heterocycloalkylene, C6-C 10 The compound comprises one or more aryl or 5-10 member heteroarylenes, where each C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 alkoxy, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 alkylynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C3-C 10 Cycloalkylene, 3-10 member heterocycloalkylene, C6-C 10 Aryl and 5-10 membered heteroarylenes are optionally given 1, 2, or 3 R 9 It is independently replaced by, n is an independent integer between 1 and 10. m is an independent integer between 1 and 10. R 9These are independently halogens, C1-C4 alkyls, C2-C4 alkenyls, C2-C4 alkynyls, C1-C4 haloalkyls, C1-C4 alkoxys, C1-C4 haloalkoxys, C1-C4 heteroalkyls, -C(O)H, -C(O)OH, -CN, and C3-C 10 Cycloalkyl, 3-10 member heterocycloalkyl, C6-C 10 Aryl, 5-10 member heteroaryl, -C(O)(C1-C4alkyl), -C(O)O(C1-C4alkyl), -C(O)NH2, -C(O)NH(C1-C4alkyl), -C(O)N(C1-C4alkyl)2, -NH2, -NH(C1-C4alkyl), -N(C1-C4alkyl)2, -NH(C2-C4 alkylene)-OH, -NH(C2-C4 alkylene)-O-(C1-C4alkyl), -OH, -O(C1 -C4alkyl), -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)-(5-10 member heteroaryl), - O(C6-C 10 Ariel), - SH, S(O)2OH, -S(O)2(C1-C4alkyl), -S(O)2NH2, -S(O)2NH(C1-C4alkyl), or -S(O)2N(C1-C4alkyl)2, or two R 9 Along with the atoms to which they are attached, C3-C 10 An imaging agent according to any one of embodiments 68 to 75, which forms a cycloalkyl or 3- to 10-membered heterocycloalkyl ring.

[0289] Embodiment 77. The linker is 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 The compounds include cycloalkylenes, 3-10 member heterocycloalkylenes, or combinations thereof, each comprising C1-C6 alkylene, C1-C6 alkoxy, C1-C6 heteroalkylene, and C3-C 10 Cycloalkylenes and 3- to 10-membered heterocycloalkylenes may optionally have 1, 2, or 3 R groups. 9 They are independently substituted by n, m, and R 9 The imaging agent according to Embodiment 76, as defined in Embodiment 36.

[0290] Embodiment 78. The linker is X1~X 16 An imaging agent according to any one of embodiments 68 to 77, wherein at least one of the amino acids is conjugated with a peptide.

[0291] Embodiment 79. An imaging agent according to any one of Embodiments 68 to 77, wherein the linker is conjugated with the N-terminus of the peptide.

[0292] Embodiment 80. The imaging agent according to Embodiment 79, wherein the peptide further comprises -NH2 at the C-terminus.

[0293] Embodiment 81. An imaging agent according to any one of Embodiments 68 to 77, wherein the linker is conjugated to the C-terminus of the peptide.

[0294] Embodiment 82. The imaging agent according to Embodiment 80, wherein the peptide further comprises a C1-C8 alkyl-C(O)- at its N-terminus.

[0295] Embodiment 83. The peptide has CH3(CH2) at its N-terminus. p The imaging agent according to Embodiment 80, further comprising C(O)-, where p is an integer from 0 to 7.

[0296] Embodiment 84. The imaging agent according to Embodiment 80, wherein the peptide further comprises CH3(CH2)6C(O)- at its N-terminus.

[0297] Embodiment 85. The imaging agent according to any one of Embodiments 68 to 84, wherein the amino acid sequence is cyclically formed by an amide bond, the amide bond being (1) head-tail between the N-terminus and the C-terminus, (2) head-side chain between the N-terminus and the internal COOH, (3) side chain-tail between the internal NH2 and the C-terminus, or (4) side chain-side chain between the internal NH2 and the internal COOH, and the amide bond is attached to an amino acid that is not bound to either a linker or a label.

[0298] Numerous embodiments of this disclosure have been described. However, it will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are also within the scope of the following claims. [Examples]

[0299] Example 1: Peptides conjugated to imaging components can be used as imaging agents to visualize disease and API / drug product features in vivo. The series of figures presented aims to demonstrate the potential of peptide agents in advanced biomedical applications. Figure 1, an example relating to endometriosis, shows that compounds 492, 494, and 493 were used in animals 2–4, respectively, demonstrating that these imaging and diagnostic agents can be used as valuable tools for imaging subjects to detect the presence of diseased tissue, thereby visualizing endometriotic lesions in animals. Ex vivo analysis, similar to live-in vivo imaging, is shown for the same mice subjected to necropsy.

[0300] C57BI / 6 albino mice were imaged using spectral imaging (Living Image 4.7.3, IVIS Spectrum, Perkin Elmer). Mice were hypnotized using isoflurane according to standard procedures. Adaptive fluorescence background subtraction, spectral separation, Cy7 epi-illumination, and spectral emission scanning from 757–781 nm were used. At each time point, the threshold for endometriotic mice was set and normalized using the threshold for normal mice.

[0301] These agents can also be used to evaluate key characteristics of active pharmaceutical ingredients (APIs) or drug products. Figure 2 shows the in vivo distribution of compound 491 using labeled peptide compound 492, demonstrating that these agents are specific and are either barely present or undetectable in other organ systems. This is further supported by Figure 15, which shows that compound 492 is detected only in endometriotic tissue and not in normal uterine tissue. Figure 3 shows intravaginal administration of compound 494 and its detection at lesion sites. This has been successfully demonstrated with other labeled peptides, as well as other administration routes, including but not limited to SQ, IV, and PO administration, all of which have different pharmacokinetic profiles, as viable options. The in vitro experiment in Figure 4 supports the visual observation in Figure 3, showing that the peptide is permeable through vaginal tissue and that intravaginal administration of the peptide takes approximately 3 hours to pass through human vaginal tissue. This is consistent with imaging performed by animal models using the imaging agents of this disclosure, supporting the accuracy of the imaging agents.

[0302] The drugs and peptides described herein have shown promise in monitoring disease progression or evaluating the effectiveness of therapeutic interventions. As shown in Figures 3 and 4, the ability to administer these drugs via various routes provides flexibility in the imaging, diagnosis, and / or delivery of these therapeutic agents. As shown in Figure 6, time points were taken between 20 minutes and 72 hours, and imaging material was observed up to 48 hours after blood clearance in subjects with endometriosis, whereas imaging material was not visualized after blood clearance in subjects without endometriosis. In Figure 7, peptides were used to track disease progression and / or treated lesions, thereby providing a quantitative assessment of treatment effectiveness and disease burden over time. Figure 7 shows the imaging, diagnosis, and efficacy effects of the drug obtained from animals that received two SQ doses of compound 492 with several weeks apart. Final autopsy reports of ex vivo lesion analysis were consistent with live visualization by drug administration. Tracking the reduction of lesions in the same mice after two SQ administrations several weeks apart has shown that daily administration is not necessary for efficacy.

[0303] Example 2: Identification of effective doses and administration regimens This embodiment aims to identify an approximate effective dose and determine the optimal dosing regimen, as shown in Figures 8 to 10. Figures 8 to 21 utilize compound 491 or 492 as examples, but are repeated and supported by many of the compounds of this disclosure.

[0304] Figure 8 shows the lesion weights obtained from endometriotic mice administered compound 491 daily for 3 weeks (left graph) or 6 weeks (right graph). The treatment improved with longer treatment durations, especially when administered at lower doses. This provides valuable guidance for optimizing treatment plans to achieve maximum therapeutic outcomes, especially when utilizing lower dose levels.

[0305] Figure 9 shows the pharmacokinetic exposure of compound 491 at 1 mg / kg in mice analyzed in serum. Compared with the data in Figures 6-8, the pharmacokinetic effect and lesion-specific persistence of compound 491 surpasses its pharmacokinetic presence in serum, demonstrating the drug's sustained potential for imaging, diagnosis, and treatment.

[0306] Figure 10 supports this finding using a cell proliferation assay. A single dose of SEQ ID NO: 491 exerted a pharmacodynamic effect that lasted up to 5 days after treatment, longer than its serum pharmacokinetic effect. 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 per 50 μL of assay medium. After incubation overnight at 37°C and 5% CO2, the medium was aspirated, and each well was supplemented with fresh Prigrow III medium supplemented with 10% FBS and 1% PS containing the peptide of interest at different concentrations. Cells were treated with the peptide for only 1 day, and then fresh medium was added to each well daily for the next 4 days. To determine the effect of the peptide, the medium was mixed with Presto Blue and turned pink within 4–6 hours. Fluorescence in each well was measured at 540nm and 590nm using a Biotek HTX microplate reader. IC was measured using a Graph Pad Prism 9.0. 50 The graphs were calculated to provide a quantitative measure of the peptide's effect on cell proliferation. A single dose of compound 491 has a sustained pharmacodynamic effect (illustrated 5 days after treatment). This suggests that while increased dosing frequency may potentially enhance the results, it is not necessary for efficacy.

[0307] Example 3: In vitro characterization of the drug These studies aim to demonstrate that diagnostic peptides remain cell-permeable even when conjugated to imaging components and can have visualized signals without quenching from conjugation, intracellular location, or peptide binding to β-catenin targets. Furthermore, the imaging components do not interfere with the ability of the diagnostic peptides to localize in the cytoplasm, bind to β-catenin in the cytoplasm, translocate from the cytoplasm to the inner membrane, or disrupt the binding of that β-catenin to the membrane. Cy7-labeled compound 491 (compound 492) can bind to target β-catenin, be successfully translocated by the inner membrane-bound target, and can be imaged using the peptide-conjugated component without signal disruption from its conjugation, peptide, or target binding. It is rapidly taken up into cells and retained in diseased tissue for longer than in serum. These in vitro datasets support in vivo imaging studies and pharmacodynamic investigations shown in other figures.

[0308] As shown in Figures 11 and 12, compound 492 directly binds to the intracellular target β-catenin, is cell-permeable, and binds to the target within the cell's cytoplasm. This binding does not occur in the nucleus and does not appear to interfere with membrane binding. To determine whether compound 491 is cell-permeable, the cellular localization of compound 492, its binding to its binding partner (β-catenin), and its visualization by fluorophores, human endometriosis 12z cells were incubated with compound 492 for 1 hour. Membrane, cytoplasmic, and nuclear fractions were extracted. BCA analysis was performed to determine the protein concentrations. Equal amounts of protein obtained from all fractions together with compound 492 were electrophoresed on a Native PAGE gel. These results support the mechanism of action by showing that both compound 492 and the β-catenin-compound complex can be visualized and separated on the native gel, and that compound 492 was present in the membrane fraction and cytoplasmic fraction but not in the nuclear fraction.

[0309] To determine the timing of compound 491 binding to β-catenin in the cytoplasm, 0.5 μM of compound 492 was administered to human endometriosis 12z cells at different time points, i.e., once at 1 hour, 3 hours, 5 hours, 7 hours, 1 day, 2 days, and 3 days. After administration, the cells were washed with PBS buffer and the cytoplasmic fraction was extracted. Electrophoresis was performed on natural gel and imaging was performed. The results in Figure 14 suggest that compound 492 binds to β-catenin within 1 hour of incubation, increases signal intensity over 24 hours, and shows a stable signal over several days. These findings are consistent with previous experiments, namely, 1) FP experiments where binding occurred after 1 hour and persisted for at least 24 hours (data not shown) and (2) imaging experiments of rodent model lesions where, although rodents without endometriosis showed no signs of the imaging agent after blood clearance (Figure 6), binding persisted in the lesions for at least 48 hours (Figure 6). Furthermore, this is consistent with proliferation results that showed compound 491, with only a single dose, produced a sustained pharmacodynamic effect that reduced cell proliferation.

[0310] The results in Figure 12 show that compound 492 binds to β-catenin within one hour of incubation, and the signal remains stable for at least three days. These findings are consistent with the inventors' previous experiments, namely (1) FP experiments in which binding occurs after one hour and persists for at least 24 hours, and (2) imaging experiments of rodent model lesions in which binding persists beyond blood clearance in the lesions. This is consistent with animal imaging results from mice showing that a single dose of compound 492 specifically persists in disease lesions.

[0311] To visualize the effect of compound 491 on β-catenin in the cytoplasmic fraction, human endometriosis (12z cells) were administered 0.5 μM of compound 492 at various time points, i.e., 1 hour, 3 hours, 5 hours, 7 hours, 1 day, 2 days, and 3 days after treatment, and analyzed. After administration, cells were washed with PBS buffer, and the cytoplasmic fraction was extracted. Electrophoresis was performed on a natural gel, and imaging was performed using Cy7. Subsequently, the gel was transferred, and Western blotting was performed using a nitrocellulose membrane and probed with a β-catenin antibody. Western blotting was imaged using chemiluminescence. The results in Figure 13 suggest that β-catenin protein begins to decrease after binding of compound 492 occurs in the cytoplasm.

[0312] To visualize the effect of compound 491 on β-catenin in the membrane fraction, human endometriosis 12z cells were administered 0.5 μM of compound 492 at different time points, i.e., 1 hour, 3 hours, 5 hours, 7 hours, 1 day, and 2 days after treatment, and analyzed. After administration, cells were washed with PBS buffer, and the non-cytoplasmic / non-nuclear fraction was extracted. Electrophoresis was performed on a natural gel, and imaging was performed using Cy7. Subsequently, the gel was transferred, and Western blotting was performed using a nitrocellulose membrane and probed with β-catenin antibody. Western blotting was imaged using chemiluminescence. The results in Figure 13 suggest that the β-catenin protein remains stable in the membrane fraction.

[0313] These observations suggest that the peptide may inherently possess flexibility, in that, even though only one of the binding sites physically interacts with compound 491, the interaction with one binding site (TCF4) may influence the allosteric confirmation of the other binding site (BCL9). This flexibility could support the relocalization and / or supportive binding of β-catenin from the cytoplasm to the membrane, providing support for its Wnt4 rescue activity and mechanism of action.

[0314] Example 4: Species homology Furthermore, this specification demonstrates that peptides can exhibit high homology across species (in this example, compound 491 exhibits over 95% homology across various animal species), and therefore are applicable to all animal and mammalian species in imaging, diagnostic, and therapeutic interventions. As shown in the data in Table 3, the binding characteristics of compound 491 to β-catenin show substantial similarity across various mammalian species. Thus, other imaging agents utilizing compound 492 and compound 491 can be effectively used in animal studies to visualize the progression or elimination of endometriotic disease using therapeutic agents or other chemical compounds. This offers significant advantages in the use of animal cohorts, and is highly useful in heterogeneous diseases such as endometriosis, for many reasons, including the ability to visualize efficacy in real time or the efficacy of therapeutic agents within the same animal without requiring animal slaughter.

[0315] Table 3 shows the binding data across all mammals. As shown in Table 3, the binding of compound 491 to β-catenin in mice, rats, dogs, and humans is nearly identical.

[0316] [Table 3]

[0317] Example 5: WNT4 activity is provided for both efficacy and safety due to the allosteric conformation (confirmation) and ring flexibility of the β-catenin target. This embodiment aims to demonstrate the allosteric conformational change of β-catenin, where binding at a single site induces a structural change throughout the entire molecule, enabling its transition from the cytoplasm to its interaction with the cell membrane. β-catenin is known to inherently possess several structures and can exist either intramembranely or cytoplasmically. Because the TCF4 and BCL9 binding sites are located on opposite sides of the large protein, it was previously assumed that binding to a small molecule could not simultaneously affect them. However, Table 4 illustrates that drug and / or peptide binding to β-catenin can affect both sites similarly, demonstrating the inherent full protein flexibility of β-catenin through conformational adaptation. This flexibility allows cytoplasmic β-catenin to adopt conformations that promote membrane localization, similar to the action of Wnt4 agonists. Consequently, this demonstrates the binding ability of β-catenin, which, due to its flexibility, can take on forms that bind to both the cytoplasm and the membrane. The ability to relocalize β-catenin to the membrane and / or support the binding of β-catenin to the membrane enables the efficacy of the drug, while retaining β-catenin within the membrane allows for both the safety and mechanism of action of the peptide. This activity is shown in Figure 15, which shows the cellular localization of the peptide, with most of it remaining in the membrane after reaching equilibrium, suggesting the Wnt4 activity of the peptide. Figure 16 quantifies the observation in Figure 15.

[0318] Drug binding to one site of β-catenin promotes allosteric binding of the drug to that site, leading to a confirmational change in allosteric conformation, which may affect both the BCL9 binding site and the TCF4 binding site, as shown in Table 4.

[0319] [Table 4-1]

[0320] [Table 4-2]

[0321] Example 6: On-target effect and downstream target This embodiment demonstrates the on-target effects and mechanism of action of a drug, particularly compound 491, and its impact on the β-catenin signaling pathway, which may lead to imaging, diagnosis, and treatment of diseases caused by β-catenin dysregulation. Since endometriotic degeneration was observed in mice treated with compound 491 for 3 or 6 weeks (Figure 8), the on-target changes in the resulting excised endometrial lesions were investigated. Figures 17–21 show the effects of administering compound 491 to animals on endometriotic lesions extracted after treatment. These include decreased epithelial proliferation (Figure 17), increased epithelial apoptosis (Figure 18), increased recruitment of activated macrophages (Figure 19), decreased targeted downstream β-catenin RNA (Figure 20), and decreased targeted downstream β-catenin protein (Figure 21). These figures support the mechanism of action and target activity of these peptides, and these sets of experiments have been repeated with many of the compounds in this disclosure.

[0322] Furthermore, Figure 22 shows that diseased tissue (in this example, endometriosis) in an endometriosis mouse model can be imaged using a labeled peptide that targets the downstream target protein MMP2.

[0323] Example 7: Peptide pKa values ​​and pH differences in endometrial cells promote peptide specificity. The specificity of the peptide to diseased cells has been demonstrated. This is supported by the understanding that abnormal cells exhibit different intracellular pH (pHi) compared to normal cells, increasing permeability in diseased cells and decreasing permeability in normal cells. The inventors tested endometriosis cells and normal cells with an intracellular pH indicator dye (Invitrogen®, catalog number P35372) and found that the pH of normal cells was 7.2 and the pH of endometriosis cells was 4.5. To evaluate whether the membrane permeability of compound 491 changes with pH fluctuations, PAMPA was performed over pH 3 to pH 10 according to published literature. The inventors confirmed that the permeability of compound 491 differs significantly from high to low permeability depending on the pH. As two examples, the pKa distributions of compound 491 and compound 430 showed pKa values ​​of 6.7 and 5.2, respectively, both of which fall between the pH of endometriosis cells (4.5) and the pH of normal cells (7.2). In short, these data suggest that these compounds exhibit dramatic changes in cell permeability based on their protonation state in response to different pH environments, thereby promoting selectivity in endometriosis cells.

[0324] Example 8: The peptides disclosed herein do not exhibit off-target activity. Compound 491 was tested across Panlabs SAFETYScan, a safety panel with 78 targets used in drug safety assessments to predict clinical adverse events. Compound 491 showed no off-target activity whatsoever, even at the highest concentrations tested, including the transporters hERG, CaV, and NaV for the assessment of cardiac arrhythmias.

[0325] Example 9: The peptides disclosed herein do not affect the HPG axis, clinical chemistry, or hematology. To visualize potential off-target effects on the reproductive system, the uterus, ovaries, and peritoneal wall were visualized by H&E after treatment with compound 491. No toxicity or necrotic off-target effects were observed in these organs (uterus and ovaries, Figure 23). Furthermore, autopsy revealed that compound 491 did not alter the uterine cycle / HPG axis or uterine weight (data not shown). Standard animal clinical chemistry parameters (Table 5) and hematological findings (not shown) after treatment with compound 491 were within the normal range. Further toxicological testing confirmed that no adverse effects were observed even at the maximum viable dose (more than 2000 times the expected clinical dose exposure).

[0326] [Table 5]

[0327] In short, preliminary data establish a robust foundation, demonstrating that peptides from our library can function as effective diagnostic agents because they (1) directly bind to β-catenin even when conjugated to imaging components, (2) are cell-permeable even when conjugated to imaging components, (3) possess a detectable signal that is not weakened by peptide conjugation, (4) exhibit a sustained half-life in lesions after blood clearance, (5) are not absorbed by normal cells, (6) accurately detect lesions in in vivo live imaging compared to ex vivo autopsy reports, (7) can quantify disease burden and therapeutic efficacy, (8) demonstrate pH-selective targeting of disease cells based on pKa characteristics, (9) exhibit neither toxicity nor off-target activity, and (10) are non-hormonal. Therefore, our main hypothesis is that novel non-hormonal peptides specific to endometriosis cells, conjugated with iron-based imaging components, can function as safe targeting MRI imaging agents. The imaging agent is expected to reliably, specifically, and sensitively detect endometriosis using a non-invasive method.

[0328] Example 10: Pharmacokinetic (PK) analysis confirms the blood clearance of peptides. PK studies were performed in healthy CD1 mice, and the absorption, distribution, and efflux of compound 491 in plasma were profiled by LC / MS (Table 6). Compound 491 was detectable up to 24 hours after administration, at which point it was removed from the bloodstream, similar to the imaging results. In subjects with endometriosis, the compound was specifically retained in the lesions and remained visible after plasma clearance, compared to subjects without endometriosis, where there was no detectable amount of the compound after removal from the bloodstream (Figures 1 and 6). In summary, these data support the imaging capability of labeled compound 491 for visualizing endometriotic lesions, extending beyond its presence in plasma.

[0329] [Table 6]

[0330] Example 11: Evaluation of pharmacokinetics, therapeutic activity, and target binding of diagnostic compounds The inventors tested several compounds in a library that specifically bind to β-catenin and investigated their relationship to therapeutic dynamics and pharmacokinetics in plasma and lesions. These experiments aimed to investigate whether the compounds needed to possess high therapeutic activity to exhibit diagnostic efficacy.

[0331] Reporter assays and target binding were performed according to the protocol in Table 4. Protein binding was measured in five human plasma samples (final 10 μM) and incubated at 37°C for 4 hours using a Rapid Equilibrium Dialysis instrument (Thermo Scientific®). Samples were prepared for LC-MS / MS analysis from both sides of the dialysis machine, with propranolol hydrochloride used as a positive control. Protein binding percentage was calculated as follows: Protein binding (%) = (area p - area b / area p) × 100. Plasma stability was analyzed by pre-incubating human plasma (990 μL) in a water bath at 37°C and 75 rpm for 5 minutes. 10 μL of the compound was added. 50 μL aliquots were collected in 1.5 mL tubes containing 150 μL of warfarin (200 ng / mL) at 0, 0.5, 2, 3.5, 4, 4.5, and 5 minutes, with duplicate collections. The tubes were incubated in a water bath at 37°C and 75 rpm for a maximum of 120 minutes. The samples were then centrifuged at 17000 × g for 10 minutes at 4°C. 100 μL of the supernatant was transferred to a 96-well plate containing 100 μL of water, vortexed, and then spun down at 3750 rpm for 10 minutes. Pharmacokinetic analysis was performed by administering 1 mg / kg of the compound to CD1 mice, with blood samples collected at the following time points: before administration and at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration. After centrifugation, the collected plasma was analyzed by LC-MS / MS, and the peptide t1 / 2 and concentration (ng / ml) were determined based on a standard curve. The pharmacodynamic effects of each peptide were analyzed by administering low doses of 0.325 mg / kg or 1 mg / kg / day via IP over a 3-week period. The results are shown in Table 8.

[0332] A key characteristic was the peptide's binding affinity to its target. Importantly, however, all compounds exhibiting some degree of plasma stability were specifically retained within disease lesions for longer periods than they were in the bloodstream, allowing for imaging of the disease after plasma clearance. Compounds with longer half-lives showed low to undetectable plasma levels within 24 hours, but were significantly retained within lesions, indicating their potential as effective diagnostic compounds. Plasma degradation was variable and inversely proportional to protein binding, which, while not theoretically constrained, suggested that intracellular protein or target binding protected the compounds from degradation. While not theoretically constrained, once a compound entered a cell, it could also be protected from plasma proteases by either intracellular localization or binding to an intracellular target, suggesting that target binding likely contributes to reduced degradation in lesions. Regardless of the strength of therapeutic efficacy, retention within lesions exceeding plasma levels supports their potential as diagnostic candidates. While not bound by theory, rapid clearance from the bloodstream may contribute to reduced therapeutic efficacy, but it still enables effective diagnostic performance. Therefore, compounds with and without strong therapeutic efficacy remain strong diagnostic candidates due to their clearance from the bloodstream and specific uptake by diseased cells.

[0333] Example 12: Conjugation of diagnostic peptides to iron-based moieties Peptides were conjugated onto functionalized iron oxide nanoparticles to enable MRI detection. In this example, iron oxide nanoparticles smaller than 100 nm were used because these small particles offer a good chance of targeting cells and fall below the 100 nm range where immune system recognition is less likely. Furthermore, since abnormal cells are surrounded by vascular endothelial cells with enhanced permeability through potentially leaky junctions, an enhanced permeability effect (EPR) is observed with smaller nanoparticles.

[0334] The examples presented here describe peptide conjugation using a method involving either functionalized iron oxide nanoparticles or [iron]DOTA, enabling visualization by MRI imaging. For the iron nanoparticles in Figure 24A, the functionalized iron oxide nanoparticles and conjugation kit were procured from Ocean Nanotech (San Diego, CA). A Malvern Zetasizer (Malvern Panalytical, Worcestershire, UK) was used to confirm the hydrodynamic size, PDI, and zeta potential. To enable the conjugation of peptides containing cysteine ​​at the N-terminus with amine-functionalized iron oxide nanoparticles, the nanoparticles were reacted with Sulfo-SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl) to promote the formation of a stable amide bond. Free thiol groups present on the peptide were reacted with the intermediate functionalized iron oxide nanoparticles to produce a peptide-iron oxide conjugate. See Figure 24B. To enable peptide conjugation via carboxyl-functionalized iron oxide nanoparticles, the carboxyl groups on the nanoparticles are activated using carbodiimide chemistry, including 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. To conjugate the peptide to [iron]DOTA, the DOTA moiety is first conjugated to the peptide via solid-phase peptide synthesis (SPPS) targeting either the N-terminus, C-terminus, or a specific side chain, ensuring the functionality and stability of the final complex. Next, iron ions are introduced into the DOTA-peptide conjugate to promote efficient chelation of iron within the DOTA cage. The resulting iron-conjugated peptide is purified using high-performance liquid chromatography (HPLC) to remove all unreacted components and by-products, followed by complete characterization of the complex. Example 13: Further embodiment of imaging. To enable the imaging disclosed herein, fluorophores were synthesized on peptides (Table 7).

[0335] [Table 7]

[0336] [Table 8-1]

[0337] [Table 8-2]

[0338] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains. Publications cited herein and materials from which they are cited are incorporated specifically by reference.

[0339] Those skilled in the art will be able to recognize or confirm, by mere routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

Claims

1. An imaging agent comprising a peptide or peptide mimetic conjugated to an imaging component by a linker, (a1) The peptide or peptide mimetic is of formula I: R−X １ −X ２ −X ３ −X ４ −X ５ −X ６ −X ７ −X ８ −X ９ −X １０ −X １１ −X １２ −X １３ −X １４ −X １５ −X １６ (Formula I) The formula includes an amino acid sequence, in which, R is NH ２ acetylated, stearic acid, palmitic acid, myristic acid, lauric acid, C １ -C ８ Hydrocarbons, C １ -C ８ It is a fatty acid, or null, X １ These are M, G, β-alanine, norleucine, norvaline, or null. X ２ is W, N-methyl W, R, Y, F, citrulline, or K. X ３ These are P, W, N-methyl-W, N-ethyl-W, N-methyl A, N-ethyl A, L, Pip, Aib, Y, or F. X ４ is E, Q, N, or D, X ５ is S, α-methyl S, K, D, Orn, T, or E, X ６ is I, Chg, H, or L, X ７ is L or I, X ８ is D, N, E, or Q, X ９ is D, E, K, Q, or Orn, X １０ is H or methyl-H, X １１ is V, α-methyl V, Chg, L, I, or norvaline. X １２ is Q, Aib, S, R, or N, X １３ These are R, K, citrulline, Orn, D, or E. X １４ These are V, I, L, or norvaline. X １５ is W, Y, or F, X １６ is R, G, or null, or (a2) The peptide or peptide mimetic is of formula II: X １ -X ２ -X ３ -X ４ -X ５ -X ６ -X ７ -X ８ -X ９ -X １０ -X １１ -X １２ -X １３ -X １４ -X １５ -X １６ (Formula II) The formula includes an amino acid sequence, in which, X １ is M or null, X ２ is S, I, G, T, A, L, or null. X ３ is R, K, or null, X ４ These are positively charged amino acids, citrulline, Orn, D, E, 8-aminooctanoic acid, or aminocarboxylic acids having 4 to 12 carbon atoms. X ５ M, norleucine, Orn, D, E, K, H, R, K, 8-aminooctanoic acid, an aminocarboxylic acid having 4 to 12 carbon atoms, or null. X ６ is W, Y, F, or N-methyl A, X ７ is F, I, L, Chg, Cha, or Tie, X ８ is L, I, or A, X ９ is L, I, or A, 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, X １３ is G, GABA, or null. X １４ is V, I, L, norvaline, or null. X １５ is W, Y, F, or null, X １６ is R, G, or null, (b) an imaging agent in which the linker is null or the linker conjugates with the imaging component and the peptide or peptide mimetic.

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

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

4. The imaging agent according to claim 2, wherein the imaging component 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 aforementioned imaging component is １８ F, ６４ Cd, ６８ Ga, ９９ｍ The imaging agent according to claim 2, comprising at least one radionuclide selected from the group consisting of Tc.

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

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

9. The linker is an amino acid, -NH(CH ２ ), ｎ C(O)O-, -(OCH ２ CH ２ ), ｍ -, -O-, -NH-, C １ -C ６ alkylene, C １ -C ６ haloalkylene, C １ -C ６ alkoxy, C １ -C ６ heteroalkylene, C ２ -C ６ alkenylene, C ２ -C ６ alkynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C ３ -C １０ cycloalkylene, 3- to 10-membered heterocycloalkylene, C[[ID=ip=39]] ６ -C １０ aryl, or 5- to 10-membered heteroarylene, or one or more combinations thereof, wherein each C １ -C ６ alkylene, C １ -C ６ haloalkylene, C １ -C ６ alkoxy, C １ -C ６ heteroalkylene, C ２ -C ６ alkenylene, C ２ -C ６ alkynylene, alkylamino, alkylthio, alkylcarbonyl, alkoxycarbonyl, alkylsulfonyl, C ３ -C １０ cycloalkylene, 3- to 10-membered heterocycloalkylene, C ６ -C １０ aryl, and 5- to 10-membered heteroarylene are optionally independently substituted with 1, 2, or 3 R ９ . n is an independent integer between 1 and 10. m is an independent integer between 1 and 10. R ９ These are independently halogen, C １ -C ４ Alkyl, C ２ -C ４ Alkenil, C ２ -C ４ Alkinyl, C １ -C ４ Haloalkyl, C １ -C ４ Alkoxy, C １ -C ４ Haloalkoxy, C １ -C ４ Heteroalkyl, -C(O)H, -C(O)OH, -CN, C ３ -C １０ Cycloalkyl, 3-10 member heterocycloalkyl, C ６ -C １０ Aryl, 5-10 member heteroaryl, -C(O)(C １ -C ４ Alkyl), -C(O)O(C １ -C ４ Alkyl), -C(O)NH ２ , -C(O)NH(C １ -C ４ Alkyl), -C(O)N(C １ -C ４ Alkyl) ２ , -NH ２ ,-NH(C １ -C ４ Alkyl), -N(C １ -C ４ Alkyl) ２ ,-NH(C ２ -C ４ Alkylene)-OH,-NH(C) ２ -C ４ Alkylene)-O-(C) １ -C ４ Alkyl), -OH, -O (C １ -C ４ Alkyl), -O (C １ -C ４ Haloalkyl), -O(C) ２ -C ４ Alkilen)-NH ２ , -O(C ２ -C ４ Alkilen)-NH-(C) １ -C ４ Alkyl), -O (C ２ -C ４ Alkylene)-N-(C) １ -C ４ Alkyl) ２ , -O(C １ -C ４ Alkylene)-C(O)OH,-O(C １ -C ４ Alkylene)-C(O)O-(C １ -C ４ Alkyl), -O (C ２ -C ４ Alkenyl), -O(C １ -C ４ Alkylene) - (C ６ -C １０ Aryl), -O(C １ -C ４ Alkylene)-(5-10 member heteroaryl), － O(C) ６ -C １０ Ariel), － SH, S(O) ２ OH, -S(O) ２ (C １ -C ４ Alkyl), -S(O) ２ NH ２ , -S(O) ２ NH(C) １ -C ４ Alkyl), or -S(O) ２ N(C) １ -C ４ Alkyl) ２ Either or two R ９ C ３ -C １０ The imaging agent according to claim 1, which forms a cycloalkyl or 3- to 10-membered heterocycloalkyl ring.

10. The linker is cysteine, -NH(CH ２ ) ｎ C(O)-, -C(O)(CH ２ ) ｎ NH-,-(OCH ２ CH ２ ) ｍ -, -O-, -NH-, C １ -C ６ Alkylene, C １ -C ６ Alkoxy, C １ -C ６ Heteroalkylene, C ３ -C １０ This includes cycloalkylenes, or 3- to 10-membered heterocycloalkylenes, or combinations thereof, where each C １ -C ６ Alkylene, C １ -C ６ Alkoxy, C １ -C ６ Heteroalkylene, C ３ -C １０ Cycloalkylenes and 3- to 10-membered heterocycloalkylenes may optionally have 1, 2, or 3 R groups. ９ They are independently substituted by n, m, and R ９ The imaging agent according to claim 9, as defined in claim 36.

11. The aforementioned linker is X １ ~X １６ The imaging agent according to claim 1, wherein at least one of the amino acids is conjugated with the peptide or peptide mimetic.

12. The imaging agent according to claim 1, wherein the linker is conjugated with the N-terminus of the peptide or peptide mimetic.

13. The peptide or peptide mimetic has -NH at its C-terminus. ２ The imaging agent according to claim 12, further comprising:

14. The imaging agent according to claim 1, wherein the linker is conjugated with the C-terminus of the peptide or peptide mimetic.

15. The peptide or peptide mimetic has a C at its N-terminus. １ -C ８ The imaging agent according to claim 14, further comprising alkyl-C(O)-.

16. The peptide or peptide mimetic has CH at its N-terminus. ３ (CH ２ ) ｐ The imaging agent according to claim 14, further comprising C(O)-, wherein p is an integer from 0 to 7.

17. The peptide or peptide mimetic has CH at its N-terminus. ３ (CH ２ ) ６ The imaging agent according to claim 14, further comprising C(O)-.

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

497.

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

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

21. The imaging agent according to claim 1, wherein the peptide or peptide mimetic is cyclic or bicyclic.

22. The imaging agent according to claim 21, wherein the peptide or peptide mimetic is cyclized by a Cys-Cys disulfide bond, an amide bond, using hydrocarbon stapling, or click chemistry.

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

24. A method for detecting endometriotic lesions in a subject, (a) Administering a formulation containing the imaging agent described in any one of claims 1 to 23 to the subject, (b) Imaging the subject to determine the presence of the imaging agent Methods that include...

25. (c) Administering a second β-catenin-binding peptide or peptide mimetic to the subject, (d) Wait for at least one week, then repeat steps (a) and (b) The method according to claim 24, further comprising:

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

27. The method according to claim 26, wherein the image is a video, a still image, a real-time video, or any combination thereof.

28. The method according to claim 27, wherein the subject or the image is visualized or displayed by the imaging agent during a surgical procedure performed on the subject.

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

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

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

33. The method according to claim 24, wherein imaging includes imaging the entire body.

34. The second β-catenin-binding peptide or peptide mimetic is formula I: R−X １ −X ２ −X ３ −X ４ −X ５ −X ６ −X ７ −X ８ −X ９ −X １０ −X １１ −X １２ −X １３ −X １４ −X １５ −X １６ (Formula I) The formula includes an amino acid sequence, in which, R is NH ２ acetylated, stearic acid, palmitic acid, myristic acid, lauric acid, C １ -C ８ Hydrocarbons, C １ -C ８ It is a fatty acid, or null, X １ These are M, G, β-alanine, norleucine, norvaline, or null. X ２ is W, N-methyl W, R, Y, F, citrulline, or K. X ３ These are P, W, N-methyl-W, N-ethyl-W, N-methyl A, N-ethyl A, L, Pip, Aib, Y, or F. X ４ is E, Q, N, or D, X ５ is S, α-methyl S, K, D, Orn, T, or E, X ６ is I, Chg, H, or L, X ７ is L or I, X ８ is D, N, E, or Q, X ９ is D, E, K, Q, or Orn, X １０ is H or methyl-H, X １１ is V, α-methyl V, Chg, L, I, or norvaline. X １２ is Q, Aib, S, R, or N, X １３ These are R, K, citrulline, Orn, D, or E. X １４ These are V, I, L, or norvaline. X １５ is W, Y, or F, X １６ is R, G, or null. The method according to claim 24.

35. The second β-catenin-binding peptide or peptide mimetic has the amino acid sequence Oct-W-NMeA-Q-Orn ＊ - ILDE ＊ H-Chg-QRVWG-NH ２ The method according to claim 34, comprising (Sequence ID 491).

36. The second β-catenin-binding peptide or peptide mimetic is given by formula II: X １ -X ２ -X ３ -X ４ -X ５ -X ６ -X ７ -X ８ -X ９ -X １０ -X １１ -X １２ -X １３ -X １４ -X １５ -X １６ (Formula II) The formula includes an amino acid sequence, in which, X １ is M or null, X ２ is S, I, G, T, A, L, or null. X ３ is R, K, or null, X ４ These are positively charged amino acids, citrulline, Orn, D, E, 8-aminooctanoic acid, or aminocarboxylic acids having 4 to 12 carbon atoms. X ５ M, norleucine, Orn, D, E, K, H, R, K, 8-aminooctanoic acid, an aminocarboxylic acid having 4 to 12 carbon atoms, or null. X ６ is W, Y, F, or N-methyl A, X ７ is F, I, L, Chg, Cha, or Tie, X ８ is L, I, or A, X ９ is L, I, or A, 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, X １３ is G, GABA, or null. X １４ is V, I, L, norvaline, or null. X １５ is W, Y, F, or null, X １６ is R, G, or null. The method according to claim 24.

Images