Peptide-linked drug delivery systems

Abstract

The present disclosure relates to a systemically administered peptide delivery platform that biodistributes to the kidney or urinary tract. The present disclosure further relates to a method of treating a kidney or urinary tract disorder in a subject in need thereof.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 128,509, filed December 21, 2020, and U.S. Provisional Patent Application No. 63 / 254,754, filed October 12, 2021, the contents of each of which are incorporated by reference in their entirety into this specification.

[0002] Statement regarding federally funded research This invention was made with Government support under Grant CA222802 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Kidney and urinary tract diseases result in significant morbidity and mortality. For example, urothelial carcinoma, the most common type of bladder cancer (BC), is one of the leading cancers in the United States. Most BC is non-muscle invasive and superficial in nature. Standard treatment involves instilling chemotherapy / immunotherapeutic agents into the bladder after initial surgical resection of the tumor. These drugs are often administered via catheterization of the urethra into the bladder (also known as intravesical therapy (ITT)). Despite evidence of clinical efficacy of ITT, disease recurrence rates remain high, up to 50%. This may be caused by incomplete drug delivery, as the administered drug can only be retained in the bladder for a limited time. Furthermore, urothelial cancer can recur throughout the urothelium (renal pelvis, ureter, and bladder). ITT delivers drugs only to the bladder, and therefore cannot reach tumors in either the ureter or renal pelvis. ITT also requires invasive catheterization, which can cause pain, infection, urinary symptoms, and reduced patient compliance, ultimately leading to treatment discontinuation. There remains a need to identify systems that overcome the drug delivery barriers of current ITT to treat kidney and urinary tract diseases without the need for invasive procedures or surgery. Such systems should provide systemically administered therapy to the urinary system without systemic toxicity and provide extended contact time of the drug with the urinary system, providing more effective treatment for diseases such as urothelial cancer. Summary of the Invention

[0004] In certain embodiments, the present disclosure provides a compound represented by the following formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein the peptide is a peptide that targets renal clearance.

[0005] In certain aspects, the disclosure provides a method of treating cancer comprising administering to a patient in need thereof a compound or composition of the disclosure. [Brief description of the drawings]

[0006] [Figure 1A] Bdd exhibits remarkable urinary disposal properties. (Figure 1A) Traditional intravesical chemotherapy (ITC) is invasive and drug delivery is limited to the bladder. Bdd is engineered to carry multiple negative charges that minimize off-target delivery and facilitate renal excretion after intravenous (iv) administration. When used as a drug carrier, Bdd promotes drug deposition throughout the urothelial system and provides continuous drug excretion for more comprehensive treatment. [Figure 1B] (Figure 1B) A table showing the components and net charges of BDD and its D-configured (Bdd), neutrally charged (BKD), positively charged (BKK), and pegylated (PEG3DD) counterparts. [Figure 1C] (Figure 1C) Synthesis scheme showing radiolabeling of peptides with 89Zr for comparative PK and biodistribution studies. [Figure 1D] (FIG. 1D) Representative μPET / CT images of BALB / c mice acquired 1, 4, and 24 hours after administration of different 89Zr-radiolabelled peptides (20 μCi, 20 μg in 100 μL PBS) via tail vein injection (n=4 / group). Whole-body images were acquired with or without emptying the animals' bladders. [Figure 1E] (Figure 1E) Bar graph showing the amount of radioactivity in urine samples (20 μL) collected from animals (n = 5) at different time intervals after administration of 89Zr-Bdd. [Figure 1F] (FIG. 1F) Comparison of renal clearance of radiolabeled peptides. Plot showing percentage of injected dose (ID%) (per cc) in the kidneys of animals over time. Percentage of ID / cc was calculated according to the radioactivity measured in the region of interest (ROI) of the acquired PET images. [Figure 1G](FIG. 1G) Comparison of pharmacokinetic (PK) profiles of radiolabeled peptides (n=4 / analog). Blood samples (20 μL) were collected at various time intervals after injection of peptide or free 89Zr into animals. The results (radioactivity measured in blood samples) were fitted to a two-compartment model to determine t1 / 2α (half-life of distribution phase) and t1 / 2β (half-life of elimination phase). Other PK parameters are available in FIG. 7A. [Figure 1H] (FIG. 1H) End point biodistribution study of 89Zr-Bdd. After administration of peptide (20 μCi, 20 μg in 100 μL PBS), animals were euthanized at different time intervals (n=4 / time point). The amount of peptide (radioactivity) in harvested organs was determined. Results (corrected for decay) were expressed as ID%. (Student's t test, **p<0.01, and ***p<0.001). [Figure 2A] Bdd efficiently distributes conjugated cyanine 5.5 fluorophores to the urinary system. (Figure 2A) Synthesis scheme of cyanine 5.5 labeled peptide (Cy-peptide) analogs. [Figure 2B] (Figure 2B) Representative merged fluorescence / white light images of SCID mice acquired 1 and 4 h after tail vein injection of different Cy-peptide analogs (0.5 nmol, 150 μL) or free Cyanine 5.5 (n=4 / group). [Figure 2C] (FIG. 2C) Plot comparing the amount of fluorophore in urine (% of injected dose) based on measured fluorescence. (Lower panel) Fluorescence images of urine samples (20 μL) collected from animals 1 h after peptide or fluorophore administration. [Figure 2D] (Figure 2D) Representative ex vivo merged fluorescent / bright light images of organs harvested from animals 4 h after tail vein injection of different Cy-peptides (n=4 / group). [Figure 2E] (FIG. 2E) Bar graph comparing peptide distribution in harvested organs (n=4 / group) based on total fluorescence intensity (Student's t test, *p<0.05, **p<0.01, and ***p<0.001). [Figure 3A]Bdd as a carrier of chemotherapeutic drugs. (Figure 3A) Bdd does not induce any innate immune response. 24 hours after intravenous administration of Bdd peptide (5 mg / kg), no increase in inflammatory cytokine concentrations was detected in the plasma of female BALB / c mice (n=3 / group). LPS was used as a positive control, and the concentrations of each cytokine were measured by ELISA kits. [Figure 3B] (Figure 3B) Cellular uptake of cyanine 5.5-labeled Bdd (Cy-Bdd). Representative fluorescence microscopy images of human UMUC-3 BC cells and mouse Renca renal adenocarcinoma cells incubated with Cy-Bdd (0.5 nmol) for 6 and 24 h. Dapi (9 μM) and LysoTracker-GFP (1 μM) were used for nuclear (blue) and organelle (green) staining, respectively, and were added to the cells 30 min prior to imaging. Scale bar is 25 μm. [Figure 3C] (FIG. 3C) Comparison of the potency of different chemotherapeutic agents (DM1, GEM, MIT, CIS, and DOX). UMUC-3 and Renca cells were incubated with various concentrations of drugs for 72 hours before measuring cell viability. Dose-response curves were plotted and the half-maximal inhibitory concentration (IC50 value) of each drug was calculated using Graph Pad Prism 6.0 software. [Figure 3D] (FIG. 3D) Conjugation of DM1 to Bdd. The cleavable linker SPDP was first conjugated to the peptide N-terminus on the solid phase. DM1 was then added to the cleaved peptide in a solution mixture of PBS and NMP. [Figure 3E] (FIG. 3E) Plot showing the percentage of cumulative DM1 released from DM1-Bdd over time in PBS in the absence and presence of GSH (1 mM). The amount of drug released was quantified using HPLC analysis (absorbance detected at 254 nm). [Figure 3F] (FIG. 3F) Conjugation of aldox to Bdd. Peptides supplemented with N-terminal cysteine ​​were incubated with aldox in PBS (pH=7.4) for 30 min and then purified by HPLC under neutral conditions. [Figure 3G](FIG. 3G) Plot showing the percentage of cumulative DOX active metabolite released from aldox-Bdd (100 μM) over time in PBS buffer at different pH values. The amount of drug released was quantified using HPLC analysis (absorbance detected at 480 nm). [Figure 3H] (FIG. 3H) DM1-Bdd shows similar cytotoxicity compared to free drug against mouse bladder (MB49), human bladder (UMUC-3 and T24), and mouse kidney (Renca) cancer cell lines. Plot of relative cell viability versus drug concentration. [Figure 3I] (FIG. 3I) Aldox-Bdd is more potent than free aldox. Plot of relative cell viability against drug concentration. [Figure 4A] Therapeutic efficacy of DM1-Bdd in treating bladder cancer. (FIG. 4A) Bar graph comparing the time BALB / c mice (n=20 / group) had to void spontaneously after intravenous or intravesical administration of PBS (80 μL). Bladders were emptied before the start of the experiment. Each animal was isolated to monitor urination patterns. [Figure 4B] (FIG. 4B) Comparing the nephrotoxicity of DM1-Bdd with other chemotherapeutic agents. Bar graphs showing the concentrations of NGAL and KIM-1, renal injury biomarkers, in urine collected from animals 1, 3, and 7 days after treatment with PBS, DM1 (0.75 mg / kg), DM1-Bdd (drug content 0.75 mg / kg), MIT (0.75 mg / kg), CIS (0.75 and 10 mg / kg), or GEM (0.75 mg / kg) via tail vein injection. (Student's t test, *p<0.05, **p<0.01, and ***p<0.001). [Figure 4C](FIG. 4C) Immunochemical staining for NGAL and KIM-1 was also performed. Representative microscopic images of kidney sections from BALB / c mice intravenously administered PBS, DM1 (0.75 mg / kg), DM1-Bdd (drug content 0.75 mg / kg), or CIS (10 mg / kg) as a positive control. Organs were harvested 3 days after drug treatment and stained with H&E. Black arrows indicate multifocal degeneration of the renal tubular epithelium after treatment with CIS. Red and green arrows indicate renal tubular epithelial cells immunoreactive for NGAL and KIM-1, respectively, after CIS treatment. Scale bar is 50 μm. [Figure 4D] (FIG. 4D) Brightfield and fluorescent images of UMUC-3 / GFP-Luc cells stably transduced with lentivirus carrying both GFP and firefly luciferase genes. Scale bar, 80 μm. [Figure 4E] (FIG. 4E) Orthotopic xenograft model. Representative images of bladders collected from female NSG mice (n=3) 1 week after implantation of UMUC-3 / GFP-Luc cells (4×104 cells / animal). Black arrows indicate tumors growing in the lamina propia. Scale bar, 500 μm. [Figure 4F] (FIG. 4F) Representative merged bioluminescence / brightfield images of tumor-bearing animals after weekly treatment for 3 weeks (n=10 / group) with intravenous PBS (150 μL), intravenous DM1 (0.75 mg / kg, 150 μL), intravenous DM1-Bdd (drug content 0.75 mg / kg, 150 μL), intravesical MIT (1 mg / mL, 50 μL), intravesical DM1 (0.75 mg / kg, 50 μL), or intravesical DM1-Bdd (0.75 mg / kg, 50 μL). Images were acquired weekly to monitor and compare tumor growth in each treatment group. [Figure 4G] (Figure 4G) Representative photographs of bladders excised from each animal group (n=3 additional recruitments / treatment group) 1 week after completion of the treatment cycle. [Figure 4H](FIG. 4H) Kaplan-Meier cumulative survival plots of animals receiving different drugs (n=14 / group). Significant differences in survival between animals treated with intravenous DM1-Bdd and other groups were assessed using the Mantel-Cox log-rank test and Benjamini-Hochberg adjusted p-values. [Figure 4I] (FIG. 4I-J) Representative images of bladder sections from animals in each treatment group (n=3 / group). At the end of 3 weeks of treatment, organs were harvested, then paraffin-embedded, sectioned, and stained with H&E (FIG. 4I) and Ki67 (proliferation marker) (FIG. 4J). [Figure 4J] (FIG. 4I-J) Representative images of bladder sections from animals in each treatment group (n=3 / group). At the end of 3 weeks of treatment, organs were harvested, then paraffin-embedded, sectioned, and stained with H&E (FIG. 4I) and Ki67 (proliferation marker) (FIG. 4J). [Figure 5A] Therapeutic efficacy of DM1-Bdd in treating renal cancer. (FIG. 5A) Bright field and fluorescent images of Renca cells stably transduced with lentivirus carrying both GFP and firefly luciferase genes. Scale bar, 80 μm. [Figure 5B] (FIG. 5B) Syngeneic xenograft model. Representative images of histological analysis of kidneys harvested from female BALB / c mice (n=3) 1 week after implantation of mouse Renca cells (4×103 cells / animal) (black arrow) in the renal capsule. Scale bar is 1 mm. [Figure 5C] (Figure 5C) Representative merged bioluminescence / brightfield images of animals bearing Renca / GFP-Luc tumors after weekly treatment for 3 weeks with intravenous PBS (150 μL), intravenous DM1 (0.75 mg / kg, 150 μL), intravenous DM1-Bdd (drug content 0.75 mg / kg, 150 μL), or intravesical DM1 (0.75 mg / kg, 50 μL) (n=10 / group). [Figure 5D] (Figure 5D) Representative photographs of kidneys excised from animals (an additional n=4 / group) after completion of the treatment cycle. [Figure 5E]Longitudinal comparison of bioluminescence signal in the region of interest (ROI = kidney) (Figure 5E), body weight (Figure 5F), and survival time (Figure 5G) between animals receiving different treatments (n = 14 / group). Significant differences in survival time between animals treated with intravenous DM1-Bdd and drugs were assessed using the Mantel-Cox log-rank test and Benjamini-Hochberg adjusted p-values. [Figure 5F] Longitudinal comparison of bioluminescence signal in the region of interest (ROI = kidney) (Figure 5E), body weight (Figure 5F), and survival time (Figure 5G) between animals receiving different treatments (n = 14 / group). Significant differences in survival time between animals treated with intravenous DM1-Bdd and drugs were assessed using the Mantel-Cox log-rank test and Benjamini-Hochberg adjusted p-values. [Figure 5G] Longitudinal comparison of bioluminescence signal in the region of interest (ROI = kidney) (Figure 5E), body weight (Figure 5F), and survival time (Figure 5G) between animals receiving different treatments (n = 14 / group). Significant differences in survival time between animals treated with intravenous DM1-Bdd and drugs were assessed using the Mantel-Cox log-rank test and Benjamini-Hochberg adjusted p-values. [Figure 5H] (FIG. 5H) Representative kidney sections from animals in each treatment group (an additional n=4 / group). Sections were stained with H&E. Green, yellow, black, and blue arrows indicate the presence of pigmented macrophages, focal calcification, interstitial fibrosis, and mononuclear cell infiltration, respectively. Scale bars are 2 mm and 50 μm. [Figure 6A] DM1-Bdd exhibits a safe toxicity profile. (FIG. 6A) Representative microscopic images of blood smears collected from female BALB / c mice after intravenous administration of PBS, DM1 (0.75 mg / kg), DM1-Bdd (drug content 0.75 mg / kg), or CIS (10 mg / kg) as a positive control once a week for 3 weeks. Black arrows indicate polychromatic macrocytes. Scale bar is 10 μm. [Figure 6B](FIG. 6B) Selected hematological results obtained one week after completion of the different treatment courses. (RBC=red blood cells, and WBC=white blood cells). (Student's t test, *p<0.05, **p<0.01, and ***p<0.001). [Figure 6C] (FIG. 6C) Comparison of selected serum biochemical analytes, including liver enzyme activity (ALP, ALT, and AST), muscle enzyme activity (AST and CK), and nitrogenous waste clearance (BUN / CREA ratio). (ALP=alkaline phosphatase, ALT=alanine aminotransferase, AST=aspartate aminotransferase, CK=creatine kinase, BUN=blood urea nitrogen, CREA=creatine). [Figure 6D] (FIG. 6D) Histopathological analysis of major organs (liver, spleen, heart, lungs, and kidneys) from animals receiving different drug treatments. Black arrows indicate increased hepatocyte mitotic activity in the liver. Arrows indicate enhanced extramedullary hematopoiesis (EMH) in the liver and spleen. The area between the white arrows indicates depletion of red blood cells and EMH elements in the red pulp of the spleen. Red arrows highlight the presence of large foamy macrophages in the alveoli. Yellow and green arrows indicate necrotic sloughed debris of flattened renal tubular cells and dying cells contained within the lumen, respectively. Scale bar, 30 μm. [Figure 7A] (FIG. 7A) Table comparing pharmacokinetic parameters of radiolabeled peptides (n=4 / analog). After tail vein injection of peptide or free 89Zr (20 μCi, 20 μg in 100 μL PBS) into animals, blood samples (20 μL) were collected at various time intervals. The results (radioactivity measured in blood samples) were fitted to a two compartment model to determine PK parameters. (k10=elimination rate constant, k12 and k21=transport rate constants, t1 / 2α=half-life of distribution phase, t1 / 2β=half-life of elimination phase, C0=threshold drug concentration for elimination, Vd=apparent volume of distribution, CL=total clearance rate, AUC=area under the curve, MRT=mean residence time, and Vss=steady-state volume of distribution). [Figure 7B](FIG. 7B) End point biodistribution study of 89Zr-Bdd. After administration of the peptide, animals were euthanized at different time intervals (n=4 / time point). The amount of peptide (radioactivity) in harvested organs was determined. Results (corrected for decay) were expressed as % of injected dose per gram of tissue (ID% / g). (Student's t test, *p<0.05, and ***p<0.001). [Figure 8A] (Figure 8A) Body weight changes in NSG mice bearing bladder tumors and treated weekly for 3 weeks with intravenous PBS (150 μL), intravenous DM1 (0.75 mg / kg, 150 μL), intravenous DM1-Bdd (drug content 0.75 mg / kg, 150 μL), intravesical MIT (1 mg / mL, 50 μL), intravesical DM1 (0.75 mg / kg, 50 μL), or intravesical DM1-Bdd (0.75 mg / kg, 50 μL). [Figure 8B] (Figure 8B) Comparison of bioluminescence signals in the region of interest (ROI = bladder) between different drugs. [Figure 8C] (Figure 8C) Representative images of excised bladders from three animals receiving different drug treatments. [Figure 8D] (FIGS. 8D-E) Plots comparing bladder volume (FIG. 8D) and weight (FIG. 8E). (Student's t test, *p<0.05, **p<0.01, and ***p<0.001). [Figure 8E] (FIGS. 8D-E) Plots comparing bladder volume (FIG. 8D) and weight (FIG. 8E). (Student's t test, *p<0.05, **p<0.01, and ***p<0.001). [Figure 8F] (FIG. 8F) Representative images of bladder sections from animals in each treatment group (n=3 / group). Organs were harvested at the end of 3 weeks of treatment, then paraffin-embedded, sectioned, and stained with anti-GFP antibody to identify GFP-expressing tumor cells. [Figure 8G] (FIG. 8G) Representative images of bladder sections from three animals that lacked gross and histological evidence of tumors following intravenous DM1-Bdd treatment. Animals were sacrificed 210 days after tumor implantation, and organs were harvested, paraffin embedded, sectioned, and stained with H&E, Ki67 (proliferation marker), and anti-GFP antibodies. [Figure 9A] (FIG. 9A) Representative merged bioluminescence / brightfield images of NSG mice bearing orthotopically implanted UMUC-3 / GFP-Luc bladder tumors after weekly treatment for 3 weeks (n=10 / group) with intravenous PBS (150 μL), intravenous DOX (5 mg / kg, 150 μL), intravenous aldox-Bdd (drug content 5 mg / kg, 150 μL), or intravesical DOX (5 mg / kg, 50 μL). Images were acquired weekly to monitor and compare tumor growth in each treatment group. [Figure 9B] (FIG. 9B) Comparison of bioluminescence signals in the region of interest (ROI=bladder) between different treatment groups. [Figure 9C] (FIG. 9C) Kaplan-Meier cumulative survival plots of animals administered different drugs (n=10 / group). Significant differences in survival between animals treated with intravenous aldox-Bdd and other groups were assessed using the Mantel-Cox log-rank test and Benjamini-Hochberg adjusted p-values. [Figure 9D] (FIG. 9D) Body weight change in NSG mice bearing bladder tumors and treated with PBS, intravenous DOX, intravenous aldox-Bdd, or intravesical DOX once a week for 3 weeks. [Figure 9E] (FIG. 9E) Representative photograph of a mouse treated with intravenous DOX (5 mg / kg) after administration of only two doses of the drug. [Figure 10] Extended results of complete blood count analysis. Table showing hematological results after weekly intravenous administration of PBS used as a positive control, DM1 (0.75 mg / kg), DM1-Bdd (drug content 0.75 mg / kg), or CIS (10 mg / kg) in BALB / c mice for 3 weeks. (RBC = red blood cells, HGB = hemoglobin, HCT = hematocrit, MCV = mean corpuscular volume, MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular hemoglobin concentration, RDW = red blood cell distribution width, RET = reticulocytes, PLT = platelets, PDW = platelet distribution width, MPV = mean platelet volume, WBC = white blood cells, NEUT = neutrophils, LYMPH = lymphocytes, MONO = monocytes, EO = eosinophils, and BASO = basophils). [Figure 11] Extended Results of Serum Biochemical Analysis. Table showing the results of all biochemical analytes measured after weekly intravenous administration of PBS as a positive control, DM1 (0.75 mg / kg), DM1-Bdd (drug content 0.75 mg / kg), or CIS (10 mg / kg) in BALB / c mice for 3 weeks. (BUN=Blood urea nitrogen, CREA=Creatinine, ALP=Alkaline phosphatase, ALT=Alanine aminotransferase, AST=Aspartate aminotransferase, GGT=Gamma-glutamyltransferase, BIL=Bilirubin, TP=Total protein, ALB=Albumin, GLOB=Globulin, A / G=Albumin / Globulin, P=Phosphate, Ca=Calcium, GLU=Glucose, CHOL=Cholesterol, TRIG=Triglyceride, CK=Creatine kinase, TCO2=Total carbon dioxide, Na=Sodium, K=Potassium, and CL=Chloride). [Figure 12] High magnification images of major organ sections (liver, spleen, lungs, and kidneys) from animals administered intravenous DM1 (0.75 mg / kg) and intravenous CIS (10 mg / kg) weekly for 3 weeks as positive controls. Sections were stained with H&E. Black arrows indicate increased hepatocyte mitotic activity in the liver. Blue arrows indicate enhanced extramedullary hematopoiesis (EMH) in the liver and spleen. The area between the white arrows indicates depletion of red blood cells and EMH elements in the red pulp of the spleen. Red arrows highlight the presence of large foamy macrophages in the alveoli. Yellow and green arrows indicate necrotic sloughed debris of flattened renal tubular cells and dying cells contained within the lumen, respectively. Scale bar, 10 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Different approaches have been offered to improve intravesical chemotherapy (ITC) treatment. To extend the treatment period, UGN-102, a thermosensitive hydrogel, was designed to transform into a semi-solid drug depot inside the bladder and slowly release mitomycin (MIT). GemRis, a semi-permeable silicone tube containing gemcitabine (GEM) that acts as an osmotic pump, was also developed to control the release of GEM. Other treatments are currently being tested in clinical trials for patients who do not respond to BCG. Oportuzumab monatox is an antibody-protein conjugate that targets tumor cells expressing EpCAM. Adstiladrin is a non-replicating adenoviral vector that encodes the human IFNα-2b gene. The resulting IFNα-2b protein, synthesized and expressed in large quantities, showed antitumor activity through inhibition of angiogenesis and induction of apoptosis in human bladder cancer cells. However, all the aforementioned approaches are invasive and require catheterization and / or surgical procedures. Alternatively, renal clearance-capable nanoparticles can be used as drug carriers. However, they are known to be non-specifically trapped by the reticuloendothelial system, leading to high off-target accumulation in the liver.

[0008] Advances in phage display have led to the discovery of many bioactive peptides targeting the urinary system (URS). For example, a galectin-3 targeting peptide, G3-C12, has been used to deliver the angiotensin-converting enzyme inhibitor captopril. Another peptide, (KKEEE)3K, was used to carry ciprofloxacin. These peptides were pharmacologically active. They targeted the kidney primarily via binding to cell surface receptors and prolonged local retention after delivery. They were not applied to BC treatment. Indeed, without chemical modification, peptides are not good drug candidates or carriers. Peptides exhibit unfavorable pharmacokinetics (PK), can be rapidly degraded by protease enzymes, and eliminated by renal filtration.

[0009] The present invention takes advantage of the rapid renal clearance of peptides to dispose of treatment to the URS. In certain embodiments, the present disclosure provides a small (e.g., 12 amino acid), negatively charged peptide (e.g., Bdd) that can bypass the reticuloendothelial system and other organs and is preferentially (e.g., exclusively) excreted in the urine with minimal reabsorption. In certain embodiments, the present disclosure provides an alternative to ITCs to minimize off-target accumulation in other organs, facilitate drug delivery to the URS, and extend bladder retention time to provide a comprehensive and more effective treatment of BC (Figure 1a).

[0010] The present disclosure relates to a systemically administered peptide delivery platform that biodistributes to the kidney or urinary tract. In certain embodiments, an active moiety is linked to the peptide delivery platform. The present disclosure further relates to a method of treating a kidney or urinary tract disease in a subject in need thereof. In some embodiments, the disease is acute kidney disease. In some embodiments, the disease is chronic kidney disease.

[0011] In certain embodiments, the peptide delivery platform comprises a negatively charged peptide that can accumulate temporarily in the kidney without off-target delivery to other organs. The peptide is gradually cleared in the urine, making it useful as a drug delivery platform to provide continuous drug flow for the treatment of kidney or urinary tract diseases such as BC. The peptide delivers systemically administered drugs to the urinary system while prolonging bladder retention time to provide more effective treatment.

[0012] In certain embodiments, the peptides have multiple negative charges that can promote rapid renal clearance. Thus, drug delivery to the bladder via renal clearance is a sequential event. Compared to drug administration via catheterization, the peptides can enhance infiltration of therapy throughout the urinary system with longer residence time in the bladder, resulting in a more effective treatment that is non-invasive. In certain embodiments, the peptides are used as a drug delivery system for BC treatment using mertansine (DM1), a highly cytotoxic microtubule inhibitor. DM1 is too toxic to be used alone, but has been approved as a pharmacophore for antibody-drug conjugates such as T-DM1. In certain embodiments, the peptides are used to deliver DM1 to treat BC as well as other cancers of the urinary tract.

[0013] In certain embodiments, the peptides are used as a drug delivery system for the treatment of urinary tract infections, for example with antibiotics. In certain embodiments, the peptides are used as a drug delivery system for the treatment of kidney stones. In certain embodiments, the peptides are used as a drug delivery system for the treatment of overactive bladder. In certain embodiments, the peptides are used as a drug delivery system for the treatment of urinary incontinence. In certain embodiments, the peptides are used as a drug delivery system for the treatment of interstitial cystitis. In certain embodiments, the peptides are used to deliver imaging agents to the bladder.

[0014] In certain aspects, the peptides are water-soluble, biologically inactive, and non-immunogenic. After intravenous injection, the peptides can be eliminated exclusively via renal clearance, with minimal accumulation in other organs, including the heart, liver, and spleen.

[0015] In certain aspects, the present disclosure provides a compound represented by the following formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein the peptide is a peptide that targets renal clearance.

[0016] In certain embodiments, the peptide that targets renal clearance comprises the sequence: [ka] wherein one of X, Y, and Z is a β-amino acid residue; two of X, Y, and Z are independently α-amino acid residues each having at least one side chain that includes a carboxylic acid group; each α-amino acid residue may independently be of D or L stereochemistry; and m is from 2 to 10.

[0017] In certain embodiments, the peptide has a zeta potential of about -30 mV to about +20 mV at physiological pH. In certain embodiments, the peptide has a zeta potential of about -20 mV to about 0 mV at physiological pH. In certain embodiments, the peptide has a zeta potential of about -5 mV to about 0 mV at physiological pH.

[0018] In certain embodiments, the linker comprises one or more groups selected from amide, imide, thiourea, thioether, disulfide, alkyl, aryl, polyether, hydrazone, ester, carbonate, ketal, and silyl ether. In certain embodiments, the active moiety is a therapeutic agent or an imaging agent.

[0019] In certain embodiments, the peptide that targets renal clearance comprises the sequence: [ka] wherein one of X, Y, and Z is a β amino acid residue; two of X, Y, and Z are independently α amino acid residues each having at least one side chain comprising a carboxylic acid group; each α amino acid residue may independently be of D or L stereochemistry; m is 2 to 10; the linker comprises one or more groups selected from amide, imide, thiourea, thioether, disulfide, alkyl, aryl, polyether, hydrazone, ester, carbonate, ketal, and silyl ether; and the active moiety is a therapeutic or imaging agent.

[0020] In certain embodiments, the compound is represented by the following formula (IA): [ka] or a pharma- ceutically acceptable salt thereof.

[0021] In certain embodiments, the β-amino acid residue does not comprise an ionizable side chain. In certain embodiments, the β-amino acid residue is a β-alanine residue. In certain embodiments, X is a β-alanine residue. In certain embodiments, each α-amino acid residue is independently selected from an aspartic acid residue and a glutamic acid residue. In certain embodiments, at least one α-amino acid residue is a non-natural amino acid residue. In certain embodiments, the non-natural α-amino acid residue has at least two side chain carboxylic acid groups. In certain embodiments, the non-natural α-amino acid residue is selected from a 2-aminoethane-1,1,2-tricarboxylic acid residue and a 2-aminopropane-1,2,3-tricarboxylic acid residue. In certain embodiments, each Y and Z is an aspartic acid residue. In certain embodiments, each Y and Z is a D-aspartic acid residue. In certain embodiments, X, Y, and Z are each independently selected from a β-alanine residue, an aspartic acid residue, and a glutamic acid residue. In certain embodiments, m is 4.

[0022] In certain embodiments, the linker is [ka] The present invention includes a group selected from the group consisting of

[0023] In certain embodiments, the linker comprises a group derived from N-succinimidyl 3-(2-pyridyldithio)propionate, or succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

[0024] In certain embodiments, the active moiety is a therapeutic agent. In certain embodiments, the therapeutic agent is selected from anti-cancer agents, antibiotics, agents for treating overactive bladder, agents for treating urinary incontinence, agents for treating interstitial cystitis, and agents for treating kidney stones. In certain embodiments, the therapeutic agent is 13-cis-retinoic acid, 2-chlorodeoxyadenosine, 5-azacytidine, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, actinomycin-D, adriamycin, aldesleukin, alemtuzumab, alitretinoin, all-trans retinoic acid, alpha interferon, altretamine, amethopterin, amifostine, anagrelide, anastrozole, arabinosylcytosine, arsenic trioxide, amsacrine. , aminocamptothecin, aminoglutethimide, asparaginase, azacitidine, bacillus Calmette-Guerin (BCG), bendamustine, bevacizumab, bexarotene, bicalutamide, bortezomib, bleomycin, busulfan, leucovorin calcium, citrovorum factor, capecitabine, canertinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, cortisone, cyclophosphamide, cytarabine, darbepoetin alfa, dasati Nib, daunomycin, decitabine, denileukin diftitox, dexamethasone, dexasone, dexrazoxane, dactinomycin, daunorubicin, decarbazine, docetaxel, doxorubicin, doxil, aldoxorubicin, doxifluridine, edrecolomab, eniluracil, epirubicin, epoetin alfa, erlotinib, everolimus, exemestane, estramustine, etoposide, filgrastim, fluoxymesterone, fulvestrant, flavopiride , floxuridine, fludarabine, fluorouracil, flutamide, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin, granulocyte-colony stimulating factor, granulocyte-macrophage-colony stimulating factor, hexamethylmelamine, hydrocortisone hydroxyurea, ibritumomab, ibritumomab tiuxetan, interferon alpha, interleukin-2, interleukin-11, isotretinoin, ixabepilone, idarubicin, imatinib mesylate,Ifosfamide, irinotecan, lapatinib, lenalidomide, letrozole, leucovorin, leuprolide, liposomal Ara-C, lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine, mertansine, mesna, methotrexate, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pemetrexed, panitumumab, PEG-interferon, pegaspargase, pegfilgrastim, PEG-L-asparaginase, pentostatin, plicamycin, prednisolone Nisolone, prednisone, procarbazine, raloxifene, rituximab, romiplostim, ralitrexed, sapacitabine, sargramostim, satraplatin, sorafenib, sunitinib, semustine, streptozocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus, temozolamide, teniposide, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, trastuzumab emtansine, tretinoin, trimitrexate, arubicin, vincristine, vinblastine, vindestine, vinorelbine, vorinostat, and zoledronic acid.

[0025] In certain embodiments, the therapeutic agent is an anti-cancer agent, hi certain embodiments, the anti-cancer agent is selected from mertansine, doxorubicin, dasatinib, cisplatin, mitomycin, gemcitabine, and paclitaxel.

[0026] In certain embodiments, X is a β-alanine residue, Y and Z are D-aspartic acid residues. In certain embodiments, X is a β-alanine residue, Y and Z are D-aspartic acid residues, and m is 4. In certain embodiments, X is a β-alanine residue, Y and Z are D-aspartic acid residues, m is 4, the linker comprises a disulfide group, and the active moiety is mertansine.

[0027] In certain embodiments, the compound is [ka] or a salt thereof, wherein B is a β-alanine residue and D is an aspartic acid residue in the D or L configuration.

[0028] In certain embodiments, the compound is [ka] or a salt thereof, wherein B is a β-alanine residue and D is an aspartic acid residue in the D or L configuration.

[0029] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of the present invention. In certain embodiments, the composition is formulated for intravenous administration.

[0030] In certain embodiments, the disclosure provides a method of treating cancer, urinary tract infection, overactive bladder, urinary incontinence, interstitial cystitis, or kidney stones, comprising administering to a patient in need thereof a compound or composition of the present invention. In certain embodiments, the disclosure provides a method of treating cancer, comprising administering to a patient in need thereof a compound or composition of the present invention. In certain embodiments, the cancer is a kidney or urinary tract cancer. In certain embodiments, the cancer is bladder cancer. In certain embodiments, the bladder cancer is non-muscle invasive bladder cancer. In certain embodiments, the bladder cancer is urothelial cancer. In certain embodiments, the compound is administered intravenously.

[0031] Pharmaceutical Compositions The compositions and methods of the present disclosure may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal, such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition, for example, comprising a compound of the present disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions, such as water or physiologically buffered saline, or other solvents or vehicles, such as glycols, glycerol, oils, such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes such as injection or implantation that avoid transport or diffusion through epithelial barriers), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients may be selected, for example, to provide delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition can be in dosage unit form, such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilisates for reconstitution, powders, solutions, syrups, suppositories, injections, etc. The composition can also be present in a transdermal delivery system, such as a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0032] A pharma- ceutically acceptable carrier can contain a physiologically acceptable agent that acts, for example, to stabilize, increase the solubility, or increase the absorption of a compound, such as a compound of the present disclosure. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose, or dextran, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or excipients. The choice of a pharma- ceutical carrier that includes a physiologically acceptable agent depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-2 microemulsifying drug delivery system. The pharmaceutical composition (preparation) can also be a liposome or other polymer matrix, in which, for example, a compound of the present disclosure can be incorporated. For example, liposomes, which include phospholipids or other lipids, are non-toxic, physiologically acceptable, metabolizable carriers, and are relatively simple to make and administer.

[0033] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0034] The phrase "pharmacologically acceptable carrier" as used herein means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can serve as pharma-ceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) glycols, such as propylene glycol, (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer, and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0035] The pharmaceutical composition (or preparation) can be administered to a subject by any of a number of routes of administration, including, for example, orally (e.g., as a drench, tablet, capsule (including sprinkle capsule and gelatin capsule), bolus, powder, granule, paste for application to the tongue, such as in an aqueous or non-aqueous solution or suspension), absorption through the oral mucosa (e.g., sublingually), subcutaneously, transdermally (e.g., as a patch applied to the skin), and topically (e.g., as a cream, ointment, or spray applied to the skin). The compounds can also be formulated for inhalation. In certain embodiments, the compounds can simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found, for example, in U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and patents cited therein.

[0036] The formulations can be conveniently presented in unit dosage form and can be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the host treated, the particular mode of administration. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form is generally the amount of compound that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0037] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the present disclosure, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0038] Formulations of the disclosure suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophilisates, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, and the like, each containing a predetermined amount of a compound of the disclosure as an active ingredient. The composition or compound may also be administered as a bolus, electuary, or paste.

[0039] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, and the like), the active ingredient may be mixed with one or more pharma- ceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid, (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia, (3) humectants, such as glycerol, (4) agar, calcium carbonate, The pharmaceutical composition may be mixed with any of the following: disintegrating agents such as potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents such as modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also include buffering agents. Similar types of solid compositions may also be used as fillers for soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, and the like.

[0040] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents, or dispersants. Molded tablets may be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.

[0041] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art of pharmaceutical formulation. They can also be formulated to provide delayed or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain an opacifying agent, and can be compositions that optionally release the active ingredient only, or preferentially, in a delayed manner in a certain part of the gastrointestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0042] Liquid dosage forms useful for oral administration include pharma- ceutically acceptable emulsions, lyophilisates for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active ingredients, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrin and its derivatives, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (specifically cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0043] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0044] Suspensions may contain, in addition to the active compounds, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0045] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharma- ceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0046] The ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0047] Powders and sprays can contain, in addition to the active compounds, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0048] Transdermal patch has the additional advantage of providing controlled delivery of the compound of the present disclosure to the body.Such dosage forms can be made by dissolving or dispersing active compound in suitable medium.Absorption enhancers can also be used to increase the flux of compound across the skin.The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing compound in a polymer matrix or gel.

[0049] The phrases "parenteral administration" and "administered parenterally" as used herein refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, spinal, and intrathoracic injection and infusion. Pharmaceutical compositions suitable for parenteral administration include one or more active compounds in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into a sterile injectable solution or dispersion immediately prior to use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0050] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0051] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersing agents. Prevention of microbial activity may be ensured by including various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the composition. In addition, extended absorption of the injectable pharmaceutical form may be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0052] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of drugs.This can be achieved by using liquid suspensions of crystalline or amorphous substances that are poorly water-soluble.In this case, the absorption rate of the drug depends on its dissolution rate, which in turn can depend on the crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0053] Injectable depot forms are prepared by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0054] For use in the methods of the present disclosure, the active compounds may be provided as such or as pharmaceutical compositions containing, for example, 0.1-99.5% (more preferably 0.5-90%) of the active ingredient in combination with a pharma- ceutically acceptable carrier.

[0055] The introduction method may also be provided by a rechargeable or biodegradable device. Recently, various delayed release polymeric devices have been developed and tested in vivo for the controlled delivery of drugs, including protein-based biopharmaceuticals. A wide variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants for sustained release of compounds at specific target sites.

[0056] Actual dosage levels of the active ingredient in a pharmaceutical composition may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0057] The selected dosage level will depend on a wide variety of factors, including the activity of the particular compound or combination of compounds employed, or their esters, salts, or amides, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound being employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0058] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian could start the dosage of the pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. By "therapeutically effective amount" is meant the concentration of the compound sufficient to induce the desired therapeutic effect. It is generally understood that the effective amount of the compound varies according to the subject's weight, sex, age, and medical history. Other factors that affect the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent administered together with the compound of the present disclosure. A larger total dose can be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0059] In general, a suitable daily dose of an active compound used in the compositions and methods of the present disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors mentioned above.

[0060] If desired, the effective daily dose of the active compound can be administered as 1, 2, 3, 4, 5, 6 or more separate doses administered at appropriate intervals throughout the day, optionally in unit dosage form.In certain embodiments of the present disclosure, the active compound can be administered two or three times a day.In a preferred embodiment, the active compound is administered once a day.

[0061] The patient receiving this treatment may be any animal in need, including primates, specifically humans in general; other mammals such as horses, cows, pigs, sheep, cats, and dogs; farm animals; and pets.

[0062] In certain embodiments, the compounds of the present disclosure may be administered alone or in combination with another type of therapeutic agent.

[0063] The present disclosure includes the use of pharma- ceutically acceptable salts of the compounds of the present disclosure in the compositions and methods of the present disclosure.In certain embodiments, the contemplated salts of the present disclosure include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkyl ammonium salts.In certain embodiments, the contemplated salts of the present disclosure include, but are not limited to, L-arginine, benthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts.In certain embodiments, the contemplated salts of the present disclosure include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.In certain embodiments, contemplated salts of the present disclosure include 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptan ... Acid salts of protonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0064] Pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates can also be prepared. The source of such solvates can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or exogenous to such solvent.

[0065] Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0066] Examples of pharma- ceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0067] definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. In general, the terms and techniques used in connection with chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0068] Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, "Principles of Neural Science", McGraw-Hill Medical, New York, NY (2000), Motulsky, "Intuitive Biostatistics", Oxford University Press, Inc. (1995), Lodish et al., "Molecular Cell Biology, 4th ed.", WH Freeman & Co., New York (2000), Griffiths et al., "Introduction to Genetic Analysis, 7th ed.", WH Freeman & Co., NY (1999), and Gilbert et al., "Developmental Biology, 6th ed.", Sinauer Associates, Inc., Sunderland, MA (2000).

[0069] Chemical terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by "The McGraw-Hill Dictionary of Chemical Terms", Parker S., Ed., McGraw-Hill, San Francisco, CA 1985.

[0070] All of the above and any other publications, patents, and published patent applications referenced in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including its specific definitions, will control.

[0071] The term "agent" is used herein to refer to a compound (organic or inorganic compound, mixture of compounds, etc.), a biopolymer (nucleic acid, antibodies including portions thereof, as well as humanized, chimeric, and human antibodies and monoclonal antibodies, proteins or portions thereof, e.g., peptides, lipids, carbohydrates), or an extract made from biological materials such as bacteria, plants, fungi, or animal (especially mammalian) cells or tissues. Agents include, for example, agents whose structures are known and agents whose structures are unknown. The ability of such agents to inhibit AR or promote AR degradation may make them suitable as "therapeutic agents" in the methods and compositions of the present disclosure.

[0072] "Patient," "subject," or "individual" are used interchangeably and refer to either a human or non-human animal. These terms include mammals such as humans, primates, livestock (including cows, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0073] "Treating" a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. As used herein and as understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of disease in a stable (i.e., not worsening) state of disease, prevention of disease spread, delay or slowing of disease progression, improvement or relief of disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment.

[0074] The term "preventing" is art-recognized and, when used in reference to conditions such as local recurrence (e.g., pain), diseases such as cancer, complex syndromes such as heart failure, or any other medical condition, is well understood in the art and includes administration of a composition that reduces the frequency or delays the onset of symptoms of a medical condition in a subject compared to subjects not receiving the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a patient population receiving a prophylactic treatment compared to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population relative to an untreated control population, for example, by a statistically and / or clinically significant amount.

[0075] "Administering" or "administration" of a substance, compound, or agent to a subject can be performed using one of a wide variety of methods known to those skilled in the art. For example, the compound or agent can be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, intraocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through skin ducts). The compound or agent can also be suitably introduced by reloadable or biodegradable polymeric or other devices, e.g., patches and pumps, or formulations that provide extended, delayed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0076] The appropriate method of administering a substance, compound, or agent to a subject also depends, for example, on the age and / or physical condition of the subject, and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, the compound or agent is administered to the subject orally, e.g., by ingestion. In some embodiments, the orally administered compound or agent is in an extended or delayed release formulation or is administered using such a delayed or delayed release device.

[0077] As used herein, the phrase "co-administration" refers to any form of administration of two or more different therapeutic agents such that the previously administered therapeutic agent remains effective in the body while the second agent is administered (e.g., the two agents are effective in the patient at the same time, which may include the synergistic effect of the two agents). For example, different therapeutic compounds can be administered simultaneously or sequentially, either in the same formulation or in separate formulations. Thus, an individual undergoing such treatment can benefit from the combined effect of the different therapeutic agents.

[0078] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is an amount of the drug or agent that will have the intended therapeutic effect when administered to a subject. The full therapeutic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. A therapeutically effective amount may be administered in one or more administrations. The exact effective amount required for a subject depends, for example, on the subject's size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. Those skilled in the art can easily determine the effective amount for a given situation by routine experimentation.

[0079] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, "optionally substituted alkyl" refers to the case where the alkyl may be substituted, as well as the case where the alkyl is unsubstituted.

[0080] It is understood that the substituents and substitution patterns of the compounds of the present disclosure can be selected by those skilled in the art to provide chemically stable compounds that can be easily synthesized from readily available starting materials by techniques known in the art, as well as those methods described below.When a substituent is itself substituted with more than one group, it is understood that these multiple groups can be on the same carbon or on different carbons, as long as a stable structure is obtained.

[0081] As used herein, the term "optionally substituted" refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent, including, but not limited to, hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -OP(O)(O-alkyl)2, or -CH2-OP(O)(O-alkyl). Preferably, "optionally substituted" refers to the replacement of one to four hydrogen radicals in a given structure with a substituent as described above. More preferably, one to three hydrogen radicals are replaced by such a substituent. It is understood that the substituent may be further substituted.

[0082] As used herein, the term "alkyl" refers to any alkyl group having a C1-C 10 Straight chain alkyl group or C1-C 10 It refers to saturated aliphatic groups, including but not limited to branched alkyl groups. Preferably, the "alkyl" group refers to a C1-C6 straight chain alkyl group or a C1-C6 branched chain alkyl group. Most preferably, the "alkyl" group refers to a C1-C4 straight chain alkyl group or a C1-C4 branched chain alkyl group. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl, and the like. The "alkyl" group may be optionally substituted.

[0083] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0084] The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0085] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0086] The term "alkoxy" refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0087] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

[0088] The term "alkyl" refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight-chain or branched-chain alkyl group has 30 or fewer carbon atoms in its backbone (e.g., C for straight chain). 1~30 , C for branched chain 3~30 ), more preferably 20 or less.

[0089] Furthermore, the term "alkyl," as used throughout the specification, examples, and claims, is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups, such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0090] "C x~y " or "C x ~C yThe term " when used in conjunction with a chemical moiety, such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups containing x to y carbons in the chain. CO alkyl indicates a hydrogen in the group's terminal position, or a bond if the group is internal. For example, C 1~6 Alkyl groups contain 1 to 6 carbon atoms in the chain.

[0091] The term "alkylamino," as used herein, refers to an amino group substituted with at least one alkyl group.

[0092] The term "alkylthio," as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0093] The term "amide," as used herein, refers to the group: [ka] In the formula, R 9 and R 10 each independently represents hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0094] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, as well as salts thereof, e.g., [ka] "The term '" refers to a portion that can be represented by In the formula, R 9 , R 10 , and R 10 each independently represents a hydrogen or a hydrocarbyl group, or R 9 and R 10together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0095] The term "aminoalkyl," as used herein, refers to an alkyl group substituted with an amino group.

[0096] The term "aralkyl," as used herein, refers to an alkyl group substituted with an aryl group.

[0097] The term "aryl" as used herein includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of the rings being aromatic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0098] The term "carbamate" is art-recognized and refers to the group: [ka] In the formula, R 9 and R 10 independently represent hydrogen or a hydrocarbyl group.

[0099] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocycle group.

[0100] The term "carbocycle" includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. Carbocycles include bicyclic molecules in which one, two, or more atoms are shared between two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of carbocycle, as valences permit. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" may be substituted at any one or more positions capable of carrying a hydrogen atom.

[0101] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocycle group.

[0102] The term "carbonate" is art-recognized and refers to the group -OCO2-.

[0103] The term "carboxy," as used herein, refers to a group represented by the formula -CO2H.

[0104] The term "ester" as used herein refers to the group -C(O)OR 9 In the formula, R 9 represents a hydrocarbyl group.

[0105] The term "ether" as used herein refers to a hydrocarbyl group linked to another hydrocarbyl group through an oxygen. Thus, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers can be either symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which can be represented by the general formula alkyl-O-alkyl.

[0106] The terms "halo" and "halogen" as used herein mean halogen and include chloro, fluoro, bromo, and iodo.

[0107] The terms "hetaralkyl" and "heteroaralkyl," as used herein, refer to an alkyl group substituted with a hetaryl group.

[0108] The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic single ring structures, preferably 5-7 membered rings, more preferably 5-6 membered rings, in which the ring structure contains at least one heteroatom, preferably 1-4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of the rings is heteroaromatic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0109] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0110] The term "heterocyclylalkyl," as used herein, refers to an alkyl group substituted with a heterocycle group.

[0111] "Heterocyclyl", "heterocycle", and "heterocyclic" refer to a substituted or unsubstituted non-aromatic ring structure, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, in which the ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of which is heterocyclic, e.g., the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0112] The term "hydrocarbyl" as used herein refers to a group that does not have a=O or =S substituents and is typically bonded through a carbon atom having at least one carbon-hydrogen bond and a primarily carbon backbone, but may optionally contain heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl for purposes of this application, while substituents such as acetyl (having a=O substituent on the linking carbon) and ethoxy (linked through an oxygen rather than a carbon) are not. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, alkynyl, and combinations thereof.

[0113] The term "hydroxyalkyl," as used herein, refers to an alkyl group substituted with a hydroxy group.

[0114] The term "lower", when used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups in which there are 10 or fewer atoms, preferably 6 or fewer atoms, in the substituent. "Lower alkyl", for example, refers to an alkyl group containing 10 or fewer carbon atoms, preferably 6 or fewer carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively, whether they appear alone or in combination with other substituents such as the enumerated hydroxyalkyl and aralkyl (where, for example, atoms in an aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0115] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings." Each of the rings of a polycycle can be substituted or unsubstituted. In certain embodiments, each ring of a polycycle contains from 3 to 10 atoms, preferably from 5 to 7 atoms, in the ring.

[0116] The term "sulfate" is art-recognized and refers to the group -OSO3H, or a pharma- ceutically acceptable salt thereof.

[0117] The term "sulfonamide" is art-recognized and refers to a group represented by the general formula: [ka] In the formula, R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0118] The term "sulfoxide" is art-recognized and refers to the group --S(O)--.

[0119] The term "sulfonate" is art-recognized and refers to the group SO3H, or a pharma- ceutically acceptable salt thereof.

[0120] The term "sulfone" is art-recognized and refers to a -S(O)2- group.

[0121] The term "substituted" refers to a moiety having a substituent replacing a hydrogen on one or more carbons of the backbone. It will be understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is subject to the permissible valences of the substituted atom and substituent, and results in a stable compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and can be the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. The substituents may include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that the moieties substituted on the hydrocarbon chain can themselves be substituted, where appropriate.

[0122] The term "thioalkyl," as used herein, refers to an alkyl group substituted with a thiol group.

[0123] The term "thioester" as used herein refers to the group -C(O)SR 9 Or -SC(O)R 9 Refers to, In the formula, R 9 represents hydrocarbyl.

[0124] The term "thioether" as used herein is equivalent to an ether, where the oxygen is replaced by a sulfur.

[0125] The term "urea" is art-recognized and may be represented by the general formula: [ka] In the formula, R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0126] The term "modulate" as used herein includes inhibiting or suppressing a function or activity (such as cell proliferation), as well as enhancing a function or activity.

[0127] The phrase "pharmacologically acceptable" is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers, and other materials and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0128] "Pharmaceutically acceptable salt" or "salt" is used herein to refer to an acid addition salt or a base addition salt that is suitable or compatible for the treatment of a patient.

[0129] The term "pharmaceutically acceptable acid addition salt" as used herein means any non-toxic organic or inorganic salt of any base compound represented by formula I. Illustrative inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, and salicylic acid, as well as sulfonic acids such as p-toluenesulfonic acid and methanesulfonic acid. Either mono- or di-acid salts can be formed, and such salts can exist in either hydrated, solvated, or substantially anhydrous form. In general, acid addition salts of compounds of formula I are more soluble in water and various hydrophilic organic solvents, and generally exhibit higher melting points compared to their free base forms. The selection of an appropriate salt will be known to one of skill in the art. Other pharma-ceutically unacceptable salts, such as oxalates, may be used, for example, in the isolation of compounds of formula I for laboratory use or for subsequent conversion to a pharma-ceutically acceptable acid addition salt.

[0130] The term "pharmaceutically acceptable basic addition salt" as used herein means any non-toxic organic or inorganic base addition salt of any acid compound represented by formula I or any of its intermediates. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine, and picoline, or ammonia. The selection of appropriate salts will be known to those skilled in the art.

[0131] Many of the compounds useful in the methods and compositions of the present disclosure have at least one stereocenter in their structure. This stereocenter can be in R or S configuration, and the R and S designations are used according to the rules described in Pure Appl.Chem.(1976),45,11-30. The present disclosure contemplates all stereoisomeric forms, including enantiomeric and diastereoisomeric forms, of the compounds, salts, prodrugs, or mixtures thereof (including all possible mixtures of stereoisomers). See, for example, WO01 / 062726.

[0132] Furthermore, certain compounds containing alkenyl groups can exist as Z (zusammen) or E (entgegen) isomers, and in each case the present disclosure includes both mixtures and the separate individual isomers.

[0133] Some of the compounds may also exist in tautomeric forms. Such forms, although not explicitly shown in the formulae set forth herein, are intended to be included within the scope of the present disclosure.

[0134] "Prodrug" or "Pharmaceutically acceptable prodrug" refers to a compound that is metabolized in the host after administration, e.g., hydrolyzed or oxidized to form a compound of the present disclosure (e.g., a compound of formula I). ​​Typical examples of prodrugs include compounds that have a biologically labile or cleavable (protecting) group on the functional moiety of an active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce an active compound. Examples of prodrugs that use esters or phosphoramidates as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patent Nos. 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of the present disclosure are metabolized to produce a compound of formula I. The present disclosure includes within its scope prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

[0135] The phrase "pharmaceutically acceptable carrier" as used herein means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, useful in formulating a drug for pharmaceutical or therapeutic use.

[0136] The term "log of solubility", "LogS", or "logS", as used herein, is used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound significantly affects its absorption and distribution properties. Low solubility often leads to low absorption. The LogS value is the unit stripped logarithm (base 10) of solubility, measured in moles / liter. EXAMPLES

[0137] The present disclosure, having generally been described above, will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the present disclosure and are not intended to limit the present disclosure.

[0138] Materials and Methods Chemicals and Supplies-2(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBt) were purchased from Vivitide (Gardner, MA). All protected amino acids, rink amide MBHA resin, and N-methylmorpholine (NMM) were supplied by Gyros Protein Technologies (Tucson, AZ). Polyethylene glycol (PEG3) was obtained from Creative PEGWorks (Durham, NC). Trifluoroacetic acid (TFA), piperidine, thioanisole, anisole, 1,2-ethanedithiol, methyl-tert-butyl ether, N,N-diisopropylethylamine (DIPEA), dimethylformamide (DMF), acetonitrile (ACN), N-methyl-2-pyrrolidone (NMP), sodium carbonate, Sephadex G25, L-glutathione reduced (GSH), lipopolysaccharide (LPS), and cis-dichlorodiammineplatinum(II) (cisplatin) were purchased from Sigma Aldrich (Saint-Louis, MO). Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was supplied by Invitrogen (Carlsbad, CA). p-SNC-deferoxamine (DFO) was from Macrocyclics Inc. (Plato, TX). N2'-Deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1) and aldoxorubicin HCl (aldox) were from MedKoo Biosciences (Morrisville, NC). Doxorubicin (DOX) and gemcitabine (GEM) were obtained from LC Laboratories (Woburn, MA). Mitomycin (MIT) was supplied by Selleckchem (Houston, TX). Cyanine 5.5 NHS ester was purchased from Lumiprobe Corporation (Hallandale Beach, FL) and luciferin was purchased from Caliper LifeScience (Hopkinton, MA).

[0139] Peptide synthesis - All peptides were synthesized on a solid-phase peptide synthesizer (PS3, Gyros Protein Technologies, Tucson, AZ) using the N-α-Fmoc method on Rink amide resin as previously described. Side-chain protected amino acids (0.4 mmol) were attached to the resin (385 mg, 0.1 mmol) by stepwise elongation using NMM as the base, HBTU / HOBt as coupling reagents, and piperidine (20% v / v in DMF) as the deprotecting agent. Prior to peptide cleavage, a β-alanine was incorporated at the peptide N-terminus as a spacer for further conjugation of desired moieties including fluorophores (cyanine 5.5), chelators (DFO), or linkers (SPDP).

[0140] Immunogenicity assay - Female BALB / c mice were treated with a single dose of Bdd or LPS (5 mg / kg) via tail vein injection (n=3 / group). Mice receiving PBS were used as negative controls. Blood samples were collected 4 hours later via retro-orbital sinus puncture technique, and concentrations of innate immune and inflammatory cytokines IL1β, IL2, IL6, IL10, TNF-α, and INF-γ in plasma were measured with a commercially available sandwich enzyme-linked immunosorbent assay kit. ELISA assays were performed according to the manufacturer's instructions (Invitrogen, Carlsbad, CA).

[0141] Methods for incorporating different functionalities into peptides - Cyanine 5.5 NHS ester (50 mg, 1.4 eq.) in DMF (4 mL), DFO (25 mg, 1 eq.) in DMSO (4 mL), or SPDP (25 mg, 1 eq.) in NMP (4 mL) were added to the resin (0.05 mmoL, 1 eq.) and reacted overnight at room temperature. For conjugation of fluorophores and chelators, the reaction was carried out in the presence of an organic base (DIPEA, 1 mL). The peptides were then removed from the resin using a cleavage cocktail (5 mL) containing TFA / thioanisole / 1,2-ethanedithiol / anisole (90:5:3:2) for 4 h and precipitated in methyl-tert-butyl ether. The resulting peptides (Cy-peptide, DFO-peptide, and SPDP-peptide) were purified to greater than 98% purity using reverse-phase high-performance liquid chromatography (rp-HPLC, Agilent, Santa Clara, CA) and characterized by MALDI-TOF analysis (Tufts Medical School, Boston, MA) to confirm their molecular weights.

[0142] Synthesis of cleavable drug-peptide conjugates - DM1 (1 mg, 1 equiv.) was added to SPDP-peptide (10 mg, 3 equiv.) in a cosolvent of NMP (100 μL) and phosphate-buffered saline (PBS; 10 mM, pH 7, 100 μL) and allowed to react for 2 days at room temperature. The DM1-peptide was then purified by rp-HPLC. To conjugate aldox, a thiol-reactive side (cysteine) was introduced at the peptide N-terminus. Aldox (2 mg, 1 equiv.) was added to peptide (10 mg, 3 equiv.) in PBS (10 nM, 1 mL, pH 7.4) and allowed to react for 30 min. The resulting drug-peptide was pH sensitive and therefore purified by rp-HPLC in neutral conditions (mobile phase A: PBS, mobile phase B: 90% ACN in PBS). Size-exclusion chromatography (TipTop C-18 column) was used to remove salt content. All final drug-peptide conjugates were characterized using MALDI-TOF analysis and analyzed using DM1 (ε = 3,700 cm) in 5% (v / v) PBS in methanol. -1 M -1) or aldox (ε = 13,000 cm -1 M -1 ) was quantified by UV absorbance according to a defined extinction coefficient.

[0143] Radiochemistry - 89 zirconium( 89 Zr) was supplied by 3DImaging LLC (Little Rock, AR). 89 Zr-oxalate (500 μCi) was first neutralized with an equal volume of sodium carbonate solution (2 M) and then added to the DFO-peptide (0.2 mg, 250 μL). After incubation at room temperature for 1.5 h, the radiolabeled peptide ( 89 Zr-peptide) was purified by size-exclusion chromatography using Sephadex G-25 gel to remove free zirconium.

[0144] Pharmacokinetic studies: In BALB / c mice (Jackson Laboratory, Bar Harbor, ME), 89 Zr-peptide or free 89 Zr (20 μCi, 100 μL) was administered via tail vein injection (n=4 / condition). Blood samples (20 μL) were collected at various time intervals using a retro-orbital sinus puncture technique. Radioactivity was measured in a Wallac Wizard 2 gamma counter (Perkin-Elmer, Waltham, MA). 89 Zr-peptide and free 89 The pharmacokinetic model and parameters of Zr were estimated by fitting the data for compartment model selection using PKSolver 2.0 software.

[0145] μPET / CT imaging and biodistribution study - BALB / c mice were treated with different 89Zr-peptide analogs (20 μCi, 100 μL) were injected intravenously. μPET / CT imaging was performed in animals that had and had not collected urine prior to the first imaging (n=4 / analog / condition). Whole-body images were acquired 1, 4, and 24 hours after injection using an Inveon μPET / CT scanner (Siemens Medical Solutions, Malvern, PA). μPET / CT maximum energy projections were processed using Amide v1.0.4 and Inveon Research Workplace software. Radioactivity in different regions of interest (ROIs) was determined. For endpoint biodistribution studies, mice were 89 Mice were euthanized 0.17, 1, 2, 5, and 7 days after treatment with Zr-peptides (n=3 / peptide analog / time point). Radioactivity in harvested organs was measured using a Wallac Wizard 2 gamma counter. Results were corrected for radioactive decay and expressed as percentage of injected dose (ID%) or percentage of injected dose / gram of tissue (ID% / g).

[0146] Fluorescence imaging - Female SHO mice (Charles River Laboratories, Wilmington, MA) were administered free cyanine 5.5 or Cy-peptide analog (0.5 nmol cyanine 5.5 content as measured by UV absorbance at 680 nm) in PBS (150 μL) via tail vein injection (n=4 / group). Real-time fluorescence imaging was performed using an in vivo Xtreme Imaging System (Bruker, Billerica, MA). Whole-body fluorescence images were acquired 1 and 4 hours after injection using appropriate excitation (670 nm) and emission (750 nm) filters. Animals were then euthanized. Organs were excised and ex vivo fluorescence imaging was performed. Imaging was also performed on urine samples (50 μL) collected from separate animals 1 hour after intravenous injection of free dye or Cy-peptide analog (n=4 / treatment). Fluorescence / bright light images were processed and fluorescence intensity in different ROIs was measured using Bruker MI software. All data were corrected to eliminate organ or fluid autofluorescence.

[0147] The cell line-MB49 was supplied by EMD Millipore Corporation (Temecula, CA). UMUC-3, T24, and Renca were obtained from ATCC (Manassas, VA). Each cell line was cultured according to the company's instructions. Mycoplasma testing (Lonza, Basel, Switzerland) was performed regularly to ensure the absence of contamination. Both UMUC-3 and Renca cell lines were further transduced with GlowCell 16 FLuc-F2A-GFP lentivirus (Biosettia, San Diego, CA), which carries both firefly luciferase and green fluorescent protein (GFP) genes. Briefly, cells were cultured in 6-well plates (0.25 × 10 6 cells / well) and inoculated with virus (2 × 10 7 The cells were incubated with 100 mM NaCl (1.1 IU / well) for 3 days. To ensure >95% cell purity, cell lines were analyzed and sorted for GFP expression using flow cytometry. Successful transduction was further confirmed by imaging using an EVOS FL Auto fluorescence microscope (Life Technology, Carlsbad, CA).

[0148] Cell viability and cytotoxicity assays - cancer cells (3 × 10 3 Cells were seeded overnight on flat-bottom 96-well plates (100 μL / well). Different concentrations of DM1, gemcitabine (GEM), mitomycin (MIT), cisplatin (CIS), doxorubicin (DOX), aldoxorubicin (aldox), or drug-loaded peptides (DM1-peptide and aldox-peptide) were then added to the cells for 72 h and then washed twice with PBS (400 μL). CellTiter Glo reagent (Promega, Madison, WI) was added to each well (50 μL). Luminescence generated was recorded using a microplate reader (Tecan US Inc., Morrisville, NC). Dose-response curves were plotted and the half-maximal inhibitory concentrations (IC 50Values) were calculated using Graph Pad Prism 6.0 software.

[0149] In vitro drug release test - DM1-peptide conjugate (drug content 10 μM) was incubated in PBS buffer (800 μL) in the presence of reducing agent GSH (1 mM). At different time intervals (0, 2, 4, 6, 8, 12, 24, and 48 h), small amounts of solution (100 μL) were removed for HPLC analysis using a C18 analytical column. The amount of DM1 active metabolite released over time was measured (absorbance detected at 254 nm) and then quantified. The experiment was performed independently and in triplicate. A similar experiment was performed to quantify the release of aldox. Aldox-peptide conjugate (drug content 100 μM) was incubated in PBS buffer (800 μL) with different pH (7.4 and 5.5) in glass vials coated with silica to avoid non-specific adsorption on the reaction vessel surface. The amount of drug released over time was then measured (absorbance detected at 480 nm) as described above. For accurate quantification, all results were normalized relative to DOX controls incubated in similar conditions.

[0150] Animal care - All animals used in this project were housed in pathogen-free, barrier-free rooms and maintained at controlled temperature (72 ± 2 °F) with a daily 12-h light-dark cycle. All procedures performed on mice were approved by the Weill Cornell Medical Center Institutional Animal Care and Use Committee (protocol number 2019-0003) and were consistent with the recommendations of the American Veterinary Medical Association and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were allowed to acclimate for at least 7 days before any experiments were performed. NSG mice are immunocompromised and susceptible to infections. Therefore, animals were fed a diet containing sulfatrim antibiotics (Envigo, Indianapolis, IN). Animals used for fluorescence imaging were fed an AING 93 non-fluorescent diet (Envigo, Indianapolis, IN).

[0151] Therapeutic Efficacy of Drugs - Bdd conjugates for treating BC-animals were orthotopically implanted into the tumor as previously described. Briefly, urine from 7-9 week old female NSG mice (Jackson Laboratory, Bar Harbor, ME) was removed through a sterile 24G pediatric venous catheter (Dublin, Ireland). Trypsin solution (0.125%, 80 μL) was then delivered into the bladder. Subsequently, UMUC-3 / GFP-Luc cells (4 × 10) in culture medium (50 μL) were cultured in the bladder. 4Cells) were transferred to and seeded in the bladder. Tumor progression was monitored by bioluminescence imaging. Luciferin (3 mg) in PBS (100 μL) was given to the animals (via intraperitoneal injection) 15 minutes before imaging. Once tumor progression was confirmed in the bladder (based on bioluminescence signal), animals were randomly assigned (n=14 / group) for weekly treatment with PBS, DM1, or DM1-Bdd (drug content 0.75 mg / kg) in saline (150 μL) via tail vein injection for 3 weeks. Separate groups of animals were assigned (n=14 / group) for treatment with intravesical DM1, DM1-Bdd (drug content 0.75 mg / kg) in saline (50 μL). Treatment efficacy was evaluated based on tumor growth inhibition (bioluminescence imaging) and long-term survival. Additional mice were recruited for histopathological analysis (n=4 / group). The animals were immediately euthanized one week after completion of the treatment schedule. The bladders were harvested and stored in neutral buffered formalin (10%). The same experimental conditions were used to compare the therapeutic outcomes between animals treated with PBS, aldox, or aldox-Bdd via tail vein injection or intravesical administration (drug content 5 mg / kg).

[0152] Evaluation of DM1-Bdd for renal cancer treatment - Renca / GFP-Luc cells (4 × 10 3 Cells) were orthotopically implanted into the kidney capsule of 7-9 week-old female BALB / c mice (Jackson Laboratory, Bar Harbor, ME) as previously described. Tumor progression was confirmed by bioluminescence imaging. Animals were randomly assigned to four groups (n=14 / treatment) for weekly treatment with PBS, DM1, or DM1-Bdd (drug content 0.75 mg / kg) via tail vein injection, or DM1-Bdd (drug content 0.75 mg / kg) via intravesical administration. Animals were monitored for tumor growth inhibition and survival as described above. Additional mice were used for histopathological analysis (n=4 / group).

[0153] Histological analysis - tissue samples were fixed in formalin, dehydrated in ethanol, and embedded in paraffin. Tissue sections (5 μm) were stained with hematoxylin and eosin Y (H&E). For immunohistochemistry, bladder sections were deparaffinized and then rehydrated before overnight incubation with anti-Ki67 or anti-GFP antibodies (Abcam, Cambridge, UK). Slides were then counterstained with hematoxylin. High-resolution images were acquired using an Aperio 9 Digital Pathology slide scanner (Leica Biosystems, Weltzar, Germany).

[0154] Necropsy - 7-9 week-old female BALB / c mice (n=4 / group) were treated once a week with PBS, DM1, DM1 peptide (drug content 0.75 mg / kg), or cisplatin (10 mg / kg) via tail vein injection (150 μL). At the end of 3 weeks of treatment, mice were euthanized. Organs / tissues were harvested and fixed in 10% neutral buffered formalin for 2 days. Bones were decalcified in formic acid solution (Surgipath Decalcifier I; Leica Biosystems, Weltzar, Germany). Samples were then embedded in paraffin, sectioned (5 μm), and stained with H&E for examination by an ACVP board-certified anatomical pathologist.

[0155] Nephrotoxicity Study - PBS, DM1, DM1-Peptide, MIT, GEM, CIS (drug content 0.75 mg / kg), or high dose of CIS (10 mg / kg) were administered to BALB / c mice via the tail vein (n=3 / group). Levels of acute kidney injury biomarkers, neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule 1 (KIM-1) in urine samples were measured using ELISA assays according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). Complementary histopathological analysis was performed on the kidneys of animals treated with PBS, DM1, DM1-Bdd (drug content 0.75 mg / kg), or high dose of CIS (10 mg / kg). All animals (n=3 / group) were euthanized at the end of treatment, and kidneys were collected and fixed in 10% neutral buffered formalin. Tissues were then processed, embedded in paraffin, sectioned, and stained with H&E. Immunohistochemistry (IHC) for renal injury (NGAL and KIM-1) was also performed.

[0156] Hematology and Biochemistry - Blood samples were collected via cardiac puncture. Complete blood counts including red blood cell, white blood cell, reticulocyte, and platelet counts were performed using an IDEXX Procyte DX hematology analyzer (iDEXX, Westbrook, ME) with automated differential white blood cell count. Blood smears were prepared, stained with modified Wright's stain, and examined in a blinded fashion by a board-certified veterinary clinical pathologist (TS) and analyzed using a photometer CoolSNAP HQ. 2 Imaging was performed using a Nikon Eclipse TE2000-U fluorescence microscope equipped with a camera (Nikon Corporation, Tokyo, Japan). Blood was centrifuged (1,500 × G) for 15 min to obtain serum for biochemical analysis using a Beckman Coulter AU680 analyzer (Beckman Coulter, Brea, CA).

[0157] Statistical Analysis - Statistical analysis was performed using Graph Pad Prism 7.0 software and R v4.0.5 (R Foundation for Statistical Computing, Vienna, Austria) software. All data are presented as mean ± standard deviation and significance was assigned at *p<0.05, **p<0.01, and ***p<0.001. Two-tailed Student's t-test was used to determine significant differences between groups. For survival evaluation, Mantel-Cox log-rank test was performed to compare survival curves of animals treated with intravenous DM1-Bdd or aldox-Bdd with other treatments. P values ​​were adjusted for multiple comparisons using the Benjamini & Hochberg method. All p values ​​are two-tailed with statistical significance assessed at the 0.05 alpha level. 95% (exact) confidence intervals were calculated for all parameters to assess the precision of the estimates obtained.

[0158] Example 1: Bdd can be eliminated exclusively via renal clearance Bdd was designed in the D-configuration to avoid degradation by protease enzymes in the blood circulation. It is composed of multiple D-aspartic acid (d) and β-alanine (B) residues (Figure 1b). The aspartic acids contributed to an overall negative charge, preventing nonspecific uptake by major organs and facilitating renal clearance of the peptide. The B residues served as linkers to avoid the formation of secondary structures. To investigate how charge affects the in vivo behavior of Bdd, a panel of Bdd analogues that were L-configured (BDD), neutral (BKD), positively charged (BKK), and pegylated forms (PEG3DD) were synthesized for comparative studies. The peptide was then coupled to a long-lived radioisotope (t) probe, allowing for pharmacokinetic (PK) and long-term biodistribution (BD) studies using micropositron emission tomography / computed tomography (μPET / CT) imaging. 1 / 2 =78 hours), 89-dircuronium ( 89 Radiolabeled peptides ( 89Zr-peptides were synthesized by first conjugating the deferoxamine (DFO) chelator to the peptide on the solid phase after amino acid elongation. 89 Zr was complexed with the resulting DFO-peptide conjugate in solution under basic conditions (FIG. 1c).

[0159] The results are: 89 Zr-Bdd and 89 We showed that Zr-BDD exhibited minimal off-target delivery (Figure 1d). Radioactivity was not detected in any major organs other than the kidney. Both peptides were rapidly excreted in the urine within 1 h after intravenous administration, as confirmed by the absence of radioactivity in the bladder, which was catheterized and emptied prior to imaging. Results also showed that as much as 80% of the total injected dose (ID) was 89 Zr-Bdd (Figure 1e) was present in the first urine samples collected from animals injected with Zr-Bdd, suggesting that the peptide was filtered through the glomerulus with minimal reabsorption. 89 Zr-BKK was delivered to the liver in addition to the URS (Fig. 1d). Renal uptake was also compared between the peptide analogs. They all showed reduced renal accumulation over time, except for an initial increase for PEG3DD (Fig. 1f). To determine the PK profiles, the experimental data were fitted to a two-compartment model. The results showed that all peptide analogs were significantly increased in the free 89 It was shown that it exhibited a shorter half-life compared to Zr (Figure 1g). 89 Zr-Bdd had a terminal half-life of 0.53 hours and rapid plasma clearance (Figure 7a). 89 An endpoint biodistribution study of Zr-Bdd was performed. The results were in good agreement with the imaging and PK studies. Four hours after intravenous injection, no peptide was detected in major organs or in the blood circulation (Figure 1h and Figure 7b). Despite rapid clearance, 89 15.1% of Zr-Bdd IDs were found in the kidney, decreasing to 2.1% after 7 days.

[0160] Example 2: As a carrier for hydrophobic molecules Most chemotherapeutic agents are hydrophobic molecules that exhibit unfavorable PK and BD, resulting in off-target delivery and undesirable toxicity. However, the rapid renal clearance of Bdd could be beneficial to facilitate drug delivery to the URS. To demonstrate this, a hydrophobic cyanine 5.5 fluorophore (Cy) was covalently attached to a peptide analogue as a drug model (Fig. 2a), and the resulting conjugates (Cy-peptide) were compared for in vivo delivery. Both Cy-Bdd and Cy-BDD could be rapidly eliminated via renal clearance. They reached the bladder of the animals 1 h after intravenous injection (Fig. 2b), and presented as much as 70–75% ID in the urine (Fig. 2c). Meanwhile, Cy-BKD, Cy-BKK, Cy-PEG3DD, and free Cy were mainly taken up by the liver. Ex vivo imaging of harvested organs was also performed (Fig. 2d). As expected, accumulation of Cy-Bdd and Cy-BDD in the kidney was minimal compared to the other conjugates (Figure 2e). 89 Unlike Zr-Bdd, trace amounts of Cy-Bdd and Cy-BDD were taken up by the stomach, liver, and intestine. 89 This is likely due to the replacement of Zr-DFO with hydrophobic Cy. Overall, Bdd could be used to deliver hydrophobic molecules, such as Cy, to the URS while maintaining the global UDD properties.

[0161] Example 3: As a carrier for chemotherapeutic agents The excellent UDD properties of Bdd prompted further investigation of the delivery of chemotherapeutic agents for NMIBC treatment. Many peptides are immunogenic. First, it was confirmed that the Bdd peptide did not induce an innate immune response. There was no increase in inflammatory cytokine levels (IL-1β, IL-2, IL-6, IL-10, TNF-α, and INF-γ) in the plasma of BALB / c mice 4 hours after intravenous injection (Fig. 3a). Using fluorescent Cy-Bdd, we confirmed that the peptide could be taken up into human BC (UMUC-3) and mouse kidney cancer (Renca) cells (Fig. 3b). Overlap of peptide (red) and lysosomal (green) fluorescence was observed, suggesting that cellular uptake of the peptide occurred primarily via endocytosis. As a proof of principle, emtansine (DM1), a potent microtubule inhibitor, was selected as a drug candidate. Compared with other conventional chemotherapy agents, DM1 is more potent than cisplatin (CIS), doxorubicin (DOX), and mitomycin (MIT), and is as effective as gemcitabine (GEM) against UMUC-3, with IC 50 values ​​in the nanomolar (nM) range (Fig. 3c). The drug was also effective against the Renca cell line. Next, DM1-Bdd conjugates were synthesized by linking DM1 to Bdd via a cleavable disulfide linker (Fig. 3d), allowing drug release in a reducing environment, such as in the presence of intracellular glutathione (GSH) (Fig. 3e). We also established chemistry for conjugating other chemotherapeutic agents, such as aldoxorubicin (aldox), to Bdd (Fig. 3f). aldox is a DOX derivative modified with a hydrazone linker known to be sensitive to acidic lysosomes and tumor microenvironments. Drug release from the aldox-Bdd conjugates was pH-dependent (Fig. 3g). With regard to cytotoxicity, both DM1-Bdd and aldox-Bdd exhibited similar potency to the corresponding free drugs against different murine (MB49) and human (UMUC-3 and T24) BC cell lines (Fig. 3h-i).

[0162] Example 4: A more effective alternative to traditional ITC Unlike intravesical (it) administration, animals did not have to urinate immediately after intravenous injection (Figure 4a). DM1-Bdd administered through intravenous injection should extend the bladder residence time of the drug. This, together with the unique UDD properties, should provide a more effective (compared to ITC) and safer (compared to systemic chemotherapy) treatment option when using Bdd as a drug carrier. However, Bdd may accumulate transiently in the kidney (Figure 1d and h). Therefore, before evaluating the therapeutic efficacy of DM1-Bdd, we evaluated the renal tolerance to a single injectable dose in healthy mice. The results showed that DM1-Bdd slightly increased the levels of tubular injury markers, kidney injury molecule 1 (KIM-1), and neutrophil gelatin enzyme-associated lipocalin (NGAL), in the urine 1 day after injection (Figure 4b). The increase was minimal and transient, returning to basal levels within 3 days. Histological examination 3 days after drug administration did not reveal any abnormalities in the kidneys, and there was no increase in immunoreactivity in tissue sections for KIM-1 and NGAL when compared to PBS (Figure 4c). In contrast, other intravenous chemotherapeutic agents, including DM1, MIT, CIS, and GEM, showed continued induction of KIM-1 and / or NGAL at this time point. Dilation and degeneration of the renal tubular epithelium in animals treated with high doses of intravenous CIS (positive control) were also found (Figure 4c).

[0163] DM1-Bdd was then evaluated for treating mice bearing orthotopic human UMUC-3 tumors. Before UMUC-3 cells were implanted into the bladder of the animals, they were stably transduced with a dual luciferase and GFP reporter (Figure 4d). This allowed for monitoring of disease progression by bioluminescence imaging. It was also confirmed that established tumors growing in the lamina propia were essentially non-invasive and confined to the urinary bladder submucosa (Figure 4e). Compared to the clinically used ITC (intravegedicinal mit), both intravenous DM1 and DM1-Bdd were more effective in inhibiting tumor growth (Figures 4f-g and Figures 8a-e) and prolonging animal survival (Figure 4h). Intravenous injection of DM1-Bdd provided better treatment outcomes when compared to intravesical administration. According to imaging data acquired over three weekly treatment courses, the antitumor activity of intravenous DM1-Bdd and DM1 was similar (Figure 4f). However, those animals treated with intravenous DM1-Bdd showed a significant improvement in overall survival (36% vs. 0% alive after 100 days). In a separate experiment, the treatment effect on tumor reduction was confirmed by histology. Tumors in animals treated with intravenous DM1-Bdd were smaller (Figure 4i). Immunohistochemistry revealed fewer GFP-positive cells and a lower percentage of cells expressing a proliferation marker (Ki67), confirming inhibition of tumor cell growth and proliferation (Figure 4j and Figure 8f). More importantly, DM1-Bdd treatment was curative for 21% of animals. Surviving mice lacked gross and histological evidence of tumors (using GFP and Ki67 immunostaining), suggesting they were disease-free after 210 days (Figure 8g). Bdd is a versatile delivery platform that can carry different chemotherapeutic agents. Aldox-Bdd was also able to extend the survival time of animals compared to PBS and free DOX (Figures 9a-c). However, it was less effective than DM1-Bdd in improving overall survival (Fig. 9c).Surprisingly, animals treated with free DOX had a shorter life expectancy compared to PBS controls, and all animals lost weight continuously during DOX treatment (Fig. 9d and e).They ultimately died before receiving the final dose, suggesting drug-induced toxicity and mortality.

[0164] Example 5: Anatomical Flexibility DM1-Bdd for treating kidney cancer was evaluated using a syngeneic mouse model for the study, which involved surgical implantation of Renca cells (stably transduced with GFP and Luc) into the right renal capsule of BALB / c mice (Fig. 5a). Tumor growth was aggressive. Substantial sized tumor masses developed as early as 1 week after implantation (Fig. 5b). Imaging studies showed that intravenous DM1-Bdd was not only able to inhibit tumor progression, as shown by a decrease in bioluminescent signal in the majority of animals during treatment (Fig. 5c and d), but also reduced tumor size (Fig. 5e). Survival after 160 days was 50% (Fig. 5f and g). Meanwhile, all animals treated with either intravenous or intravesical DM1 died. In a separate experiment, histological examination of the kidneys of the animals was performed 1 week after completion of the treatment course (Fig. 5h). Minimal tumors were present in animals treated with intravenous DM1-Bdd, whereas tumors in animals treated with DM1 or PBS control were large and associated with mononuclear cell infiltration. Overall, the UDD approach is anatomically flexible and could be used to treat tumors located in the upper urinary tract.

[0165] Example 6: Toxicity Profile The toxicity profile of DM1-Bdd in healthy animals after completing 3 weeks of weekly treatment was evaluated. DM1-Bdd did not affect the numbers or morphological characteristics of red blood cells, white blood cells, or platelets (Figures 6a-b and 10). On the other hand, DM1 induced an inflammatory response characterized by reticulocytosis without obvious anemia, and increased percentages of neutrophils, monocytes, and platelets. The positive control CIS was also toxic, as evidenced by thrombocytopenia and lymphopenia. Serum biochemistry analyses to assess liver injury and renal function were also performed. DM1-Bdd was not hepatotoxic, with no significant changes in the release of ALP, ALT, and AST (Figures 6c and 11). Importantly, the urea nitrogen / creatinine ratio was normal, suggesting that DM1-Bdd did not affect renal clearance of nitrogenous wastes. Histopathological studies further confirmed that there was no morphological evidence of damage in the liver, spleen, heart, lungs, or kidneys of animals treated with DM1-Bdd (Fig. 6d and Fig. 12). In contrast, biochemical tests showed that both DM1 and CIS induced hepatotoxicity and nephrotoxicity, as indicated by increased ALT and AST activity and BUN / creatinine ratio (Fig. 6c). They also induced muscle damage (CK activity), which may partially explain the increased AST activity. Histological examination also showed that CIS caused degeneration and necrosis of proximal tubules, lung inflammation, and depletion of both erythrocytes and extramedullary hematopoiesis (EMH) in the spleen (Fig. 6d and Fig. 12). Animals treated with DM1 showed similar renal damage, but to a lesser extent. An increase in splenic and hepatic EMH with DM1 was also observed, which may explain the reticulocytosis observed in treated animals.

[0166] Consideration Most chemotherapeutic agents that are toxic to healthy and cancer cells are given by infusion so that they can be administered continuously at higher total doses over a longer period of time. The goal is to achieve a more effective treatment to improve patient tolerance to off-target toxicity by maintaining the plasma concentration of the drug at a certain level and prolonging tumor exposure to the drug. This study aimed to promote, rather than reduce, drug clearance as a non-invasive alternative to ITC. Many bioactive peptides have been approved for the treatment of various diseases, including cancer, diabetes, and cardiovascular disease. Without chemical modification, peptides have a short circulating half-life of a few minutes. They are rapidly degraded by protease enzymes and eliminated by renal filtration. This study recognized that the rapid renal clearance of peptides could be advantageous as drug carriers to dispose of most systemically administered drugs in the urine to treat NMIBC and reduce unwanted systemic side effects. In the present invention, a UDD approach was introduced by using a bioinert, negatively charged peptide (Bdd) with minimal uptake by the reticuloendothelial system and other organs, and exclusively excreted in the urine. Bdd was used to deliver DM1, a microtubule inhibitor. DM1 was selected because it was 100-fold more potent than commonly used ITC drugs, including MIT, DOX, and CIS, against a panel of BC cell lines. Furthermore, conjugation of the drug to the Bdd peptide did not compromise cytotoxicity.

[0167] In terms of therapeutic efficacy, intravenously administered peptide-DM1 conjugates (DM1-Bdd) improved overall survival in mice with BC compared to conventional intravesical MIT (Figure 4h). It was also more effective compared to the same treatment given by intravesical (Figure 4f-i). The improved efficacy was expected given that animals did not need to urinate immediately after intravenous injection (Figure 4a). Accelerating renal clearance could reduce the off-target toxicity of the drug. DM1-Bdd did not induce unwanted toxicity (Figure 6). In contrast, animals treated with DM1 or CIS showed evidence of liver and kidney damage. The flexibility of the UDD approach to reduce drug-induced toxicity was also demonstrated by using aldox-Bdd for BC treatment. Unlike free DOX, which caused death, improved survival was observed in those animals treated with aldox-Bdd (Figure 9). However, compared to DM1-Bdd, aldox-Bdd treatment only delayed cancer progression but did not eliminate tumors in individual animals. The result was not unexpected, as DM1 is more potent than DOX (Figure 3c).

[0168] ITC is a localized treatment that targets tumors only in the bladder, as the drug solution cannot reach the upper urinary tract. DM1-Bdd is administered by intravenous injection. This should allow the drug to flow throughout the URS, along with rapid renal clearance. DM1-Bdd has been applied to treat kidney cancer, where it has been found that DM1-Bdd offers a significant survival benefit compared to free drug. In fact, approximately 50% of the animals lack gross or histological evidence of tumors one week after completion of the treatment course. Thus, intravenous drug administration would allow for more comprehensive targeting of the URS when used to treat BC, as tumors may extend, migrate, or recur throughout the urothelium, including the renal pelvis and ureter, where 8-12% of urothelial cancers originate. Currently, when treating patients with renal pelvis or ureter tumors, surgeons often have no choice but to remove the entire kidney and ureter to prevent disease recurrence for URS tumors (even if the tumor is noninvasive). DM1-Bdd may potentially be a kidney-sparing treatment option for patients with upper tract urothelial carcinoma.

[0169] A drawback of ITC is low patient compliance (16–30%). Patients undergoing ITC require weekly catheter insertion in a hospital / clinic by trained personnel. In contrast, intravenous DM1-Bdd treatment is noninvasive, which avoids complications associated with catheter procedures and can improve patients' quality of life and compliance. Lifelong management of BC is expensive and its high recurrence rate requires repeated treatments. Systemic administration of chemotherapeutic agents such as DM1-Bdd would likely reduce hospitalization costs.

[0170] Overall, a UDD approach was developed that can provide comprehensive treatment by delivering the drug throughout the URS, minimizing non-specific accumulation in other organs, as a more effective alternative to ITC. The developed DM1-Bdd is clinically translatable. The FDA has approved many peptides for treating different cancers. The DM1 used is an active pharmacophore already used in antibody-drug conjugates, such as Herceptin-DM1 (T-DM1), for breast cancer treatment.

[0171] Incorporation by Reference All publications and patents mentioned herein are herein incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0172] Equivalent Although specific embodiments of the present disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the present disclosure will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the present disclosure should be determined by reference to the claims, their full scope of equivalents, and the specification, together with such variations.

Claims

1. A compound represented by the following formula (I): 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof, wherein the peptide is a peptide that targets renal clearance.

2. The renal clearance targeting peptide comprises the sequence: 【Chemistry 2】 During the ceremony, one of X, Y, and Z is a β-amino acid residue; two of X, Y, and Z are independently an α-amino acid residue each having at least one side chain containing a carboxylic acid group; each α-amino acid residue may independently be of D or L stereochemistry; The compound according to claim 1, wherein m is 2 to 10.

3. The compound of claim 1 or 2, wherein the peptide has a zeta potential of about -30 mV to about +20 mV at physiological pH.

4. The compound of claim 3, wherein the peptide has a zeta potential of about -20 mV to about 0 mV at physiological pH.

5. The compound of claim 4, wherein the peptide has a zeta potential of about -5 mV to about 0 mV at physiological pH.

6. 6. The compound of any one of claims 2 to 5, wherein the linker comprises one or more groups selected from amide, imide, thiourea, thioether, disulfide, alkyl, aryl, polyether, hydrazone, ester, carbonate, ketal, and silyl ether.

7. The compound of any one of claims 2 to 6, wherein the active moiety is a therapeutic agent or an imaging agent.

8. The renal clearance targeting peptide comprises the sequence: 【Chemistry 3】 During the ceremony, one of X, Y, and Z is a β-amino acid residue; two of X, Y, and Z are independently an α-amino acid residue each having at least one side chain containing a carboxylic acid group; each α-amino acid residue may independently be of D or L stereochemistry; m is 2 to 10; the linker comprises one or more groups selected from amide, imide, thiourea, thioether, disulfide, alkyl, aryl, polyether, hydrazone, ester, carbonate, ketal, and silyl ether; The compound of any one of claims 1 to 7, wherein the active moiety is a therapeutic agent or an imaging agent.

9. The compound according to any one of claims 1 to 8, represented by the following formula (IA): 【Chemistry 4】 or a pharma- ceutically acceptable salt thereof.

10. The compound according to any one of claims 2 to 9, wherein the β-amino acid residue does not contain an ionizable side chain.

11. The compound according to any one of claims 2 to 10, wherein the β-amino acid residue is a β-alanine residue.

12. The compound according to any one of claims 2 to 11, wherein X is a β-alanine residue.

13. The compound of any one of claims 2 to 12, wherein each α-amino acid residue is independently selected from an aspartic acid residue and a glutamic acid residue.

14. The compound according to any one of claims 2 to 12, wherein at least one α-amino acid residue is a non-naturally occurring amino acid residue.

15. The compound of claim 14, wherein the non-natural α-amino acid residue has at least two side chain carboxylic acid groups.

16. 16. The compound of claim 15, wherein the unnatural α-amino acid residue is selected from 2-aminoethane-1,1,2-tricarboxylic acid residue and 2-aminopropane-1,2,3-tricarboxylic acid residue.

17. The compound according to any one of claims 2 to 16, wherein each Y and Z is an aspartic acid residue.

18. The compound according to any one of claims 2 to 16, wherein each Y and Z is a D-aspartic acid residue.

19. 3. The compound of claim 2, wherein X, Y, and Z are each independently selected from a β-alanine residue, an aspartic acid residue, and a glutamic acid residue.

20. The compound according to any one of claims 2 to 19, wherein m is 4.

21. The linker is 【Chemistry 5】 The compound of any one of claims 2 to 20, comprising a group selected from:

22. 22. The compound of any one of claims 2 to 21, wherein the linker comprises a group derived from N-succinimidyl 3-(2-pyridyldithio)propionate or succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

23. The compound of any one of claims 1 to 22, wherein the active moiety is a therapeutic agent.

24. 24. The compound of claim 23, wherein the therapeutic agent is selected from an anti-cancer agent, an antibiotic, an agent for treating overactive bladder, an agent for treating urinary incontinence, an agent for treating interstitial cystitis, and an agent for treating kidney stones.

25. The therapeutic agent is 13-cis-retinoic acid, 2-chlorodeoxyadenosine, 5-azacytidine, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, actinomycin-D, adriamycin, aldesleukin, alemtuzumab, alitretinoin, all-trans retinoic acid, alpha interferon, altretamine, amethopterin, amifostine, anagrelide, anastrozole, arabinosylcytosine, arsenic trioxide, amsacrine, aminocamptothecin, aminoglutethimide, asparaginase, azacytidine , Bacillus Calmette-Guerin (BCG), bendamustine, bevacizumab, bexarotene, bicalutamide, bortezomib, bleomycin, busulfan, leucovorin calcium, citrovorum factor, capecitabine, canertinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, cortisone, cyclophosphamide, cytarabine, darbepoetin alfa, dasatinib, daunomycin, decitabine, denileukin diftitox, dexamethasone, dexasone, dexrazoxane, dactinomycin, dau Norubicin, decarbazine, docetaxel, doxorubicin, doxil, aldoxorubicin, doxifluridine, edrecolomab, eniluracil, epirubicin, epoetin alfa, erlotinib, everolimus, exemestane, estramustine, etoposide, filgrastim, fluoxymesterone, fulvestrant, flavopiridol, floxuridine, fludarabine, fluorouracil, flutamide, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin, granulocyte-colony stimulating factor, granulocyte-macrophage - colony stimulating factors, hexamethylmelamine, hydrocortisone hydroxyurea, ibritumomab, ibritumomab tiuxetan, interferon alpha, interleukin-2, interleukin-11, isotretinoin, ixabepilone, idarubicin, imatinib mesylate, ifosfamide, irinotecan, lapatinib, lenalidomide, letrozole, leucovorin, leuprolide, liposomal Ara-C, lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine, mertansine, mesna, methotrexate,Methylprednisolone, mitomycin C, mitotane, mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pemetrexed, panitumumab, PEG-interferon, pegaspargase, pegfilgrastim, PEG-L-asparaginase, pentostatin, plicamycin, prednisolone, prednisone, procarbazine, raloxifene, rituximab, romiplostim, ralitrexed, sapacitabine, sargramostim 24. The compound of claim 23, selected from satraplatin, sorafenib, sunitinib, semustine, streptozocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus, temozolamide, teniposide, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, trastuzumab emtansine, tretinoin, trimitrexate, arubicin, vincristine, vinblastine, vindestine, vinorelbine, vorinostat, and zoledronic acid.

26. 24. The compound of claim 23, wherein the therapeutic agent is an anti-cancer agent.

27. 27. The compound of claim 26, wherein the anticancer drug is selected from mertansine, doxorubicin, dasatinib, cisplatin, mitomycin, gemcitabine, and paclitaxel.

28. X is a β-alanine residue, The compound of claim 2, wherein Y and Z are D-aspartic acid residues.

29. X is a β-alanine residue, Y and Z are D-aspartic acid residues; The compound of claim 2 , wherein m is 4.

30. X is a β-alanine residue; Y and Z are D-aspartic acid residues; m is 4; the linker comprises a disulfide group; The compound of claim 2 , wherein the active moiety is mertansine.

31. The compound is 【Chemistry 6】 or a pharma- ceutically acceptable salt thereof, wherein B is a β-alanine residue and D is an aspartic acid residue in the D or L configuration.

32. The compound is 【Chemistry 7】 or a pharma- ceutically acceptable salt thereof, wherein B is a β-alanine residue and D is an aspartic acid residue in the D or L configuration.

33. A pharmaceutical composition comprising a compound according to any one of claims 1 to 32.

34. 34. The pharmaceutical composition of claim 33, wherein the composition is formulated for intravenous administration.

35. 35. The pharmaceutical composition of claim 33 or claim 34 for treating cancer, urinary tract infections, overactive bladder, urinary incontinence, interstitial cystitis, or kidney stones.

36. 35. The pharmaceutical composition of claim 33 or claim 34 for treating cancer.

37. 37. The pharmaceutical composition of claim 36, wherein the cancer is a kidney or urinary tract cancer.

38. 38. The pharmaceutical composition of claim 37, wherein the cancer is bladder cancer.

39. 39. The pharmaceutical composition of claim 38, wherein the bladder cancer is non-muscle invasive bladder cancer.

40. 40. The pharmaceutical composition of claim 39, wherein the bladder cancer is urothelial cancer.

41. A pharmaceutical composition according to any one of claims 35 to 40, administered intravenously.

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