Cationic lipids for covalently modifying peptides

Abstract

Provided herein are compounds (e.g., peptides) comprising a cationic alkyl moiety, compositions comprising the compounds, and methods of using the compounds, for example, in the treatment of a disease, disorder, or condition.

Description

Related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 493,290, filed March 30, 2023. The entire teachings of the above application are incorporated herein by reference.

[0002] By referencing and incorporating XML materials This application incorporates the sequence list contained in the following Extensible Markup Language (XML) file, which was filed concurrently with it: a) File name: 6165.1024-001_SL.xml; created on March 29, 2024, size is 67,928 bytes.

[0003] Government support This invention was carried out with government support under approval number HL156295 granted by the National Institutes of Health (NIH). The government owns certain rights to this invention. Background Technology

[0004] Alkyl chain modification of natural peptides is one of several methods used to enhance pharmacokinetic properties and / or increase the in vivo half-life of biounstable peptides (see, for example, Menacho-Melgar et al., *Journal of Controlled Release*. 2019 Feb 10;295: 1-12). To date, most modified peptides utilize fatty acids or alkyl chains without cationic moieties, as the inherent toxicity of cationic alkyl or lipid compounds is well-documented (see, for example, Cui et al., *The Royal Society of Chemistry*. 2018. pp. 473-479). It is well known that using cationic lipid-modified peptide drugs increases their toxicity, often resulting in a narrow therapeutic window with toxic doses close to those desired for biological activity. Therefore, this approach is generally considered undesirable. Furthermore, peptide modification can lead to loss of peptide biological activity or reduced solubility.

[0005] Despite these drawbacks, the use of cationic alkyl or lipid modifications can potentially improve in vivo half-life by interacting with or binding to cell membranes and proteins. However, the same properties can also lead to increased toxicity due to hemolysis caused by tight binding to cell membrane components, caspase-induced apoptosis, mitochondrial dysfunction due to decreased membrane potential, elevated levels of reactive oxygen species (ROS), cell arrest in S phase, and / or other unknown mechanisms leading to toxicity. Therefore, the use of cationic alkyl or lipid moieties in peptide modification is limited because the potential toxicity may reduce the therapeutic index. The toxicity of cationic lipids is closely related to the presence of several amino head groups, which have been shown to kill up to 50% of cells in cultures at concentrations of µM or higher. Therefore, the application of cationic lipids is limited to killing cells with membranes that have an abnormally high negative charge compared to normal mammalian cells, such as cancer cells (see, for example, Cui et al., Royal Society of Chemistry. 2018. pp. 473-479) and bacterial cells (see, for example, Małuch et al., International Journal of Molecular Sciences 2020, 21, 8944). Summary of the Invention

[0006] This article provides compounds comprising a cationic alkyl moiety of formula (I): J-(CH2)x(CO)-(A)y-(B)z- (I), in: J is either HOOC or CH3; x is 10⁻¹⁶; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-aminobutyric acid (γAbu), γ-linked glutamic acid (γE) and α-linked glutamic acid (E); y is 2-4; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2-4; Wherein -(B)z- contains no more than 2 Dab residues, and wherein the Dap or Dab residues are linked by an α-amino group.

[0007] This article also provides the conjugated peptide of formula (II): CH3(CH2)x(CO)-(A)y-(B)z-peptide (II) in: x is 10⁻¹⁶; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-aminobutyric acid (γAbu), γ-linked glutamic acid (γE) and α-linked glutamic acid (E); y is 2-4; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2-4; in: -(B)z- contains no more than 2 Dab residues, wherein the Dap or Dab residues are linked by an α-amino group; CH3(CH2)x(CO)-(A)y-(B)z- is covalently linked to the N-terminus of the peptide or to one of the amino groups on the side chain of the peptide.

[0008] In some embodiments, at an equivalent bolus dose, the bioactivity of the conjugated peptide is equal to or higher than that of the unmodified peptide; at the same time point following bolus administration of an equivalent dose, the blood level of the conjugated peptide is equal to or higher than that of the unconjugated peptide; or a combination thereof.

[0009] This document provides compositions comprising compounds or conjugated peptides disclosed herein for use in preparing pharmaceutical compositions.

[0010] In addition, this document provides compounds, conjugated peptides, and compositions thereof for treating diseases or conditions in subjects in need.

[0011] This document also provides compositions comprising compounds or conjugated peptides of the present disclosure and one or more pharmaceutically acceptable carriers or excipients. Attached Figure Description

[0012] The foregoing will become apparent from the following more specific description of the exemplary embodiments as shown in the accompanying drawings, in which the same reference numerals refer to the same parts in different views. The drawings are not necessarily drawn to scale, but are intended to illustrate the embodiments.

[0013] Figure 1A-1B PK / PD curves of natural CNP (SEQ ID NO: 32), SEQ ID NO: 31, and SEQ ID NO: 29 (0.45, 0.33, and 0.33 µmol / kg, respectively) administered via single subcutaneous (SC) injection at 1.0 mg / kg in mice. Error bars represent the standard error of the mean. Figure 1APlasma CNP levels in CD-1 mice following subcutaneous administration of 1.0 mg / kg of buffer containing native CNP (SEQ ID NO: 32), SEQ ID NO: 31, and SEQ ID NO: 29 (0.45, 0.33, and 0.33 µmol / kg, respectively) [mean (SEM); n = 5]. Baseline CNP levels before administration were 4.5 (1.4) ng / mL in all groups [mean (SD); n = 15]. Sequences 31 and 29 not only increased baseline plasma CNP levels by more than 5-fold and 10-fold, respectively, but also showed a sustained increase in CNP levels compared to native CNP. Plasma CNP in CD-1 mice was measured using a CNP ELISA kit from Phoenix Pharmaceuticals (EKE-012-03, Burlingame, CA). Figure 1B Plasma cGMP in CD-1 mice following subcutaneous administration of 1.0 mg / kg of native CNP (SEQ ID NO: 32), SEQ ID NO: 31, and SEQ ID NO: 29. Baseline plasma cGMP levels were 13.9 (5.7) pmol / mL in all groups [mean (SD); n = 15]. SEQ ID NO: 31 and 29 not only increased baseline plasma cGMP levels by more than 10-fold but also showed a sustained increase in cGMP levels compared to native CNP. Plasma cGMP was measured using an Abcam cGMP ELISA kit (ab133052, Waltham, MA).

[0014] Figure 2A-2B Single and repeated administration of cationic alkyl-modified C-type natriuretic peptides increased the survival rate of LPS-induced sepsis and ALI animal models. Figure 2ASepsis induction: Male C57BL / 6J mice (n = 10 per group; 3 groups) were injected intraperitoneally with LPS (15 mg / kg) and treated with various test products, including cationic alkyl-modified CNPs (SEQ ID NO: 31 and 29; 0.3 mg / kg (0.1 µmol / kg) SC). Control groups received LPS treatment without any test products. Test products were administered immediately after LPS administration (marked by vertical dashed lines). Survival was monitored every 2 hours over 8–56 hours, after which surviving mice were euthanized under isoflurane anesthesia. Statistical analysis was based on the Gehan-Breslow-Wilcoxon test (n = 10, 10, and 10; controls, SEQ ID NO: 31, and SEQ ID NO: 29) using GraphPad Prism. P<0.01, P<0.05, compared to the control group. Figure 2B ALI induction: Male C57BL / 6J mice (n = 6 per group; 3 groups) were injected with LPS (20 mg / kg) via intraperitoneal injection and treated with various test products, including cationic alkyl-modified CNPs (SEQ ID NO: 31 and 29; 0.3 mg / kg (0.1 µmol / kg) IT). The control group received LPS treatment without any test product. Test products were administered immediately after LPS administration and repeated every 24 hours for a total of 3 bolus doses (marked by vertical dashed lines). Survival was monitored every 8 hours for 72 hours, after which surviving mice were euthanized under isoflurane anesthesia. Statistical analysis was based on the Gihen-Breslow-Wilkerson test using GraphPad Prism (n = 6, 6, and 6; control, SEQ ID NO: 31, and SEQ ID NO: 29). P<0.01, P<0.05, compared to the control group.

[0015] Figures 3A-3D Bulk injection of cationic alkyl-modified C-type natriuretic peptide inhibited lung injury and indicated the regression of ALI / ARDS. Neutrophil count and myeloid-derived protein (S100A8 / A9) expression are elevated in several types of inflammatory lung diseases. MPO+ cells serve as a direct measure of neutrophil presence. Therefore, in ALI animal models ( Figure 3A The expression of S100A8 and S100A9 ( Figure 3B ) and the presence of MPO+ cells in the lungs ( Figure 3C(A) and (D) are common markers of inflammation. These decreases indicate the regression of ALI / ARDS, which is consistent with the observed increase in survival (see Figure 2). Figure 3A The diagram illustrates a procedure for administering LPS (0.05 mg / kg IT) to C57BL / 6J mice, followed by treatment with various test products, including sivelestat (150 mg / kg), an inhibitor of human neutrophil elastase, as a positive control administered via IP, and cationic alkyl-modified CNP (SEQ ID NO: 31, 29, and 30; 0.3 mg / kg (0.1 µmol / kg) SC). Test products were administered immediately after LPS injection. Additionally, a normal control (NC) group without LPS administration and a control group receiving only LPS without any test products were included. Mice were euthanized under isoflurane anesthesia 24 hours later, and their lungs were harvested for analysis. This procedure was repeated due to variations in lung treatment techniques between studies. Figure 3B Lung tissue was chopped and processed in Tri-Reagent to measure gene expression levels of S100A8 and S100A9 using qRT-PCR analysis with a cDNA synthesis kit (Qiagen; Venlo, the Netherlands). Statistical analysis was based on Dunnett's test performed using GraphPad (n = 5, 8, 8, 8, 8, 8 and 8; NC, Control, PC, A [SEQ ID NO: 31], B [SEQ ID NO: 29], Control, D [SEQ ID NO: 30]). P<0.001, P<0.01, P<0.05, relative to each corresponding control group. Figure 3C (D) Lung tissue was fixed, paraffin-embedded, and sectioned. Immunohistochemical staining was performed on the sections, and the number of myeloperoxidase-positive (MPO+) cells was quantified by field of view. Statistical analysis was based on the Dunnett test performed using GraphPad (n = 5, 8, 8, 8, 8, 8 and 8; NC, Control, PC, A [SEQ ID NO: 31], B [SEQ ID NO: 29]; Control, D [SEQ ID NO: 30]). P<0.001, relative to each corresponding control group.

[0016] Figures 4A-4C Cationic alkyl-modified CNP derivatives reduced neutrophil infiltration in the lungs. ALI and ARDS are associated with increased cellularity, particularly neutrophils, in BALF. To assess the regression of ALI / ARDS in animal models, cell number was measured ( Figure 4B ) and total protein level ( Figure 4C These markers act as markers of neutrophils. A decrease in these markers indicates regression of ALI / ARDS. Figure 4A The diagram illustrates a procedure for administering LPS (0.05 mg / kg IT) to mice, followed by treatment with various test products, including cevelexat (150 mg / kg), an inhibitor of human neutrophil elastase, as a positive control (IP injection), and cationic alkyl-modified CNP (SEQ ID NO: 31, 29, and 30; 0.3 mg / kg (0.1 µmol / kg) SC). Test products were administered immediately after LPS injection. Additionally, a normal control (NC) group without LPS administration and a control group receiving only LPS without any test products were included. Mice were euthanized under isoflurane anesthesia 24 hours later, and BALF was collected. Statistical analysis was based on the Dunnett test performed using GraphPad (n = 5, 8, 8, 8, 8, 8, and 8; NC, Control, PC, A [SEQ ID NO: 31], B [SEQ ID NO: 29]; Control, D [SEQ ID NO: 30]). P<0.001, relative to each corresponding control group.

[0017] Figures 5A-5E The role of cationic alkyl-modified CNP derivatives in the inflammatory state of acute exacerbations of idiopathic pulmonary fibrosis (IPF-AE). Error bars indicate the standard error of the mean. Increased inflammatory state of the lungs is observed in IPF-AE, particularly increases in neutrophils, MCP1, and IL-6. Their reduction indicates the resolution of IPF-AE. Figure 5AThe diagram illustrates the procedure for treating C57BL / 6J mice with bleomycin (Bleo; 1.0 mg / kg IT). After 3 weeks, the mice were treated with LPS (0.025 mg / kg IT) and then with SEQ ID NO: 29 or 31 at 0.3 mg / kg (0.1 µmol / kg) SC on the day before, the day of, and the day after LPS treatment, as shown in the diagram. Additionally, a normal control (NC) group without LPS / Bleo treatment, a Bleeo group without LPS treatment, and a control group without test product treatment were also included. On the last day, the mice were sacrificed under isoflurane anesthesia, and lung tissue was then collected and weighed. Figure 5B An increased lung weight to body weight ratio is a parameter of lung injury. Figure 5C The group treated with the cationic alkyl-modified CNP derivatives showed a significant reduction in the upregulation of the neutrophil marker MPO. Figure 5D SEQ ID NO: 31 shows a significant decrease in another common inflammatory marker, MCP1. Figure 5E Compared with the NC group, IL-6 levels did not show a significant difference, indicating a reduction in inflammation compared to the control group. Statistical analysis was based on Student's t-test performed using GraphPad (n = 5, 5, 8, 8, 8; NC, Bleo, Control, B [SEQ ID NO: 29], A [SEQ ID NO: 31]). ##P<0.01 and #P<0.05, relative to the control.

[0018] Figures 6A-6B Repeated subcutaneous administration of cationic alkyl-modified CNP derivatives demonstrated significant antitumor activity in an orthotopic breast cancer mouse model using E0771 cells. Error bars represent the standard error of the mean. Figure 6A Tumor growth kinetics in 6-week-old female C57BL / 6J mice (n = 10 per group) inoculated with E0771 breast cancer cells (250,000 cells / mouse). Starting on day 4 post-inoculation, cationic alkyl-modified CNP derivatives (SEQ ID NO: 29, 30, and 31) were administered subcutaneously at a bolus dose of 0.3 mg / kg (0.1 µmol / kg) once daily for 5 days (5 days administration, 2 days rest), for 3 cycles; dose days are indicated by grid lines. Baseline tumor growth kinetics were established by administering the same dose to a control group receiving only buffer. Figure 6BAt the end of the study, the group treated with cationic alkyl-modified CNPs showed a significant reduction in tumor volume compared to the control group. However, cationic alkyl sequences without CNPs (SEQ ID NO: 14) were also tested in this model, but did not show a significant reduction in tumor volume. Therefore, it can be concluded that the conjugation of CNPs is crucial for the observed antitumor activity. Statistical analysis was based on the Dunnett test performed using GraphPad (n = 10, 10, 10, and 10; control, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31). P<0.0001, compared to the control group.

[0019] Figure 7 The binding of CNP derivatives to both natriuretic peptide receptor B (NPRB) and natriuretic peptide receptor C (NPRC). Error bars represent the standard error of the mean. CNP-based probes only (5 nM CNP-F) The CNP probe exhibits rapid rotation and induces a low fluorescence polarization (FP) signal. When human NPRB or NPRC (50 nM) is added, the CNP probe binds to these receptors and induces slow rotation and a high FP signal. In the presence of NPRA, no change in FP signal was detected, indicating that the CNP probe did not bind to NPRA. In the presence of SEQ ID. NO. 31, the low FP signal demonstrates binding to both NPRB and NPRC.

[0020] Figures 8A-8B Cationic alkyl-modified CNP derivatives, used alone or in combination with pirfenidone, showed reduced fibrosis in a mouse model of idiopathic pulmonary fibrosis (IPF). Figure 8A The diagram illustrates the procedure for inducing pulmonary fibrosis, in which male C57BL / 6J mice (6 weeks old) received bleomycin (Bleo; 1.0 mg / kg) via IT and, 7 days later, were administered SEQ ID NO: 31 via SC at 0.3 mg / kg once daily on weekdays and / or pirfenidone (100 mg / kg, orally) once daily. Additionally, this study included a normal control group (NC; n = 3) without Bleeo and a Bleeo control group (n = 7) without treatment with the test product. On day 21 following Bleeo administration, the mice were sacrificed under isoflurane anesthesia, and their lung tissue was harvested. Figure 8BPartial lung tissue was fixed in 4% PFA and stained with Azan. Alveolar area (inversely proportional to the degree of fibrosis) was measured in a blinded environment using Image J. Error bars indicate the standard error of the mean. Statistical analysis was performed on GraphPad Prism using Studden's t-test (n = 3, 7, 7, 7, and 7); NC, Bleo control, SEQ ID NO: 31, Pir, and the combination (SEQ ID NO: 31 and pirfenidone) were also included. P < 0.001, ns = not significant relative to NC. Groups treated with SEQ ID NO: 31 alone or in combination with pirfenidone showed significantly larger alveolar areas, demonstrating the presence of healthy tissue and reduced fibrosis.

[0021] Figure 9 Repeated subcutaneous administration of cationic alkyl-modified CNP derivatives as monotherapy or in combination with immune checkpoint inhibitors significantly reduced tumor volume in an orthotopic breast cancer mouse model using E0771 cells. Error bars represent the standard error of the mean. Tumor growth kinetics in 6-week-old female C57BL / 6J mice (n = 7–8 per group) orthotopically seeded with E0771 breast cancer cells (250,000 cells / mouse) in the mammary gland. Starting on day 4 post-inoculation, anti-PD1 antibody (aPD1) was administered intraperitoneally at 5 mg / kg twice weekly for 2 cycles; SEQ ID NO: 31 was administered subcutaneously at a bolus dose of 0.3 mg / kg once daily for 5 days (5 days of administration followed by 2 days of rest) for 3 cycles; or a combination of aPD1 (5 mg / kg IP, twice weekly; 2 cycles) and SEQ ID NO: 31 (0.3 mg / kg SC, once daily, 5 days of administration followed by 2 days of rest; 3 cycles) was administered; the number of days for SEQ ID NO: 31 dosing is indicated by dashed or grid lines. The control group, which received only buffer, was administered the same way as SEQ ID NO: 31 to establish baseline tumor growth kinetics. At the end of the study, the group treated with SEQ ID NO: 31 showed antiproliferative activity or a significant reduction in tumor volume compared to the control group and the group treated with the immune checkpoint inhibitor aPD1 alone. Statistical analysis was based on the Dunnett test performed using GraphPad (n = 8, 7, 8, and 7; control, aPD1, SEQ ID NO: 31, and combination (aPD1 and SEQ ID NO: 31)). P<0.05, P<0.0001, compared to the control group.

[0022] Figures 10A-10B The combination of radiation, immune checkpoint inhibitors, and repeated subcutaneous administration of a cationic alkyl-modified CNP derivative reduced the incidence of bone metastasis and significantly improved overall survival in a mouse model of orthotopic bone metastasis using breast cancer E0771 cells. Error bars indicate the standard error of the mean. Figure 10A The figure shows a diagram illustrating the procedures for in situ implantation of E0771 breast cancer cells (250,000 cells / mouse, in RPMI 1640 medium) into the left mammary gland of female C57BL / 6J mice (6 weeks old) and in situ implantation of E0771 mouse breast cancer cells (500,000 cells / mouse, in 50% Matrigel) into the femur. The corresponding timeline for each treatment applied throughout this study is also shown in the figure. Starting from tumor implantation, mice were administered SC (0.3 mg / kg / 0.1 µmol / kg in buffer, 15 mM succinate, 4% (w / v) D-mannitol, 10 mM hydroxypropyl-β-cyclodextrin, pH 4.4) to the SEQ ID NO: 31 treatment group (2, 3, 6, and 7) on days 5–9, 12–16, 19–23, and 26–29; IP (5 mg / kg) was administered to the aPD1 treatment group (3, 5, and 7) on days 5, 7, 12, and 14; and the irradiation groups (4, 5, 6, and 7) received buffer and 5 Gy X-ray irradiation at the bone site on days 5, 8, and 12, in triplicate. Survival was monitored until day 33, after which the remaining mice were euthanized as planned. Tumor size was measured using calipers at this time. Figure 10B The survival probabilities of groups 1 through 7 are shown. All groups were significantly different from the control group; therefore, group 5 (radiation + aPD1) was statistically analyzed using GraphPadPrism based on the log-rank (Mantel-Cox) test. With the addition of SEQ ID NO: 31, all combinations of the three treatments showed a significant improvement in survival (…). P<0.01). Furthermore, compared with 1 out of 5 mice in group 4 (radiation only) and 1 out of 5 mice in group 5 (radiation + aPD1), 5 out of 6 mice in group 6 (SEQ ID NO: 31 + radiation) and 6 out of 6 mice in group 7 (SEQ ID NO: 31 + radiation + aPD1) had no tumors in their bones, demonstrating that the addition of SEQ ID NO: 31 reduced the incidence of metastasis and cancer burden.

[0023] Figure 11A-11CAmputation combined with repeated subcutaneous administration of a cationic alkyl-modified CNP derivative significantly reduced the incidence of lung metastases in a mouse model of orthotopic lung metastases using osteosarcoma LM8 cells compared to amputation alone. Error bars indicate the standard error of the mean. Figure 11A The diagram illustrates the procedure for in situ implantation of LM8 osteosarcoma cells (1,000,000 cells / mouse) into the femur of male CH3 / He mice (7 weeks old). The corresponding treatment schedule for SEQ ID NO: 31 and amputation is also shown in the diagram. From tumor implantation onwards, the SEQ ID NO: 31 treatment group SC was administered 0.3 mg / kg / 0.1 µmol / kg in buffer on days 4–8, 11–15, 18–22, and 25–26. On day 7 post-inoculation, all mice underwent amputation to remove the primary tumor. Alternatively, the amputation control group also received buffer on the same day as SEQ ID NO: 31 administration. Mice were sacrificed on day 34, and lung tissue was harvested. Figure 11B and 11C Lung tissue was immersed in 4% paraformaldehyde, embedded in paraffin, and sections were stained with hematoxylin and eosin (H&E). Lung images were observed using a high-resolution microscope (Keyence, Tokyo, Japan, #BZ-X700), and lung metastases were assessed by direct counting of present metastatic lung nodules. The group undergoing amputation (SEQ ID NO: 31) had a significantly reduced number of metastatic nodules in the lungs compared to the group undergoing amputation but without additional treatment. P < 0.01. Outliers were identified using the ROUT test (Q = 1%). All statistical analyses were based on the Dunnett test performed using GraphPad (n = 7 and 7; control (amputation), combination (amputation and SEQ ID NO: 31)). P<0.01, compared to the control group.

[0024] Figure 12A-12B Repeated subcutaneous administration of cationic alkyl-modified CNP derivatives as monotherapy or in combination with immune checkpoint inhibitors significantly reduced tumor volume in a mouse model of subcutaneous colon cancer using MC38 cells. Error bars represent the standard error of the mean. Figure 12A A graph showing the dosage schedule for administering an immune checkpoint inhibitor (anti-tigit antibody) and SEQ ID NO: 31. Figure 12BTumor growth kinetics in 6-week-old male C57BL / 6J mice (n = 8–9 per group) subcutaneously inoculated with MC38 colon cancer cells (1,000,000 cells / mouse) in the right flank. Starting on day 4 post-inoculation, anti-tigit antibody was administered intraperitoneally at 5 mg / kg twice weekly for 2 cycles; SEQ ID NO: 31 was administered subcutaneously at a bolus dose of 0.3 mg / kg (0.1 µmol / kg) once daily for 5 days (5 days administration, 2 days rest) for 3 cycles; or a combination of anti-tigit antibody (5 mg / kg IP, twice weekly; 2 cycles) and SEQ ID NO: 31 (0.3 mg / kg SC, once daily, 5 days administration, 2 days rest; 3 cycles). Baseline tumor growth kinetics were established by administering the same medication as SEQ ID NO: 31 to a control group receiving only buffer. Compared with the control group, the group treated with SEQ ID NO: 31 showed a significant reduction in tumor volume and cancer burden, and the combination group (anti-tigit antibody + SEQ ID NO: 3) had a superior anti-tumor effect compared with anti-tigit antibody as a monotherapy. Statistical analysis was based on the Dunnett test performed using GraphPad (n = 9, 8, 8, and 8; control, anti-tigit antibody, SEQ ID NO: 31, combination (anti-tigit antibody and SEQ ID NO: 31)). P<0.05, compared to the anti-tigit antibody monotherapy group.

[0025] Figure 13 Based on the significant inhibition of baseline cyclic adenosine monophosphate (cAMP) levels, HeLa cells treated with cationic alkyl-modified CNP derivatives showed binding to NPR-C. In this study, HeLa cells were cultured at 37°C, 100% humidity, and 5% CO2 in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS. Cells were harvested and cultured at 10... 7Cells were suspended in ENGS at a concentration of 10 cells / mL, and 5 μL of cell suspension was added to each well of a 96-well low-adhesion plate. Cells (n = 4 wells) were then treated with 5 μL of SEQ ID NO: 31 (final concentration 10 µg / mL) in ENGS containing 1 mM IBMX for 10 min. cAMP levels were assessed using a cAMP assay kit (PerkinElmer, Waltham, MA, USA, #62AM4PEB) and a PerkinElmer plate reader, according to the manufacturer's protocol. Statistical analysis of untreated control wells (n = 4 wells) was performed using GraphPadPrism. P<0.05. The NPR-C receptor is involved in inhibiting adenylate cyclase activity, leading to a reduction in cyclic adenosine monophosphate (cAMP) production. This study concludes that baseline cAMP levels were altered (inhibited) in HeLa cells (known to express NPR-C) treated with SEQ ID NO: 31, indicating binding to NPR-C. Detailed Implementation

[0026] This disclosure provides novel compositions comprising compounds containing a single cationic alkyl chain moiety, which can be used to modify bioactive molecules (such as peptides) and exhibit surprisingly and significantly reduced toxicity upon in vivo administration (e.g., no toxicity or ataxia of the specific modifiers disclosed herein was observed at 10 µmol / kg in rats). The novel compositions comprise compounds containing a single alkyl chain linked to a chain of 2-4 cationic amino acid residues selected from diaminopropionic acid (Dap) and diaminobutyric acid (Dab), wherein no more than 2 Dab residues are present in the chain. The non-cationic linker comprises 2-4 residues independently selected from 2-aminoethoxy-2-ethoxyacetic acid (Aeea), γ-aminobutyric acid (γAbu), γ-linked glutamic acid (γE), and glutamic acid (E). The cationic alkyl chains disclosed herein can be linked to peptides to enhance their pharmacokinetic and / or pharmacodynamic activity (see, for example, Figure 1), for example, relative to natural, unmodified peptides, without the risk of additional toxicity (e.g., at bolus doses of 3.0 µmol / kg or less, Table 1). As illustrated in this specification, cationic alkyl chain-modified peptides, such as C-type natriuretic peptides (CNPs) (or derivatives thereof), can be used to increase survival rates in sepsis and / or acute respiratory distress syndrome / acute lung injury and / or pulmonary fibrosis (see Figures 2–5 and 8 and Examples 5–9 and 12 herein). Additionally, cationic alkyl chain-modified CNPs (or derivatives thereof) can be used as therapeutic agents, for example, for the treatment of cancer (see Figures 6 and 9–12 and Examples 10 and 13–16), without reaching doses that would cause toxicity / ataxia. Furthermore, this specification also exemplifies cationic alkyl chain-modified atrial natriuretic peptides, B-type natriuretic peptides, and their derivatives.

[0027] Acute lung injury and acute respiratory distress syndrome Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinical conditions characterized by acute onset of arterial hypoxia. ALI is characterized by a PaO2 / FiO2 ratio of less than 300 Torr, and for more severe ARDS, this ratio is less than or equal to 200 Torr, accompanied by bilateral radiographic infiltrates and no evidence of left atrial hypertension (see, for example, Bernard, GR et al., *Journal of Critical Care*, 1994. 9(1): 72-81; Rubenfeld, GD et al., *New England Journal of Medicine*, 2005. 353(16): 1685-93; Brun-Buisson, C. et al., *Intensive Care Medicine*, 2004. 30(1): 51-61; and Phua, J. et al., *American Journal of Respiratory and Critical Care Medicine*). Respir Crit CareMed, 2009. 179(3): 220-7. As used herein, PaO2 refers to the partial pressure of oxygen in the arteries, and FiO2 is the fraction of oxygen in inhaled air (for reference, the FiO2 of indoor air is about 0.21, and the normal PaO2 / FiO2 is about 500 Torr). Both ALI and ARDS can lead to death or pulmonary fibrosis. ARDS is an overwhelming inflammatory response of the lungs to certain primary and secondary harmful stimuli, such as pneumonia (e.g., aseptic pneumonia, viral pneumonia, bacterial pneumonia), sepsis, aspiration, inhalation injury, drowning, and lung resection surgery (see, for example, Alam, N. et al., Ann ThoracSurg, 2007. 84(4): 1085-91). ARDS is characterized by the rapid onset of respiratory failure, requiring hospitalization and respiratory support in the intensive care unit (ICU). If a patient survives ALI / ARDS, their long-term quality of life is often adversely affected by lung scarring (see, for example, Rubenfeld, GD et al., *The New England Journal of Medicine*, 2005. 353(16): 1685–93; Dowdy, DW et al., *Critical Care Medicine*, 2006. 32(8): 1115–24). To date, no effective agents have been identified for the treatment of acute lung injury (ALI) and ARDS, and therefore there is a significant need for such agents.Previous clinical trials of agents for the treatment of ALI, including glucocorticoids, surfactants, N-acetylcysteine, inhaled nitric oxide, liposomal PGE1, ketoconazole, lisofylline, salbutamol, procysteine, activated protein C, and inhaled albuterol, have all failed (see, for example, Johnson ER and Matthay MA, J Aerosol Med Pulm Drug Deliv. 2010, 23(4):243-52). Medications for the treatment of ALI or ARDS in humans remain elusive to those skilled in the art. Supportive care for ALI includes oxygen therapy to maintain an arterial partial pressure of oxygen (PaO2) above 55 mmHg or an oxygen saturation (SaO2) above 88%, and fluid management. However, care must be taken not to provide excessive oxygen (i.e., oxygen levels should be below 60%) to avoid oxygen toxicity. Furthermore, this measure does not address the underlying alveolar edema (fluid within the alveoli filled with hemoglobin and inflammatory cells). Underlying alveolar edema in ALI or ARDS is the root cause of hypoxemia.

[0028] Lung or pulmonary fibrosis Pulmonary fibrosis (PF) is a progressive scarring of lung tissue caused by a variety of conditions, including infections (i.e., sepsis or pneumonia, which can be aseptic, viral, or bacterial), environmental factors (e.g., asbestos, silica, exposure to certain gases), exposure to ionizing radiation (e.g., radiotherapy used to treat chest tumors), chronic autoimmune conditions (e.g., lupus, rheumatoid arthritis), chronic inflammatory processes (e.g., sarcoidosis, Wegener's granulomatosis), or certain medications. Interstitial lung disease (ILD) is another encompassing term for PF and will be used synonymously with PF for the purposes of this specification. Idiopathic pulmonary fibrosis (IPF) is a type of PF of unknown etiology. PF, or IPF, is an incurable type of chronic cicatricial lung disease characterized by a progressive and irreversible decline in lung function, gradually leading to shortness of breath and dry cough, affecting 5 million people worldwide (see, for example, Raghu G, Collard HR, Egan JJ et al., (2011) American Journal of Respiratory and Critical Care Medicine. 183(6): 788-824). Associated risk factors include inhaled chemicals such as smoking, viral infections, or a family history of the condition. Other symptoms may include fatigue and abnormally large, dome-shaped nails and toenails (clumping). See, for example, the National Institutes of Health's health page on idiopathic pulmonary fibrosis and Wikipedia's page on idiopathic pulmonary fibrosis. Complications may include pulmonary hypertension, heart failure, pneumonia, or pulmonary embolism.

[0029] Therefore, there is a need for safer and more effective compositions for treating ALI and / or ARDS, as well as PF, which maintain or increase plasma levels of CNP therapeutic agents while avoiding cardiovascular side effects such as hypotension. There is also a need for long-half-lived natriuretic peptide derivatives for the formulation of medicaments for treating ALI and / or ARDS. This disclosure aims to meet these needs and provide additional related advantages.

[0030] Natriuretic peptides (NPs) and natriuretic peptide receptors (NPRs) Natriuretic peptides (NPs) are peptides that induce renal sodium excretion and lower blood pressure through vasodilation by binding to natriuretic transmembrane receptors containing intracellular guanylate cyclase domains. Upon binding, guanylate cyclase activity is activated, leading to increased blood and intracellular cGMP levels and the expression of various physiological activities. Several natriuretic peptides are well known in the art. C-type natriuretic peptide (CNP; GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 32]) acts via natriuretic peptide receptor B (NPRB) and natriuretic peptide receptor C (NPRC) (see, for example, Silver MA, *Curr. Opin. Nephrol. Hypertens.*, 2006, Vol. 15, 14-21; Yoshibayashi M. et al., *Eur. J. Endocrinol.*, 1996, Vol. 135, 265-268; Itoh H and Nakao K, *Nihon Rinsho*, 1997; 55: 1923-1936; Koller JK et al.). ,Science. 1991;252: 120-123; Suga S et al., Endocrinology. 1992; 130: 229-239; and Potter LR and Hunter T. Journal of Biol. Chem. 2001; 276:6057-6060.), while atrial natriuretic peptide (ANP; SLRRSSCFGGRMDRIGAQSGLGCNSFRY; [SEQ ID NO:44]), urodilantin (URO; TAPRSLRRSSCFGGRMDRIGAQSGLGCNSFRY [SEQ ID NO: 75]), a longer version of ANP and brain natriuretic peptide (BNP; SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH; [SEQ. IN. NO.

[48] ​​CNP, BNP, ANP, and URO are peptides that have the cyclic structure required for their activity, which is achieved by the presence of disulfide bonds. In addition to their vasodilatory effects and their ability to regulate blood pressure and vascular fluid through diuresis, NPs have a variety of physiological activities. For example, ANP has been reported to inhibit bacterial infection-induced inflammation and associated endothelial barrier dysfunction (see, for example, Xing J. et al., *Journal of Applied Physiology*, 2011, 110(1), 213-224). Furthermore, CNP, BNP, ANP, and URO all bind to NPRC (lacking guanylate cyclase activity) and are subject to clearance and degradation (see, for example, Koller KJ et al.). , Science. 1991; 252:120-123; Suga S et al. , Endocrinology. 1992; 130:229-239; and Potter LR and Hunter T. Journal of Biochemistry. 2001; 276:6057-6060).

[0031] Natriuretic peptides typically must be administered in low, continuous doses because of their short half-life and the fact that high bolus doses can result in very high peak plasma concentrations (C50). maxFurthermore, the drop in blood pressure can cause drowsiness and ataxia, which are major causes of toxicity. To mitigate these harmful effects, natriuretic peptides are typically delivered via slow infusion. See, for example, Kimura et al., *Journal of Surgical Research*. 2015, 194(2); 631-637. One aspect of the disclosed invention mitigates natriuretic peptide toxicity caused by bolus administration while improving cyclic GMP response and therapeutic efficacy.

[0032] This disclosure relates to the surprising and unexpected discovery of compounds comprising a low-toxicity cationic alkyl moiety (e.g., the need for high doses before ataxia is observed). The cationic alkyl moiety of this disclosure can be covalently linked to molecules (e.g., peptides) to reduce in vivo degradation and / or prolong their presence or half-life in the blood without causing toxicity (ataxia). These benefits can occur at doses equal to or exceeding the therapeutically effective dose, and can occur at higher doses than the same peptide modified with other cationic alkyl moieties (see Table 3 in Example 3). Additionally, the disclosed compositions can exhibit enhanced biological activity (pharmacodynamics) compared to unmodified peptides.

[0033] This disclosure relates to compositions comprising compounds containing a single alkyl chain having at least two positive charges derived from diaminopropionic acid (Dap) and / or diaminobutyric acid (Dab) (defined herein as cationic alkyl), exhibiting surprisingly and non-obvious characteristics of significantly reduced toxicity (e.g., as demonstrated in rat ataxia tests) compared to those modified with cationic amino acids (including natural or non-natural D-type amino acids such as lysine or arginine) having larger or longer chain R groups. The novel compositions comprise a single alkyl chain linked to 2-4 cationic amino acid residues via a noncationic linker, the cationic amino acid residues being independently selected from diaminopropionic acid (Dap) and / or diaminobutyric acid (Dab), and wherein the cationic chain has no more than 2 Dab residues. The noncationic linker is 2-4 residues independently selected from: 2-aminoethoxy-2-ethoxyacetic acid (Aeea), γ-aminobutyric acid (γAbu), γ-linked glutamic acid (γE), and glutamic acid ε. The cationic alkyl compositions of this disclosure can be linked to peptides to enhance their pharmacokinetic properties, bioavailability, and / or pharmacodynamic activity (see Example 4). As illustrated by examples in this specification, cationic alkyl-modified peptides, such as C-type natriuretic peptides (CNPs) (or derivatives thereof), can be used to increase survival rates in sepsis, acute respiratory distress syndrome, acute lung injury, and / or pulmonary fibrosis (see, for example, Examples 5-9 and 12 and associated figures or tables). Additionally, cationic alkyl-modified CNPs (or derivatives thereof) can be used to treat or inhibit cancer (see Examples 10 and 13-16). This disclosure relates to a surprising finding that safer cationic alkyl compositions are safe at doses up to 10 µmol / kg and lower bolus doses (see Table 1 in Example 1). Additionally, the cationic alkyl moiety of this disclosure can typically be covalently linked to a therapeutic peptide to increase the peptide's in vivo stability, half-life (pharmacokinetics), and / or activity (pharmacodynamics) without causing toxicity or ataxia at bolus doses at at least twice the therapeutically effective dose and / or at doses providing significant (measurable) pharmacodynamic activity. In other words, peptide modifications using the cationic alkyl moiety of this disclosure do not cause toxicity to the peptide at doses providing significant pharmacodynamic and / or therapeutic activity compared to unmodified peptides. This disclosure also relates to cationic alkyl-linked natriuretic peptides and their derivatives or combinations thereof, which exhibit unexpectedly superior ability to increase cGMP in the blood compared to natural natriuretic peptides and lower toxicity compared to other cationic alkyl-linked natriuretic peptides. Examples of cationic alkyl-linked peptides of this disclosure include C-type natriuretic peptides (CNP), atrial natriuretic peptides (ANP), brain-type natriuretic peptides (BNP), and their corresponding derivatives, which exhibit unexpectedly superior biological activity and / or lower toxicity compared to their corresponding natural peptides or other modified natriuretic peptides known in the art.

[0034] For peptides to be considered for parenteral drug development, they must be therapeutically effective and non-toxic when administered at practical or reasonable high doses parenterally (e.g., subcutaneously, intramuscularly, by inhalation, or intravenously). For subcutaneous, intramuscular, and inhalation administration of peptides, the preferred and practical bolus dose for humans should not exceed 12 mg / human to avoid excessive injection volume and / or peptide concentrations exceeding the peptide's solubility in the injection solution. The human bolus dose is 12 mg / 70 kg (or 0.17 mg / kg), which, after allometric scaling, is converted to rat and mouse doses of 1.0 mg / kg and 2.0 mg / kg, respectively. Therefore, for a 5 kDa peptide, the molar doses in rats and mice are 0.20 µmol / kg and 0.40 µmol / kg, respectively. For a 1 kDa peptide, the molar doses in rats and mice are 1.0 µmol / kg and 2.0 µmol / kg, respectively. Therefore, in order to ensure that the cationic lipid or cationic alkyl moiety linked to the peptide in a 1:1 (molar:molar) ratio does not increase toxicity / adverse reactions at actual or reasonable therapeutic doses, the MTD or no-observed-adverse-reaction dose of the cationic alkyl moiety must be at least twice the preferred and / or actual therapeutic molar dose of the peptide in the same species.

[0035] For the purposes of this disclosure, preferred compositions of the present invention comprise compounds containing the cationic alkyl chains of the present disclosure, which in some embodiments are capable of improving the in vivo pharmacokinetics, pharmacodynamics, and / or bioavailability of modified compounds (e.g., peptides) with minimal or no observed toxicity (ataxia) in rats at bolus doses of up to 10 µmol / kg (see Table 1 in Example 1). For cationic alkyl moieties that can improve the in vivo pharmacokinetics and / or pharmacodynamics of peptides, cationic alkyl moieties that do not cause toxicity (or ataxia) when administered alone at bolus doses of at least 10 µmol / kg, 9.0 µmol / kg, 8.0 µmol / kg, 7.0 µmol / kg, 6.0 µmol / kg, or 5.0 µmol / kg are preferred (see Table 1 in Example 1). This preference will ensure that cationic lipids or cationic alkyl moieties linked to the peptide in a 1:1 molar ratio to prolong half-life and / or improve potency do not produce toxicity / adverse effects on the peptide at doses of 3.0 µmol / kg and lower. This disclosure discloses compositions comprising an alkyl cationic moiety that can be linked or covalently linked to a peptide in a 1:1 ratio without causing additional toxicity to the peptide when administered in rats at preferred and practical bolus doses of 3.0 µmol / kg or less. For example, the cationic alkyl moiety of this disclosure is covalently linked to a natriuretic peptide that itself does not cause ataxia in rats at 10 µmol / kg or less, thereby producing a conjugate that unexpectedly exhibits superior ability to increase blood cGMP and / or intracellular cGMP in vivo compared to natural peptides such as atrial natriuretic peptide (ANP), brain-type natriuretic peptide (BNP), or C-type natriuretic peptide (CNP) and their derivatives (see Table 3 in Example 3).

[0036] This disclosure describes modifications to peptides or their derivatives to prolong their half-life without increasing the risk of toxicity at therapeutic doses due to cationic alkyl modification. The modifiers disclosed are cationic lipids, which are known to be toxic. However, when two or more non-natural amino acids, such as diaminopropionic acid (Dap) or diaminobutyric acid (Dab), form the cationic moiety of a cationic alkyl modifier, and these cationic amino acids are separated from the alkyl chain by non-cationic spacers (such as 2-4 residues of Aeea, γAbu, γE, and / or E), they have unexpectedly been found to exhibit low toxicity in rats (i.e., no ataxia and / or death). This contrasts sharply with alkyl modifications of cationic amino acids (such as lysine) with larger R groups (natural or non-natural D-type) and without non-cationic spacers between the alkyl chain and the cationic amino acid. This invention allows for the modification of peptides with cationic alkyl / lipid modifications, reducing the additional toxicity of the peptides. This provides a greater safety margin, equal to or higher than the dose required for biological activity (pharmacodynamics) or a practical and reasonable bolus dose (3.0 µmol / kg or lower for parenteral administration), which is crucial in commercial drug development.

[0037] To date, most modified peptides use alkyl chains without cationic moieties because cationic lipids or cationic alkyl groups have been well-proven to have inherent toxicity. [Cui et al., 2018]. This disclosure provides cationic alkyl compositions with very limited biotoxicity compared to other cationic alkyl compositions. Additionally, the cationic alkyl compositions of this disclosure can be covalently linked to peptides at residues not critical to the peptide's biological activity, thereby increasing the in vivo half-life of the peptide composition or its blood presence after bolus administration compared to unmodified peptides. Furthermore, because the cationic alkyl moiety on the modified peptide can interact with the anionic lipid membrane of cells, coupled with the specific interaction between the peptide and its receptor, the cationic alkyl moiety can enhance the peptide's biological activity or pharmacokinetic potency. The cationic alkyl compositions of this disclosure can be linked or covalently linked to peptides (e.g., natriuretic peptides). The compositions and methods of use of this disclosure are described herein.

[0038] The following is a description of an example embodiment.

[0039] Composition Formula (I) This disclosure provides a cationic alkyl or cationic lipid compound (e.g., for covalently modifying peptides, for example, to improve therapeutic efficacy), said compound comprising a cationic moiety of formula (I): J-(CH2)x(CO)-(A)y-(B)z; (I) in: J is either HOOC or CH3; x is 10⁻¹⁶; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-aminobutyric acid (γAbu), γ-linked glutamic acid (γE) and glutamic acid (E); y is 2-4; B is independently selected from diaminopropionic acid (Dap) or diaminobutyric acid (Dab). z- is 2-4; Wherein -(B)z- has no more than 2 Dab residues in the sequence, and the bond between the residues is established by the amide bonded to the α-amino group of Dab and / or Dap; Furthermore, no clinically observable toxicity or ataxia was observed after subcutaneous bolus administration of the composition at doses of 10 µmol / kg or lower in rats.

[0040] When determining toxicity (where observations of ataxia are used as a marker of toxicity), the dose is increased until ataxia (i.e., toxicity) is observed, up to a maximum of 10 µmol / kg, which is the maximum practical bolus dose of a liquid parenteral peptide administered in a small volume injection. As used in this disclosure, “ataxia” is a clinical sign characterized by poor muscle control, resulting in clumsiness of voluntary movement. Ataxia may cause difficulty in movement, coordination, and / or eye movement.

[0041] One embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); and z is 2.

[0042] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); and z is 2.

[0043] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); y is 3; and z is 2.

[0044] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); y is 3; and z is 2.

[0045] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); and z is 2.

[0046] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); and z is 2.

[0047] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14, or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); y is 3; and z is 2. Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); y is 3; and z is 2.

[0048] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); and z is 2.

[0049] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 14; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); and z is 2.

[0050] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); y is 3; and z is 2.

[0051] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 14; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); y is 3; and z is 2.

[0052] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is γ-aminobutyric acid (γAbu); and z is 2.

[0053] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is CH3, x is 14; A is γ-aminobutyric acid (γAbu); and z is 2.

[0054] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is γ-aminobutyric acid (γAbu); y is 3; and z is 2.

[0055] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is CH3, x is 14; A is γ-aminobutyric acid (γAbu); y is 3; and z is 2.

[0056] One embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); and z is 3.

[0057] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); and z is 3.

[0058] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); y is 3; and z is 3.

[0059] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); y is 3; and z is 3.

[0060] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); and z is 3.

[0061] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); and z is 3.

[0062] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); y is 3; and z is 3.

[0063] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); y is 3; and z is 3.

[0064] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); and z is 3.

[0065] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 14; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); and z is 3.

[0066] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); y is 3; and z is 3.

[0067] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 14; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); y is 3; and z is 3.

[0068] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is γ-aminobutyric acid (γAbu); and z is 3.

[0069] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is CH3, x is 14; A is γ-aminobutyric acid (γAbu); and z is 3.

[0070] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is CH3, x is 10, 12, 14 or 16; A is γ-aminobutyric acid (γAbu); y is 3; and z is 3.

[0071] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is CH3, x is 14; A is γ-aminobutyric acid (γAbu); y is 3; and z is 3.

[0072] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); and z is 2.

[0073] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); and z is 2.

[0074] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); y is 3; and z is 2.

[0075] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); y is 3; and z is 2.

[0076] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); and z is 2.

[0077] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); and z is 2.

[0078] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); y is 3; and z is 2.

[0079] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); y is 3; and z is 2.

[0080] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); and z is 2.

[0081] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is HOOC, x is 14; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); and z is 2.

[0082] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); y is 3; and z is 2.

[0083] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 14; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); y is 3; and z is 2.

[0084] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is γ-aminobutyric acid (γAbu); and z is 2.

[0085] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is HOOC, x is 14; A is γ-aminobutyric acid (γAbu); and z is 2.

[0086] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is γ-aminobutyric acid (γAbu); y is 3; and z is 2.

[0087] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is HOOC, x is 14; A is γ-aminobutyric acid (γAbu); y is 3; and z is 2.

[0088] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); and z is 3.

[0089] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); and z is 3.

[0090] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); y is 3; and z is 3.

[0091] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu); y is 3; and z is 3.

[0092] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); and z is 3.

[0093] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); and z is 3.

[0094] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); y is 3; and z is 3.

[0095] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 14; A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-linked glutamic acid (γE); y is 3; and z is 3.

[0096] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); and z is 3.

[0097] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is HOOC, x is 14; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); and z is 3.

[0098] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); y is 3; and z is 3.

[0099] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is HOOC, x is 14; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea); y is 3; and z is 3.

[0100] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is γ-aminobutyric acid (γAbu); and z is 3.

[0101] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is HOOC, x is 14; A is γ-aminobutyric acid (γAbu); and z is 3.

[0102] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is HOOC, x is 10, 12, 14 or 16; A is γ-aminobutyric acid (γAbu); y is 3; and z is 3.

[0103] Another embodiment of this disclosure is a compound comprising the cationic portion of formula (I), wherein J is HOOC, x is 14; A is γ-aminobutyric acid (γAbu); y is 3; and z is 3.

[0104] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3 or HOOC; x is 10, 12, 14 or 16; (A)y is Aeea-Aeea-Aeea, γAbu-γAbu-γAbu, γAbu-Aeea-Aeea, γE-Aeea-Aeea, E-Aeea-Aeea; Aeea-Aeea, γAbu-γAbu, γAbu-Aeea, γE- Aeea or E-Aeea; and (B)z is Dap-Dap, Dab-Dab, Dab-Dap, Dap-Dab, Dap-Dap-Dap, Dab-Dab-Dap, Dab-Da p-Dap, Dap-Dab-Dap, Dap-Dap-Dap-Dap, Dab-Dab-Dap-Dap, Dab-Dap-Dap-Dap or Dap-Dab-Dap-Dap.

[0105] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3 or HOOC; x is 14; (A)y is Aeea-Aeea-Aeea, γAbu-γAbu-γAbu, γAbu-Aeea-Aeea, γE-Aeea-Aeea, E-Aeea-Aeea; Aeea-Aeea, γAbu-γAbu, γAbu-Aeea, γE-Aeea or E-Aeea; and (B)z is Dap-Dap, Dab-Dab, Dab-Dap, Dap-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap-Dap, Dab-Dap-Dap-Dap, or Dap-Dab-Dap-Dap.

[0106] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3 or HOOC; x is 10, 12, 14 or 16; (A)y is Aeea-Aeea-Aeea, γAbu-γAbu-γAbu, γAbu-Aeea-Aeea, γE-Aeea-Aeea or E-Aeea-Aeea; and (B)z is Dap-Dap, Dab-Dab, Dab-Dap, Dap-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap-Dap, Dab-Dap-Dap-Dap, or Dap-Dab-Dap-Dap.

[0107] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3 or HOOC; x is 14; (A)y is Aeea-Aeea-Aeea, γAbu-γAbu-γAbu, γAbu-Aeea-Aeea, γE-Aeea-Aeea or E-Aeea-Aeea; and (B)z is Dap-Dap, Dab-Dab, Dab-Dap, Dap-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap-Dap, Dab-Dap-Dap-Dap, or Dap-Dab-Dap-Dap.

[0108] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3; x is 10, 12, 14 or 16; (A)y is Aeea-Aeea-Aeea, γAbu-γAbu-γAbu, γAbu-Aeea-Aeea, γE-Aeea-Aeea, E-Aeea-Aeea; Aeea-Aeea, γAbu-γAbu, γAbu-Aeea, γE-Ae ea or E-Aeea; and (B)z is Dap-Dap, Dab-Dab, Dab-Dap, Dap-Dab, Dap-Dap-Dap, Dab-Dab-Dap, Dab-Dap -Dap, Dap-Dab-Dap, Dap-Dap-Dap-Dap, Dab-Dab-Dap-Dap, Dab-Dap-Dap-Dap or Dap-Dab-Dap-Dap.

[0109] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3; x is 14; (A)y is Aeea-Aeea-Aeea, γAbu-γAbu-γAbu, γAbu-Aeea-Aeea, γE-Aeea-Aeea, E-Aeea-Aeea; Aeea-Aeea, γAbu-γAbu, γAbu-Aeea, γE-Aeea or E-Aeea; and (B)z is Dap-Dap, Dab-Dab, Dab-Dap, Dap-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap-Dap, Dab-Dap-Dap-Dap, or Dap-Dab-Dap-Dap.

[0110] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3; x is 10, 12, 14 or 16; (A)y is Aeea-Aeea-Aeea, γAbu-γAbu-γAbu, γAbu-Aeea-Aeea, γE-Aeea-Aeea or E-Aeea-Aeea; and (B)z is Dap-Dap, Dab-Dab, Dab-Dap, Dap-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap-Dap, Dab-Dap-Dap-Dap, or Dap-Dab-Dap-Dap.

[0111] Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), wherein J is CH3; x is 14; (A)y is Aeea-Aeea-Aeea, γAbu-γAbu-γAbu, γAbu-Aeea-Aeea, γE-Aeea-Aeea or E-Aeea-Aeea; and (B)z is Dap-Dap, Dab-Dab, Dab-Dap, Dap-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap, Dab-Dap-Dap-Dap, Dab-Dap-Dap-Dap, or Dap-Dab-Dap-Dap.

[0112] In another embodiment of this disclosure, the cationic portion of formula (I) is any one of [SEQ ID. NO: 10 to 22 (see Table 1 in Example 1) or 51 to 69], wherein SEQ ID. NO: 10 to 22 and 51-69 are as follows: CH3(CH2)14(C=O)-Aeea-Aeea-Aeea-Dab-Dab [SEQ ID NO: 10], CH3(CH2)14(C=O)-γAbu-γAbu-Dab-Dab [SEQ ID NO: 11], CH3(CH2)14(C=O)-γAbu-γAbu-γAbu-Dab-Dab [SEQ ID NO: 12], CH3(CH2)14(C=O)-Aeea-Aeea-Dap ... 13], CH3(CH2)14(C=O)-Aeea-Aeea-Aeea-Dap-Dap [SEQ ID NO: 14], CH3(CH2)14(C=O)-γE-Aeea-Aeea-Dab-Dab [SEQ ID NO: 15], CH3(CH2)14(C=O)-E-Aeea-Aeea-Dab-Dab [SEQ ID NO: 16], CH3(CH2)14(C=O)-Aeea-Aeea-Aeea-Dab-Dap [SEQ ID NO: 17], (HOOC)(CH2)16(C=O)-Aeea-Aeea-Aeea-Dab-Dab [SEQ ID NO: 18], CH3(CH2)14(C=O)-Aeea-Aeea-Aeea-Dap-Dap-Dap [SEQ ID NO:19], (HOOC)(CH2)16(C=O)-Aeea-Aeea-Aeea-Dap-Dap [SEQ ID NO: 20], (HOOC)(CH2)16(C=O)-Aeea-Aeea-Dab-Dab [SEQ ID NO: 21], CH3(CH2)14(C=O)-Aeea-Aeea-Aeea-Dap-Dap-Dap-Dap [SEQ ID NO: 22], CH3(CH2)14(C=O)-Aeea-Aeea-Aeea-Dap-Dab [SEQ ID NO: 51], CH3(CH2)14(C=O)-γAbu-γAbu-γAbu-Dap-Dap [SEQ ID NO: 52], CH3(CH2)14(C=O)-γAbu-γAbu-γAbu-Dap-Dab [SEQ ID NO:53], CH3(CH2)14(C=O)-γAbu-γAbu-γAbu-Dab-Dap [SEQ ID NO: 54], CH3(CH2)14(C=O)-γE-Aeea-Aeea-Dap-Dap [SEQ ID NO:55], CH3(CH2)14(C=O)-γE-Aeea-Aeea-Dap-Dap [SEQ ID NO: 56], CH3(CH2)14(C=O)-γE-Aeea-Aeea-Dap-Dap [SEQ ID NO: 57], (HOOC)(CH2)14(C=O)-Aeea-Aeea-Aeea-Dap-Dap [SEQ ID NO: 58], (HOOC)(CH2)14(C=O)-Aeea-Aeea-Aeea-Dab-Dab [SEQ ID NO: 59], (HOOC)(CH2)14(C=O)-Aeea-Aeea-Aeea-Dap-Dap [SEQ ID NO: 60], (HOOC)(CH2)14(C=O)-Aeea-Aeea-Aeea-Dap-Dap [SEQ ID NO: 61], (HOOC)(CH2)14(C=O)-γAbu-γAbu-γAbu-Dap-Dap [SEQ ID NO: 62], (HOOC)(CH2)14(C=O)-γAbu-γAbu-γAbu-Dab-Dab [SEQ IDNO: 63], (HOOC)(CH2)14(C=O)-γAbu-γAbu-γAbu-Dap-Dab [SEQ ID NO: 64], (HOOC)(CH2)14(C=O)-γAbu-γAbu-γAbu-Dab-Dap [SEQ ID NO: 65], (HOOC)(CH2)14(C=O)-γE-Aeea-Aeea-Dap-Dap [SEQ ID NO: 66], (HOOC)(CH2)14(C=O)-γE-Aeea-Aeea-Dap-Dab [SEQ ID NO: 67], (HOOC)(CH2)14(C=O)-γE-Aeea-Aeea-Dap-Dab [SEQ ID NO: 68] or (HOOC)(CH2)14(C=O)-γE-Aeea-Aeea-Dab-Dap [SEQ ID NO: 69].

[0113] Another embodiment of this disclosure is a composition of formula (I) used for preparing a medicine, such as a pharmaceutical composition.

[0114] Another embodiment of this disclosure is a composition comprising a compound and one or more pharmaceutically acceptable carriers and / or excipients (e.g., a pharmaceutical composition), said compound comprising an alkyl cationic moiety of formula (I) (e.g., conjugated with a peptide).

[0115] Another embodiment of this disclosure is a composition comprising a compound comprising a cationic moiety of formula (I), the composition further comprising an immunomodulatory or anticancer agent formulation.

[0116] Equation (II) Another embodiment of this disclosure is a compound comprising the cationic moiety of formula (I), said compound further comprising a covalently linked peptide to obtain a conjugated peptide of formula (II): J-(CH2)x(CO)-(A)y-(B)z-peptide, The portion corresponding to formula (I) is covalently linked to the peptide via an amide bond with Dap or Dab; ​​the variables in the portion corresponding to formula (I) are limited to the same variables as in formula (I), and the conjugated peptide of formula (II) has: i) higher bioactivity than the unmodified peptide at an equivalent bolus dose (mol / kg); ii) a longer in vivo half-life and / or higher blood levels over time compared to the unmodified peptide, while maintaining its activity; and / or iii) no toxicity or ataxia when administered to rats at a bolus dose of 3.0 µmol / kg or lower.

[0117] The compound of formula (II), wherein the compound binds to the natriuretic peptide receptor, and wherein when formula (II) is administered in rats at a bolus dose of 3.0 µmol / kg or less, no adverse reactions or ataxia occur.

[0118] In another embodiment of formula (II), the cationic alkyl moiety of formula (I) is covalently linked to the N-terminus of the peptide or to a side-chain amino group (side amino group) on the peptide.

[0119] In another embodiment of formula (II), the cationic alkyl moiety of formula (I) is covalently linked to the N-terminus of the peptide.

[0120] In one embodiment of formula (II), the cationic portion of formula (I) is selected from [SEQ ID NO: 10 to 22 and 51 to 69]. In one embodiment of formula (II), the peptide portion is a natriuretic peptide or a natriuretic peptide derivative. As used herein, the term "derivative" for peptide means a natural peptide modified by substituting one or more amino acids, adding one or more amino acids, removing one or more amino acids, or any combination thereof, while retaining the biological activity of the peptide in its natural form.

[0121] In one embodiment of formula (II), the peptide portion is a natriuretic peptide [SEQ ID NO: 32, 44, 48, 75] or a natriuretic peptide derivative, wherein one or more methionine residues in the natriuretic peptide are replaced by glutamine (Q), ortholeucine (Nle), methoxybutyric acid (Mox), or leucine (L).

[0122] In one embodiment of formula (II), the peptide moiety is a natriuretic peptide [SEQ ID NO: 32, 44, 48, 75] or a natriuretic peptide derivative, wherein one or more methionine residues in the natriuretic peptide are replaced by glutamine (Q), ortholeucine (Nle), methoxybutyric acid (Mox), or leucine (L), and the cationic alkyl moiety of formula (I) is selected from [SEQ ID NO: 10 to 22 and 51 to 69].

[0123] In one embodiment of formula (II), the peptide is a natriuretic peptide derivative, wherein one or more methionine residues in the natriuretic peptide are replaced by glutamine (Q), ortholeucine (Nle), methoxybutyric acid (Mox), or leucine (L), and the portion of formula (I) is selected from [SEQ ID NO: 10 to 22 and 51 to 69].

[0124] In one embodiment of formula (II), the peptide is a natriuretic peptide derivative, wherein one or more methionine residues in the natriuretic peptide are replaced by glutamine (Q), ortholeucine (Nle), methoxybutyric acid (Mox), or leucine (L), and the portion of formula (I) is selected from [SEQ ID NO: 10 to 22].

[0125] In one embodiment of formula (II), the peptide binds to natriuretic peptide receptor B (NPRB), natriuretic peptide receptor C (NPRC), or both NPRB and NPRC.

[0126] In one embodiment of formula (II), the peptide binds to natriuretic peptide receptor A (NPRA), natriuretic peptide receptor B (NPRB), and / or natriuretic peptide receptor C (NPRC).

[0127] In one embodiment of formula (II), the peptide is a natriuretic peptide receptor B (NPRB) agonist.

[0128] In one embodiment of formula (II), the peptide is a natriuretic peptide receptor C (NPRC) agonist.

[0129] In one embodiment of formula (II), the peptide is a natriuretic peptide receptor A (NPRA) agonist.

[0130] In one embodiment of formula (II), the peptide produces physiological effects. Non-limiting examples of physiological effects that may be produced by the peptide include: antiproliferative effects, decreased endothelial permeability, inhibition of cyclooxygenase 2 (COX-2) expression, decreased blood pressure, antagonism of the renin-angiotensin-aldosterone system, inhibition of cardiac hypertrophy, sustained increase in blood cGMP, alteration of cAMP, increased survival rate in sepsis, increased survival rate in acute lung injury, increased survival rate in acute respiratory distress syndrome, reduction of MPO-positive cells, reduction of cell count in alveolar fluid or bronchoalveolar lavage fluid, reduction of protein content in alveolar fluid or bronchoalveolar lavage fluid, reduction of lung weight by body weight, reduction of monocyte chemoattractant protein-1, reduction of IL-6, reduction of TNF-α, reduction of A1008 / A9, reduction of fibrosis, reduction of tumor volume, reduction of inflammation and / or reduction of cancer burden.

[0131] In one embodiment of formula (II), the peptide is a natriuretic peptide derivative, wherein one or more methionine residues in the natriuretic peptide are replaced by glutamine (Q).

[0132] In one embodiment of formula (II), the conjugated peptide is defined by SEQ ID NO: 29-31, 33-43, 45-47, 49, 50.

[0133] In one embodiment of formula (II), the conjugated peptide is defined by SEQ ID NO: 29-31, 33-43.

[0134] In one embodiment of formula (II), the conjugated peptide is defined by SEQ ID NO: 29-31.

[0135] In one embodiment of formula (II), the conjugated peptide is defined by SEQ ID NO: 29.

[0136] In one embodiment of formula (II), the conjugated peptide is defined by SEQ ID NO: 30.

[0137] In one embodiment of formula (II), the conjugated peptide is defined by SEQ ID NO: 31.

[0138] Compounds for the preparation of pharmaceutical compositions and / or for the treatment of diseases, comprising a cationic moiety of formula (I) and / or a conjugated peptide of formula (II).

[0139] The compounds or compositions of this disclosure are effective in reducing alveolar inflammatory edema, improving blood oxygenation, and / or increasing survival rates, as clearly seen from the examples presented. Additionally, the compounds or compositions of this disclosure are effective in preventing and alleviating pulmonary fibrosis.

[0140] Another embodiment of the present invention is a compound comprising a cationic alkyl moiety or conjugated peptide of formula (I) or formula (II) for use in the preparation of a medicament (or a pharmaceutical composition) (or a method for preparing said medicament (or pharmaceutical composition)).

[0141] Another embodiment of the present invention is a compound comprising the cationic alkyl moiety of formula (I), which is used as an excipient for preparing pharmaceutical compositions.

[0142] Compounds of Formula II are used to prepare pharmaceutical compositions by adding one or more pharmaceutically acceptable carriers or excipients, such as leavening agents (e.g., sugars), buffers, stabilizers (e.g., cyclodextrins), and / or preservatives.

[0143] The compounds of Formula II are used to treat the disease or condition of a subject by administering a therapeutically effective bolus dose of 10.0 µmol / kg or less and / or 10.0 µmol / kg to 0.0001 µmol / kg, including the extreme values, to a subject in need. Another embodiment of the present invention is a pharmaceutical compound comprising a cationic moiety or conjugated peptide of formula (I) and / or formula (II), said pharmaceutical compound further comprising one or more pharmaceutically acceptable excipients.

[0144] Another embodiment of the present invention is a compound comprising a conjugated peptide of formula (II) for: a) preparing a pharmaceutical composition or b) treating a disease in a subject.

[0145] Another embodiment of the invention provides a compound comprising a cationic moiety or conjugated peptide of formula (I) and / or formula (II) for the preparation of a medicament (or pharmaceutical composition) for treating a disease affecting the lungs, liver, heart, bones and / or joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, testes, ovaries, uterus and / or blood vessels.

[0146] This disclosure also provides compounds comprising cationic moieties or conjugated peptides of formula (I) and / or formula (II) for use in treating diseases affecting the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, testes, ovaries, uterus, and / or blood vessels, wherein the method comprises parenteral administration of a pharmaceutical composition comprising a cationic moiety or conjugated peptide of formula (I) and / or formula (II) at a therapeutically effective bolus dose of 3.0 µmol / kg or less.

[0147] Another embodiment of the invention provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) for treating a disease (or a method of preparing said medicament (or pharmaceutical composition)) affecting the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, testes, ovaries, uterus and / or blood vessels, wherein said method comprises adding one or more pharmaceutically acceptable excipients to said conjugated peptide of formula (II).

[0148] In some embodiments, a drug or pharmaceutical composition for treating a disease affecting the lungs is selected from the following diseases: ALI, ARDS, COVID (e.g., COVID-19), inflammation, sepsis, fibrosis, or cancer. In some embodiments, a drug or pharmaceutical composition for treating a disease affecting the liver is selected from the following diseases: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), inflammation, fibrosis, or cancer. In some embodiments, a drug or pharmaceutical composition for treating a disease affecting the heart is selected from the following diseases: heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), acute heart failure, or congestive heart failure. In some embodiments, a drug or pharmaceutical composition for treating a disease affecting bones and / or joints is selected from the following diseases: osteoporosis, osteoarthritis, rheumatoid arthritis, inflammatory cancer, or dwarfism. In some embodiments, a drug or pharmaceutical composition for treating a disease affecting the kidneys is selected from the following diseases: chronic kidney disease (CKD), acute kidney injury (AKI), drug-induced kidney injury, inflammation / nephritis, renal fibrosis, glomerulosclerosis, or renal cancer. In some embodiments, a medicine or pharmaceutical composition for treating a disease affecting the prostate treats a disease selected from benign prostatic hyperplasia or prostate cancer.

[0149] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for treating a disease (or a treatment method for said disease) affecting the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, testes, ovaries, uterus, and / or blood vessels, wherein said treatment comprises administering a compound comprising a conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein the peptide portion of said conjugated peptide of formula (II) is a natriuretic peptide or a derivative thereof, and the portion corresponding to the cationic alkyl portion of formula (I) is selected from SEQ ID NO: 10 to 22 and 51 to 69.

[0150] In some embodiments, this disclosure provides the use of the conjugated peptide of Formula II for treating diseases, symptoms, or conditions. In some embodiments, the disease, symptom, or condition affects the lungs (e.g., selected from diseases such as ALI, ARDS, COVID (e.g., COVID-19), inflammation, sepsis, fibrosis, or lung cancer). In some embodiments, the disease, symptom, or condition affects the liver (e.g., selected from diseases such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), inflammation, fibrosis, or cancer). In some embodiments, the disease, symptom, or condition affects the heart (e.g., selected from diseases such as heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), acute heart failure, or congestive heart failure). In some embodiments, the disease, symptom, or condition affects bones and / or joints (e.g., selected from diseases such as osteoporosis, osteoarthritis, rheumatoid arthritis, inflammatory cancer, or dwarfism). In some embodiments, the disease, symptom, or condition affects the kidneys (e.g., diseases selected from: chronic kidney disease (CKD), acute kidney injury (AKI), drug-induced kidney injury, inflammation / nephritis, renal fibrosis, glomerulosclerosis, or renal cancer). In some embodiments, the disease, symptom, or condition affects the prostate, for example, benign prostatic hyperplasia or prostate cancer.

[0151] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for the treatment of ALI, ARDS, COVID, sepsis and / or fibrosis (or for the treatment of these conditions), wherein said treatment comprises administering a compound comprising a conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein the peptide moiety of said conjugated peptide of formula (II) is a natriuretic peptide [e.g., SEQ ID NO: 32, 44, 48, 75] or a derivative thereof, wherein one or more methionine residues in said natriuretic peptide are replaced by glutamine (Q), ortholeucine (Nle), methoxybutyric acid (Mox) or leucine (L), and the cationic alkyl moiety of formula (I) is [SEQ ID NO: 10 to 22 and 51 to 69].

[0152] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) for the treatment of ALI, ARDS, COVID, sepsis and / or pulmonary fibrosis (or a method for the preparation of said medicament (or pharmaceutical composition)), said preparation comprising adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein the conjugated peptide of formula (II) is selected from SEQ ID NO: 29-31, 33-43, 45-47, 49-50.

[0153] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for the treatment of ALI, ARDS, COVID, sepsis and / or fibrosis (or for the treatment of these conditions), wherein said treatment comprises administering to a subject in need a therapeutically effective bolus dose of the conjugated peptide of formula (II) at a dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is selected from SEQ ID NO: 29-31, 33-43, 45-47, 49-50.

[0154] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) for the treatment of ALI, ARDS, COVID, sepsis and / or pulmonary fibrosis (or a method of preparing said medicament (or pharmaceutical composition)), wherein the preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein the conjugated peptide of formula (II) is selected from SEQ ID NO: 29-31, 33-43.

[0155] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for the treatment of ALI, ARDS, COVID, sepsis and / or fibrosis (or for the treatment of these conditions), wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is selected from SEQ ID NO: 29-31, 33-43.

[0156] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) for the treatment of ALI, ARDS, COVID, sepsis and / or pulmonary fibrosis (or a method of preparing said medicament (or pharmaceutical composition)), wherein the preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein the conjugated peptide of formula (II) is selected from SEQ ID NO: 29-31.

[0157] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for the treatment of ALI, ARDS, COVID, sepsis and / or fibrosis (or for the treatment of these conditions), wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is selected from SEQ ID NO: 29-31.

[0158] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition)) for the treatment of ALI, ARDS, COVID, sepsis and / or pulmonary fibrosis, wherein said treatment comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is SEQ ID NO: 29.

[0159] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for the treatment of ALI, ARDS, COVID, sepsis and / or fibrosis (or for the treatment of these conditions), wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is SEQ ID NO: 29.

[0160] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparation of said medicament (or pharmaceutical composition)) for the treatment of ALI, ARDS, COVID, sepsis and / or pulmonary fibrosis, wherein said preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is SEQ ID NO: 30.

[0161] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for the treatment of ALI, ARDS, COVID, sepsis and / or fibrosis (or for the treatment of these conditions), wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is SEQ ID NO: 30.

[0162] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition)) for the treatment of ALI, ARDS, COVID, sepsis and / or pulmonary fibrosis, wherein said preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is SEQ ID NO: 31.

[0163] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for the treatment of ALI, ARDS, COVID, sepsis and / or fibrosis (or for the treatment of these conditions), wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is SEQ ID NO: 31.

[0164] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for treating a medical condition selected from one or a combination thereof, including hypooxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, said treatment comprising administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein the peptide portion of said conjugated peptide of formula (II) is a natriuretic peptide (e.g., SEQ ID NO: 32, 44, 48, 75) or a derivative thereof, wherein one or more methionine residues in said natriuretic peptide are replaced by glutamine (Q), ortholeucine (Nle), methoxybutyric acid (Mox), or leucine (L), and the portion corresponding to the cationic alkyl portion of formula (I) is selected from SEQ ID NO: 10 to 22 and 51 to 69.

[0165] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition)) for the treatment of a medical condition selected from one or a combination thereof, such as hypooxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, wherein said preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is selected from SEQ ID NO: 29-31, 33-43, 45-47, and 49-50.

[0166] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for treating one or a combination thereof selected from medical conditions of low blood oxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, said treatment comprising administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein the conjugated peptide of formula (II) is selected from SEQ ID NO: 29-31, 33-43, 45-47, and 49-50.

[0167] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition)) for the treatment of a medical condition selected from one or a combination thereof, such as hypooxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, said preparation or method comprising adding one or more pharmaceutically acceptable excipients to said conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is selected from SEQ ID NO: 29-31 and 33-43.

[0168] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for treating one or a combination thereof selected from medical conditions of low blood oxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis and / or bacteremia, wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is selected from SEQ ID NO: 29-31 and 33-43.

[0169] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition)) for the treatment of a medical condition selected from one or a combination thereof, such as hypooxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, wherein said preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is selected from SEQ ID NO:: 29-31.

[0170] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for treating one or a combination thereof selected from medical conditions of low blood oxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis and / or bacteremia, wherein the treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein the conjugated peptide of formula (II) is selected from SEQ ID NO:: 29-31.

[0171] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition)) for the treatment of a medical condition selected from one or a combination thereof, such as hypooxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, wherein said preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is SEQ ID NO:: 29.

[0172] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for treating one or a combination thereof selected from medical conditions of low blood oxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis and / or bacteremia, wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is SEQ ID NO::29.

[0173] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition)) for the treatment of a medical condition selected from one or a combination thereof, such as hypooxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis and / or bacteremia, said preparation or method comprising adding one or more pharmaceutically acceptable excipients to said conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is SEQ ID NO:: 30.

[0174] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for treating one or a combination thereof selected from medical conditions of low blood oxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, wherein the treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein the conjugated peptide of formula (II) is SEQ ID NO::30.

[0175] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition)) for the treatment of a medical condition selected from one or a combination thereof, such as hypooxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, wherein said preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is SEQ ID NO:: 31.

[0176] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for treating one or a combination thereof selected from medical conditions of hypooxygenation, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis and / or bacteremia, wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is SEQ ID NO::31.

[0177] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for treating metastatic cancer (or for treatment of metastatic cancer) located in one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus, wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein the peptide portion of the conjugated peptide of formula (II) is a natriuretic peptide (e.g., SEQ ID NO:: 32, 44, 48, 75) or a derivative thereof, wherein one or more methionine residues in said natriuretic peptide are replaced by glutamine (Q), ortholeucine (Nle), methoxybutyric acid (Mox) or leucine (L), and the portion of said conjugated peptide corresponding to the cationic alkyl portion of formula (I) is SEQ ID NO:: 10 to 22 and 51 to 69.

[0178] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition) for the treatment of metastatic cancers located in one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus), wherein said preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is selected from SEQ ID NO:: 29-31, 33-43, 45-47, 49-50.

[0179] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for the treatment (or for a treatment of) metastatic cancer located in one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus, wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is selected from SEQ ID NO:: 29-31, 33-43, 45-47, 49-50.

[0180] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition) for the treatment of metastatic cancers located in one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus), wherein said preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is selected from SEQ ID NO:: 29-31, 33-43.

[0181] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for the treatment (or for a treatment of) metastatic cancer located in one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus, wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is selected from SEQ ID NO:: 29-31, 33-43.

[0182] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition) for treating metastatic cancers located in one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus), wherein said preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is SEQ ID NO:: 29-31.

[0183] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for the treatment (or for a treatment of) metastatic cancer located in one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus, wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is SEQ ID NO:: 29-31.

[0184] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition) for treating metastatic cancer located in any one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus), wherein said preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is SEQ ID NO:: 29.

[0185] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for the treatment (or for a treatment of) metastatic cancer located in one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus, wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is SEQ ID NO:: 29.

[0186] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition) for treating metastatic cancers located in any one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus), wherein said preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is SEQ ID NO:: 30.

[0187] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for the treatment (or for a treatment of) metastatic cancer located in one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus, wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is SEQ ID NO:: 30.

[0188] Another embodiment of this disclosure provides a conjugated peptide of formula (II) for the preparation of a medicament (or pharmaceutical composition) (or a method of preparing said medicament (or pharmaceutical composition) for treating metastatic cancer located in any one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus), wherein said preparation or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), wherein said conjugated peptide of formula (II) is SEQ ID NO:: 31.

[0189] Another embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugated peptide of formula (II) for treating (or for use in the treatment of) metastatic cancer located in one or more organs selected from the lung, liver, heart, bone / joint, kidney, prostate, brain, eye, skin, muscle, blood, gastrointestinal tract, bladder, prostate, testis, ovary and / or uterus, wherein said treatment comprises administering the conjugated peptide of formula (II) to a subject of need at a therapeutically effective bolus dose of 3.0 µmol / kg or less, wherein said conjugated peptide of formula (II) is SEQ ID NO:: 31.

[0190] definition The following list of definitions explicitly defines the invention as described in this disclosure. Any terms not listed herein but appearing in this disclosure have the same meaning as understood by those skilled in the art.

[0191] As used herein, the term "alkyl" refers to a straight-chained (e.g., linear) or branched saturated hydrocarbon group. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), etc. Alkyl groups may contain 1 to about 30, 1 to about 24, 2 to about 24, 1 to about 20, 2 to about 20, 1 to about 10, 1 to about 8, 1 to about 6, 1 to about 4, or 1 to about 3 carbon atoms. For the purposes of this disclosure, the alkyl moiety is written using the formula CH3(CH2)x-CO-, HOOC(CH2)x-CO-, or -(CH2)x-, where x represents the number of methylene groups (i.e., CH2) constituting the alkyl chain, and CO represents the carbonyl group connecting the alkyl moiety to the remainder of the molecule. Substituents of the compounds disclosed herein are disclosed in groups or ranges throughout this specification. The specific intent is that this disclosure includes each individual subgroup of members of such groups and ranges. For example, the term "-(CH2)x-", where "x is 10-18" is specifically intended to be disclosed separately, but not limited to, "CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2", "CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2", "CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2", "CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2", and "CH2- ...

[0192] As used herein, “cationic alkyl,” “cationic alkyl group,” or “cationic alkyl moiety” refers to an alkyl group derived from a fatty acid covalently linked to one or more positively charged groups or portions via spacers or linkers. A spacer or linker consists of several amino acids in the chain, and each amino acid can be either non-natural (meaning not normally present in living higher organisms, such as type D) or natural (meaning normally present in living organisms). The linker can be derived from amino acids such as 2-[2-(2-(aminoethoxy)ethoxy]acetic acid (also known as 8-amino-3,6-dioxanoic acid), γ-aminobutyric acid (γAbu), or naturally occurring amino acids such as glutamic acid (E) or γ-linked glutamic acid (γE), a glutamic acid residue in which the side-chain carboxyl group (γ(gamma)) is the nitrogen moiety used to link the molecule or peptide to which it is attached, rather than the typical α-carboxyl group. The positively charged group is provided by the amino acid, whose positively charged side chain contains an amino group. As used herein, “fatty acid” refers to a molecule containing a carboxylic acid having a long alkyl chain (or aliphatic chain), which may be saturated or unsaturated. The carboxylic acid moiety is the reactive part that can form an “acylamide bond” with the linker.

[0193] As used herein, the term "acylamide" or "fatty acid amide" refers to an alkyl chain (saturated or unsaturated) having a -C(O)NH- moiety at one or both ends. For example, in "-(CH2)x-CONH-", where -(CH2)x- is the alkyl moiety and -CONH- is the amide moiety, "-(CH2)x-CONH-" is an acylamide or a fatty acid amide.

[0194] The phrase modifies that, within reasonable medical judgment, it is suitable for contact with human and lower animal tissues without causing excessive toxicity (ataxia), irritation, allergic reactions, etc., and is commensurate with a reasonable benefit / risk ratio.

[0195] As used herein, "allometric scaling" is a tool used by drug developers to predict human pharmacokinetics based on animal data. Predictive methods such as allometric scaling can provide a "peek" into how a drug will perform in humans before any clinical studies are conducted. This information is crucial for both drug developers and regulatory agencies (such as the FDA) as it provides a data-driven basis for establishing safe starting bolus doses for human use. For the purposes of this disclosure, the index used in allometric scaling is 0.7 (i.e., dose in other species = rat dose / ((rat body weight / average body weight of other species) index 0.7)). A dose of 2.0 mg / kg in mice is equivalent to approximately 1.0 mg / kg in rats, approximately 0.33 mg / kg in dogs, and 0.17 mg / kg in humans. In terms of molar-molar dose, 3.0 µmol / kg in rats is interpreted as 6.0 µmol / kg in mice, 1.0 µmol / kg in dogs, and 0.5 µmol / kg in humans. 5.0 µmol / kg in rats is interpreted as 10 µmol / kg in mice, 1.66 µmol / kg in dogs, and 0.83 µmol / kg in humans. 10 µmol / kg in rats is interpreted as 20 µmol / kg in mice, 3.33 µmol / kg in dogs, and 1.66 µmol / kg in humans. For the purposes of this specification, the claimed doses are derived from rats, and it should be understood that, after appropriate allometric scaling, they are also applicable to other species.

[0196] As used herein, “bulk,” “bulk dose,” or “bulk administration” refers to a single dose of a drug or other substance administered or applied over a short period of time (e.g., less than 10 minutes (e.g., less than 8 minutes, less than 5 minutes, less than 3 minutes, or less than 1 minute)). Administration includes one of the following: injection at any site of the body (including, but not limited to, intravascular, subcutaneous, intrathecal, or intradermal), enteric (e.g., oral, as a dosage form), inhalation (e.g., intratracheal inhalation administration, where the subject is exposed to a high aerosol concentration, causing the active pharmaceutical ingredient to deposit directly in the lower respiratory tract), or nasal administration (e.g., as an aerosol, liquid, or powder). For the purposes of this disclosure, bulk administration differs from infusion administration, which typically requires 30 minutes or more to complete.

[0197] As used in this article, the terms “acute lung injury” or “ALI” and the more severe “acute respiratory distress syndrome” or “ARDS” are pulmonary manifestations of an acute systemic inflammatory process clinically characterized by pulmonary infiltration, hypoxia, and edema, without evidence of left atrial hypertension (see, for example, Bernard, GR et al., Journal of Critical Care Medicine, 1994. 9(1): 72-81; Rubenfeld, GD et al., New England Journal of Medicine, 2005. 353(16): 1685-93; Brun-Buisson, C. et al., Critical Care Medicine, 2004. 30(1): 51-61; and Phua, J. et al., American Journal of Respiratory and Critical Care Medicine, 2009. 179(3): 220-7). ALI and ARDS are acute exacerbations of severe arterial hypoxia (low blood oxygen levels due to ventilation abnormalities) with PaO2 / FiO2 less than 300 Torr and less than or equal to 200 Torr, respectively. Signs and symptoms of ALI and ARDS typically begin within two hours of the precipitating event, but can take 1–3 days; diagnostic criteria require known damage within 7 days of the onset of the syndrome. Signs and symptoms may include shortness of breath, tachypnea, muscle fatigue and general weakness, hypotension, dry cough, and fever. ARDS is an overwhelming pulmonary inflammatory response to certain primary and secondary harmful stimuli, such as pneumonia (e.g., aseptic, viral, bacterial), sepsis, aspiration, inhalation injury, drowning, and lung resection (see, for example, Alam, N. et al., Annals of Thoracic Surgery, 2007. 84(4): 1085–91). ARDS is characterized by rapid onset of respiratory failure requiring hospitalization and respiratory support in the intensive care unit (ICU). If a patient survives ALI / ARDS, their long-term quality of life is often adversely affected by lung scarring (see, for example, Rubenfeld, GD et al., *The New England Journal of Medicine*, 2005. 353(16): 1685–93; Dowdy, DW et al., *Critical Care Medicine*, 2006. 32(8): 1115–24). To date, no effective agents have been identified for the treatment of acute lung injury (ALI) and ARDS, and therefore there is a significant need for such agents.

[0198] As used in this article, "pneumonia" refers to an infection that inflames the air sacs in one or both lungs. The air sacs may fill with fluid or pus (purulent material), causing a cough with sputum or pus, fever, chills, and difficulty breathing.

[0199] As used in this article, “COVID” or “coronavirus-induced disease” is a collective term for diseases caused by coronaviruses. Coronaviruses are a family of viruses with many varieties, and some of them cause disease or COVID. For example, coronavirus 19 (COVID 19), discovered in 2019, causes COVID, affecting the lungs and other parts of the body, and is referred to as COVID-19.

[0200] As used herein, the term "cancer" refers to a malignant mass of tissue. Malignant tumor cells can "metastasize" (i.e., spread) to various organs throughout the body, such as the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, testes, ovaries, and uterus, via the blood and lymphatic system. Affected organs may develop dysfunction and / or lesions, and such organs can be treated using the compounds or compositions disclosed herein.

[0201] As used herein, the term "amino acid" refers to an organic compound having a carboxyl group at one end and a primary amino group at the other end. In a peptide, the carboxyl and amino groups form an amide bond, also known as a peptide bond, and any two or more amino acids linked together by a peptide bond are called a peptide. The term "residue" refers to a portion of a peptide derived from an amino acid. An amino acid having an α-amino group to a carboxyl group is called an α-amino acid. These α-amino acids, as well as other types of amino acids, typically have another substituent at the α position, referred to as a "side chain" or "R group." The chemical properties of the R group significantly influence the broader chemical properties of the amino acid. In this invention, a "cationic amino acid" is preferably an amino acid having a cation on its side chain due to protonation or alkylation of an amino group. Amino acids can be natural or protein-derived, meaning they are present in nature and used to synthesize polypeptides or proteins. Amino acids that can be used in this disclosure can also be non-natural or non-naturally occurring amino acids. For the purposes of this disclosure, non-naturally occurring amino acids that can be used to construct peptides are any organic compounds with a carboxyl group at one end and a primary amino group at the other end and a molecular weight of less than 500 Da. Such bifunctional molecules can be used to form amide bonds at both ends. The amino terminus can condense with the carboxyl group of another molecule to form an amide bond, and vice versa, to form a polymer chain. Examples of non-natural amino acids in this disclosure include 2-[2-(2-(aminoethoxy)ethoxy]acetic acid (abbreviated as Aeea), diaminobutyric acid (abbreviated as Dab), and diaminopropionic acid (Dap). For the purposes of describing compounds of formula (II) of this disclosure, "cationic alkyl moiety" should refer to the part of formula (II) corresponding to formula (I).

[0202] The single-letter codes used herein are those for naturally occurring amino acids. For example, alanine is A, arginine is R, asparagine is N, aspartic acid is D, cysteine ​​is C, glutamic acid is E, glutamine is Q, glycine is G, histidine is H, isoleucine is I, leucine is L, lysine is K, methionine is M, phenylalanine is F, proline is P, serine is S, threonine is T, tryptophan is W, tyrosine is Y, valine is V, and ornithine is O. For the purposes of this disclosure, the single-letter codes for amino acids may represent any stereoisomer of the amino acid, i.e., L- or D-amino acids. For the purposes of this disclosure, γE is glutamic acid, wherein the side-chain carboxyl group (γ) is the part used to connect to the N-terminal portion of the peptide rather than the typical α-carboxyl group.

[0203] As used herein, the term “derivative” refers to a modified peptide resulting from any one or a combination of the following: 1) a peptide with the covalent addition of an amino acid, which partially or has an inherent biological activity different from that of the modified peptide; 2) a peptide whose backbone sequence is truncated or removed by one or more amino acids; and / or 3) a peptide whose backbone sequence is replaced by one or more amino acids, wherein the derivative maintains the inherent biological activity of the original peptide.

[0204] As used herein, “bioactivity” refers to the activity of a peptide as a measurable and inherent property. The bioactivity of a natural peptide is the effect it produces after binding to a receptor, prior to any modification or alteration of its structure. In other words, it is the intrinsic bioactivity of a peptide after exposure to its receptor in vitro or in vivo. When a modified peptide exhibits measurable bioactivity similar to that of the corresponding natural or unmodified peptide, the modification can be considered not to have eliminated the peptide's bioactivity. In other words, the modified peptide can be considered to have maintained the characteristic, inherent, or intrinsic bioactivity of its natural peptide.

[0205] As used herein, the term "bioavailability" refers to the proportion of a drug or other substance that, after being introduced into the body, enters the bloodstream and is capable of exerting its active effect. Increased bioavailability can be measured using a determination of blood levels of the drug at the corresponding time point after administration. One way to measure bioavailability is to measure the area under the curve of blood levels of the drug over time. In this specification, at the corresponding time point after administration, derivatives of the drug that have significantly higher blood levels compared to the parent drug (the underivative drug) exhibit higher bioavailability.

[0206] As used herein, “sleepiness” refers to clinical signs of an inability or reluctance to resume normal activity characterized by exploration and alertness. In this disclosure, somnolent rats are readily observed to exhibit little to almost no activity and / or reluctance to explore, with half-closed eyes and an arched back posture.

[0207] As used herein, “swelling” refers to the clinical sign of an observed body part becoming enlarged or congested due to blood or fluid and associated with visible redness. It is caused by fluid accumulation in tissues. Swelling can occur throughout the body (generalized) or only in a specific part of the body (localized). In this disclosure, swelling induced by cationic alkyl molecules or peptides in rats is readily observed in the paws and muzzle and is associated with skin redness.

[0208] As used herein, “actual or reasonable mass dose” refers to the therapeutic dose actually administered after allometric scaling of the human dose, particularly regarding the volume of the injection solution, which must remain in liquid form for parenteral bolus administration. A reasonable injection volume for non-intravenous parenteral bolus administration in humans is 2 mL or less. Volumes greater than 2 mL, while feasible, are not practical for non-intravenous parenteral administration in humans. Volumes greater than 2 mL may be a consequence of limited solubility of the peptide at therapeutically effective doses. Therefore, considering practical limitations in volume and solubility, therapeutic doses must be achieved within this volume parameter range; exceeding this range is considered impractical. Allometric scaling can be used to assess doses across species.

[0209] As used herein, “subject” includes humans, domesticated animals such as laboratory animals (e.g., dogs, monkeys, pigs, rats, mice, etc.), domestic pets (e.g., cats, dogs, rabbits, etc.) and livestock (e.g., pigs, cattle, sheep, goats, horses, etc.), and non-domesticated animals. In some respects, subjects are humans.

[0210] As used herein, the phrase “therapeutic effective dose” refers to a bolus dose of a therapeutic agent (i.e., a drug or therapeutic agent compound, measured in µmol / kg or mg / kg) that elicits a biological or pharmaceutical response sought by researchers, veterinarians, physicians, or other clinicians in a tissue, system, animal, individual, or human, and includes one or more of the following: (1) Altering the levels of analytes produced by tissues and / or blood that serve as biomarkers of biological response and can alleviate disease progression; (2) To prevent the disease, symptoms or condition in individuals who may be susceptible to the disease, symptoms or condition but have not yet experienced or shown the pathology or symptomatology of the disease; (3) Suppressing the disease, symptoms, or symptom in an individual experiencing or exhibiting the pathology or symptomology of a disease, symptom, or condition; and (4) Improve the pathology or symptomology of an individual who is experiencing or exhibiting a disease, symptom or condition (i.e., reverse the pathology and / or symptomology), such as reducing the severity of the disease, prolonging survival time and / or preventing death.

[0211] As used herein, the term "composition" refers to a substance, particularly a therapeutic substance, that is formulated, mixed, suspended, doped, dissolved, and / or co-crystallized with an excipient, carrier, or solvent. This disclosure relates to compositions comprising formula (I) or formula (II) formulated with one or more pharmaceutically acceptable excipients. The compositions described herein may be used according to the purposes and / or methods described herein, for example, to supply a compound of formula (I) or formula (II) for administration to a subject. Compositions include, for example, pharmaceutical compositions or medical compositions.

[0212] As used herein, the terms “peptide” and “polypeptide” refer to polymers of amino acids. A “peptide” is a polypeptide having three or more amino acids covalently linked together by amide bonds via α-amino and α-carboxyl groups. The number of amino acid residues in a peptide can range from 3 to about 100 residues. The amino acid residues in a polypeptide or peptide can be typical or atypical, and can be modified or unmodified. As used herein, the term “protein” refers to a polypeptide large enough to have a 3-dimensional structure, such as a β-barrel or α-helix. Examples of peptides suitable for inclusion in the compounds and conjugated peptides of this disclosure include, for example, any one of SEQ ID NO: 23-50.

[0213] As used herein, the terms "subcutaneous administration," "administered subcutaneously," "sc," "sc administration," "SC," and "SC administration" refer to the direct delivery of a drug, typically in liquid form, into the adipose tissue beneath the skin. Delivery is usually performed by direct injection. These injections are shallower than injections into muscle tissue. For drugs intended for slow and steady absorption into the bloodstream, providers often use subcutaneous injection.

[0214] As used herein, the terms "intravenous administration," "IV administration," and "IV injection" refer to the direct delivery of a drug, typically in liquid form, into a vein in an animal or human. Delivery is usually performed by direct injection. Intravenous administration can be used for injection, using a syringe at higher pressure; it can also be used for infusion, for example, using pressure provided by gravity.

[0215] As used herein, the terms "intramuscular administration," "IM administration," and "IM injection" refer to the direct delivery of a drug, typically in liquid form, into the muscle of an animal or human. Delivery is usually performed by direct injection. This allows the drug to be rapidly absorbed into the bloodstream. In some cases, individuals may administer IM injections themselves. In some embodiments, IM injections may be used instead of intravenous injections, for example, when certain therapeutic agents are irritating to veins, or when a suitable vein cannot be located.

[0216] As used herein, the term "nasal administration" refers to the delivery of a therapeutic agent (e.g., in the form of a gel, liquid, aerosol, gas, or powder) through topical application, as a liquid drop, or by inhalation into the nose of an animal or human (e.g., by blowing or spraying). This form of administration (depending on the formulation) can be used to deliver the therapeutic agent into the nasal cavity or lungs (depending on the device used). The therapeutic agent may not be absorbed systemically (purely topical application), may be completely absorbed systemically (purely systemic application), or may be partially absorbed both topically and systemically. Nasal sprays may include locally acting drugs that typically have minimal systemic effects.

[0217] As used herein, the terms "inhalation administration" and "administration by inhalation" refer to the delivery of a therapeutic agent (usually in the form of a gas or aerosol) via the mouth or nose (blown in). Inhalation administration allows the therapeutic agent to rapidly reach body tissues through near-instantaneous contact with the blood supplying the alveoli (air sacs) of the lungs. In laboratory animals, inhalation is similar to intratracheal administration (IT), which avoids the nasal region and thus distinguishes inhalation from nasal administration (intranasal). In this specification, intratracheal administration (IT) is similar to administration by inhalation.

[0218] As used herein, the terms “parenteral” and “non-gastrointestinal” administration refer to routes of administration that do not involve the enteral or gastrointestinal tract. Examples of parenteral administration include subcutaneous (under the skin), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intraperitoneal (infusion or injection into the peritoneum), inhalation (e.g., administration via the trachea or inhalation into the lower respiratory tract), nasal administration (through the nose), sublingual and buccal administration, intrathecal (into the spinal canal), intracerebral (into the brain), intravenous (into the ventricles), intradermal (into the skin itself), or any other route of administration that does not involve the gastrointestinal tract. As used herein, the term “enteral” means administration to any region of the digestive tract, including the mouth (oral cavity), pharynx (larynx), esophagus, stomach, small intestine, large intestine, rectum, anus, and any artificial openings in these regions.

[0219] As used herein, the term "excipient" refers to a substance formulated or mixed with the active pharmaceutical ingredient for the purpose of providing long-term stability, making formulations containing small amounts of potent active ingredients fuller (hence often referred to as "bulking agents," "fillers," or "diluents"), and / or enhancing the therapeutic effect of the active pharmaceutical ingredient in the final dosage form, such as improving drug absorption, potency, dosage, viscosity, solubility, and / or duration of action or the presence of the active pharmaceutical ingredient in the blood. The selection of a suitable excipient depends on the route of administration, dosage form, active pharmaceutical ingredient, and other factors. Excipients can include sugars, amino acids, buffers, antioxidants, chelating agents, solvents, mediators, and / or complex polymers that bind to and stabilize the active pharmaceutical ingredient in vitro and / or in vivo. While excipients were once considered "inactive" components, it should now be understood that they can sometimes be key determinants of dosage form performance. In other words, the effects of excipients on pharmacodynamics and pharmacokinetics can be very important and require extensive investigation and research. In fact, how excipients affect the delivery of the active pharmaceutical ingredient is often unpredictable.

[0220] For the purposes of this disclosure, "ataxia" means "toxicity" and is characterized by observed clinical signs of poor muscle control leading to clumsy voluntary movement and / or death. This manifests as difficulty in movement, coordination, and / or eye movement. In determining toxicity (where the observation of ataxia is used as a marker of toxicity), the bolus dose is increased until ataxia (i.e., toxicity) is observed, up to a maximum of 10 µmol / kg, which is the maximum practical bolus dose of a peptide administered parenterally in a small volume injection. As used herein, "ataxia" is also a clinical sign manifested as poor muscle control and coordination leading to uncomfortable, unsteady, or clumsy voluntary movement. Examples presented include rats with ataxia exhibiting difficulty in movement, coordination, and / or eye movement. The dose that causes ataxia and / or death is considered the toxic dose. As used herein, seeking the "maximum tolerated dose" or "MTD" refers to an in vivo safety assessment of the compound under investigation. For the purposes of this disclosure, the MTD of the test compound is the highest bolus dose to which no adverse reactions or ataxia are observed. Drowsiness is reversible and is not considered an adverse reaction. In other words, the MTD is the highest bolus dose administered to a group of animals without causing visible or observable toxicity compared to the control group. In the case of testing the MTD of a cationic alkyl moiety or a peptide-cationic alkyl conjugate, the control group is the mediator group. In this disclosure, the effects commonly observed after administration of the cationic alkyl moiety primarily include reversible swelling, changes in skin pigmentation, and reversible drowsiness.

[0221] As used herein, the terms “therapeutic index,” “TI,” and “therapeutic ratio” are quantitative measures of the relative safety of a drug. For the purposes of this disclosure, TI refers to the ratio of the highest bolus dose that does not cause adverse reactions such as ataxia or drowsiness to the highest bolus dose that does not cause observable peripheral discoloration. The highest bolus dose that does not cause adverse reactions relative to a control may also be referred to as the “No Observed Adverse Effect Level” or “NOAEL.” The highest bolus dose that does not cause observable peripheral discoloration compared to an untreated control may also be referred to as the “No Observed Response Level” or “NOEL.” Therefore, TI can also be interpreted as NOAEL / NOEL. By providing a wider safety margin for administration during disease treatment, peptides with a high therapeutic index will have better safety than peptides with a low therapeutic index. This NOAEL / NOEL ratio is a quantitative measure of relative safety, a comparison of the bolus dose that initiates a therapeutic effect (e.g., vasodilation for the purposes of the examples in this specification) to the highest bolus dose prior to the dose that causes toxicity.

[0222] For the purposes of this specification, peripheral discoloration includes redness of the skin on the extremities (e.g., hands, feet, ears, and / or lips). In rats, peripheral vasodilation is associated with redness that may or may not include swelling, while peripheral vasoconstriction is associated with pallor of the extremities. Redness or pallor is determined by side-by-side comparison of treated subjects with untreated control subjects. The Sprague Dawley rat used in the included examples is a white rat whose color changes are easily observed.

[0223] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While similar or equivalent methods and materials may be used to practice or test this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting. It is readily understood that, as generally described herein and shown in the accompanying drawings, aspects of this disclosure can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0224] Furthermore, the specific arrangements shown in the figures and / or tables should not be considered limiting. It should be understood that other embodiments may include more or fewer of the elements shown in the figures and / or tables. Further, some of the elements shown may be combined or omitted. Still further, exemplary embodiments may include elements not shown in the figures and / or tables. As used herein, in relation to measurement results, “about” means + / - 5%. As used herein, the stated ranges include endpoints, such as 0.5 mol% to 99.5 mol% including both 0.5 mol% and 99.5 mol%.

[0225] Non-limiting embodiments of this disclosure Example 1. A compound comprising a cationic alkyl moiety of formula (I): J-(CH2)x(CO)-(A)y-(B)z- (I), in: J is either HOOC or CH3; x is 10⁻¹⁶; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-aminobutyric acid (γAbu), γ-linked glutamic acid (γE) and α-linked glutamic acid (E); y is 2-4; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2-4; Wherein -(B)z- contains no more than 2 Dab residues, and wherein the Dap or Dab residues are linked by an α-amino group.

[0226] Example 2. The compound according to Example 1, wherein: J is CH3; x is 10, 12, 14, or 16; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

[0227] Example 3. The compound according to Example 1, wherein: J is CH3; x is 10, 12, 14, or 16; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-linked glutamic acid (γE); y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

[0228] Example 4. The compound according to Example 1, wherein: J is CH3; x is 10, 12, 14, or 16; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

[0229] Example 5. The compound according to Example 1, wherein: J is CH3; x is 10, 12, 14, or 16; A is γ-aminobutyric acid (γAbu). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

[0230] Example 6. The compound according to Example 1, wherein: J stands for HOOC; x is 10, 12, 14, or 16; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

[0231] Example 7. The compound according to Example 1, wherein: J stands for HOOC; x is 10, 12, 14, or 16; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-linked glutamic acid (γE). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

[0232] Example 8. The compound according to Example 1, wherein: J stands for HOOC; x is 10, 12, 14, or 16; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

[0233] Example 9. The compound according to Example 1, wherein: J stands for HOOC; x is 10, 12, 14, or 16; A is γ-linked glutamate (γE). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

[0234] Example 10. The compound according to Example 1, wherein: J is CH3; x is 14; (A)y is Aeea-Aeea-Aeea γAbu - γAbu - γAbu, γAbu -Aeea-Aeea、 γE -Aeea-Aeea、 E -Aeea-Aeea;Aeea-Aeea γAbu - γAbu, γAbu -Aeea γE -Aeea or E -Aeea; and (B)z is Dap-Dap, Dab-Dab, Dab-Dap or Dap-Dab.

[0235] Example 11. The compound according to Example 1, wherein: J is CH3; x is 14; (A)y is Aeea-Aeea-Aeea γAbu - γAbu - γAbu, γAbu -Aeea-Aeea γE -Aeea-Aeea or E -Aeea-Aeea; and (B)z is Dap-Dap, Dab-Dab, Dab-Dap or Dap-Dab.

[0236] Example 12. The compound according to Example 1, wherein the cationic alkyl moiety is selected from SEQ ID NO: 10 to 22 and 51 to 69.

[0237] Example 13. The compound according to Example 1, wherein the cationic alkyl moiety is selected from SEQ ID NO: 10 to 22.

[0238] Example 14. The compound according to any one of Examples 1 to 13, wherein the compound, when conjugated with a peptide, did not cause clinically observable ataxia after parenteral bolus administration in rats at doses of 10 µmol / kg and lower.

[0239] Example 15. A compound according to any one of Examples 1 to 14, used for modifying a peptide by covalent conjugation.

[0240] Example 16. A conjugated peptide of formula (II): CH3(CH2)x(CO)-(A)y-(B)z-peptide (II) in: x is 10⁻¹⁶; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-aminobutyric acid (γAbu), γ-linked glutamic acid (γE) and α-linked glutamic acid (E); y is 2-4; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2-4; in: -(B)z- contains no more than 2 Dab residues, wherein the Dap or Dab residues are linked by an α-amino group; CH3(CH2)x(CO)-(A)y-(B)z- is covalently linked to the N-terminus of the peptide or to one of the amino groups on the side chain of the peptide; Optionally, wherein at an equivalent bolus dose, the bioactivity of the conjugated peptide is equal to or greater than that of the unmodified peptide; and / or At the same time point following bolus administration of an equivalent dose, the blood level of the conjugated peptide was equal to or higher than that of the unconjugated peptide.

[0241] Example 17. The conjugated peptide according to Example 16, wherein the conjugated peptide binds to a natriuretic peptide receptor and has no adverse reactions or ataxia in rats at 3.0 µmol / kg and lower bolus doses.

[0242] Example 18. A conjugated peptide according to Example 16 or 17, wherein the CH3(CH2)x(CO)-(A)y-(B)z- moiety is covalently linked to the N-terminus of the peptide.

[0243] Example 19. A conjugated peptide according to any one of Examples 16 to 18, wherein the peptide is a natriuretic peptide or a natriuretic peptide derivative of SEQ ID NO: 32, 44, 48 or 75.

[0244] Example 20. A conjugated peptide according to any one of Examples 16 to 19, wherein the CH3(CH2)x(CO)-(A)y-(B)z- portion is selected from SEQ ID NO: 10 to 22 and 51 to 69.

[0245] Example 21. The conjugated peptide according to Example 20, wherein the CH3(CH2)x(CO)-(A)y-(B)z- portion is selected from SEQ ID NO: 10 to 22.

[0246] Example 22. A conjugated peptide according to any one of Examples 16 to 20, wherein the peptide is a natriuretic peptide derivative, and one or more methionine residues of the natriuretic peptide derivative are replaced by glutamine (Q), leucine (L), oroleucine (Nle) or methoxinine (Mox).

[0247] Example 23. A conjugated peptide according to any one of Examples 16 to 22, wherein the peptide is a natriuretic peptide or a derivative thereof according to SEQ ID NO:32, wherein one or more methionine residues are replaced by glutamine (Q), and the CH3(CH2)x(CO)-(A)y-(B)z- portion is selected from SEQ ID NO: 10 to 22.

[0248] Example 24. A conjugated peptide according to any one of Examples 16 to 23, wherein the conjugated peptide is selected from SEQ ID NO: 29-31, 33-43, 45-47, 49-51.

[0249] Example 25. The conjugated peptide according to Example 24, wherein the conjugated peptide is selected from SEQ ID NO: 29-31 and 33-43.

[0250] Example 26. The conjugated peptide according to Example 25, wherein the conjugated peptide is selected from SEQ ID NO: 29-31.

[0251] Example 27. The conjugated peptide according to Example 26, wherein the conjugated peptide is SEQ ID NO: 29.

[0252] Example 28. The conjugated peptide according to Example 26, wherein the conjugated peptide is SEQ ID NO: 30.

[0253] Example 29. The conjugated peptide according to Example 26, wherein the conjugated peptide of formula (II) is SEQ ID NO:31.

[0254] Example 30. A conjugated peptide according to any one of Examples 16 to 29, wherein the conjugated peptide binds to natriuretic peptide receptor B (NPRB), natriuretic peptide receptor C (NPRC), or a combination thereof.

[0255] Example 31. A conjugated peptide according to any one of Examples 16 to 30, wherein the conjugated peptide is an NPRB agonist.

[0256] Example 32. A conjugated peptide according to any one of Examples 16 to 31, wherein the conjugated peptide is an NPRC agonist.

[0257] Example 33. A conjugated peptide according to any one of Examples 16 to 32, wherein the conjugated peptide produces a physiological effect selected from the following: sustained increase in blood cGMP, alteration of cAMP, alteration of blood pressure, increase in sepsis survival, increase in acute lung injury survival, increase in acute respiratory distress syndrome survival, reduction of MPO-positive cells, reduction in the number of cells in alveolar fluid or bronchoalveolar lavage fluid, reduction in the amount of protein in alveolar fluid or bronchoalveolar lavage fluid, reduction in endothelial permeability, reduction in lung weight by body weight, reduction in monocyte chemoattractant protein-1, reduction in IL-6, reduction in TNF-α, reduction in A1008 / A9, reduction in fibrosis, reduction in tumor volume, reduction in metastasis, reduction in inflammation, antiproliferative effect, reduction in cancer burden, inhibition of cyclooxygenase 2 (COX-2) expression, antagonism of the renin-angiotensin-aldosterone system, inhibition of cardiac hypertrophy or combinations thereof.

[0258] Example 34. The compound according to any one of Examples 1 to 15 or the conjugated peptide according to any one of Examples 16 to 33, used to prepare a pharmaceutical composition.

[0259] Example 35. A compound or conjugated peptide for use according to Example 34, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers or excipients.

[0260] Example 36. A compound or conjugated peptide for use according to Example 35, wherein one or more pharmaceutically acceptable carriers or excipients comprise a bulking agent, a buffer, a stabilizer, a preservative, or a combination thereof.

[0261] Example 37. A compound according to any one of Examples 1 to 15 or a conjugated peptide according to any one of Examples 16 to 33, used to treat a disease or condition in a subject in need.

[0262] Example 38. A compound or conjugated peptide for use according to Example 37, wherein the compound or conjugated peptide is selected from: a) Any one of SEQ ID NO: 29-31, 33-43, 45-47, 49-51, or b) Any one of SEQ ID NO: 29-31, 33-43, 45-47, or c) Any one of SEQ ID NO: 29-31, 33-43, or d) Any one of SEQ ID NO: 29-31, or e) SEQ ID NO: 29 or f) SEQ ID NO: 30, or g) SEQ ID NO: 31.

[0263] Example 39. A compound for use according to any one of Examples 37 or 38, wherein the compound or the conjugated peptide comprises SEQ ID NO: 31.

[0264] Example 40. A compound or conjugated peptide for use according to any one of Examples 37 to 39, wherein the disease or condition affects the lungs (e.g., ALI, ARDS, COVID, inflammation, sepsis, fibrosis, or cancer), liver (e.g., non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), inflammation, fibrosis, or cancer), heart (e.g., heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), acute heart failure, or congestive heart failure), bones / joints (e.g., osteoporosis, osteoarthritis, rheumatoid arthritis, inflammation, cancer, or dwarfism), kidneys (e.g., chronic kidney disease (CKD), acute kidney injury (AKI), drug-induced kidney injury, inflammation / nephritis, renal fibrosis, glomerulosclerosis, or renal cancer), prostate (e.g., benign prostatic hyperplasia or prostate cancer), brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, testes, ovaries, uterus, and / or blood vessels).

[0265] Example 41. A compound or conjugated peptide for use according to any one of Examples 37 to 39, wherein the disease or symptom is pre-metastatic cancer or post-metastatic cancer.

[0266] Example 42. The compound or conjugated peptide for use according to Example 41, wherein the cancer is a cancer selected from any one or more of the following organs: lung, pleura, liver, heart, bone / joint, kidney, prostate, breast, brain, eye, skin, muscle, blood, blood vessels, gastrointestinal tract, bladder, testis, ovary and / or uterus.

[0267] Example 43. A compound or conjugated peptide for use according to any one of Examples 37 to 39, wherein the disease or symptom is pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), or COVID in the subject of need.

[0268] Example 44. A compound or conjugated peptide for use according to any one of Examples 37 to 39, wherein the disease or symptom is fibrosis.

[0269] Example 45. A compound or conjugated peptide for use according to any one of Examples 37 to 44, wherein the treatment comprises administering to the subject a therapeutically effective bolus dose of 10.0 µmol / kg or less and / or 10.0 µmol / kg to 0.0001 µmol / kg, including the end value.

[0270] Example 46. A compound or conjugated peptide for use according to any one of Examples 37 to 45, wherein the compound is administered to the subject as a single therapy or in combination with one or more other agents or treatments.

[0271] Example 47. The compound or conjugated peptide for use according to Example 46, wherein one or more additional agents or treatments are selected from immune checkpoint inhibitors, surgery / amputation, radiation, chemotherapy or combinations thereof.

[0272] Example 48. The compound for use according to any one of Examples 37 to 46, wherein the compound is applied subcutaneously, by infusion, by inhalation, by nasal spray, orally, by eye drops, and / or by topical application.

[0273] Example 49. A compound or conjugated peptide for use according to any one of Examples 37 to 42, wherein the compound or the conjugated peptide is applied to the subject subcutaneously, by infusion, by inhalation, by nasal spray, orally, by eye drops, and / or by topical application.

[0274] Example 50. A composition comprising a compound according to any one of Examples 1 to 15 or a conjugated peptide according to any one of Examples 16 to 33, and one or more pharmaceutically acceptable carriers or excipients.

[0275] Example 51. The composition according to Example 49, wherein one or more pharmaceutically acceptable carriers or excipients comprise a leavening agent, a buffer, a stabilizer, a preservative, or a combination thereof.

[0276] Example Example 1: Clinically observable effects of various cationic alkyl moieties at concentrations of 3.0, 5.0, or 10 µmol / kg. Adverse reaction tests showed that the toxicity of cationic alkyl moieties containing Dap and / or Dab as cationic residues was significantly lower than that of those containing... It has the toxicity of the cationic alkyl moiety of cationic amino acids with a long R group.

[0277] Data in Table 1 were generated according to the experimental protocol below. Animal care and treatment: Male Sprague-Dowley rats (8 to 10 weeks old, Charles River, Hollister, CA) were housed in pairs (n = 3 / group) in disposable polypropylene cages with rodent cob bedding at the PharmaIN Animal Facility. Animals had free access to food (LabDiet Pico Rodent #5053; Animal Specialties, Woodburn, OR) and water. Temperature (68–74℉) and humidity (30–60%) were maintained within controlled ranges using a 12-hour light / dark cycle. Drug administration and observation: Each rat received a subcutaneous injection of sterile aqueous buffer containing cationic alkyl-modified agents (SEQ ID NO: 1–22; listed in Table 1 below) adjusted to pH 4.5–5 at an initial bolus dose of 5.0 µmol / kg and an injection volume of 1.0 mL / kg. The bolus dose was adjusted based on observations, and animals were closely monitored for lethargy, ataxia, swelling, or discoloration during the first 4 hours post-injection, and hourly thereafter. As used herein, “ataxia” refers to a clinical sign of poor muscle control resulting in clumsy voluntary movement. It may cause difficulty in movement, coordination, and eye movement. As used herein, “lethargy” refers to a clinical sign of inability or reluctance to resume normal activity characterized by exploration and alertness. Lethargic rats are readily observed to exhibit little to almost no activity and / or reluctance to explore, half-closed eyes, and an arched posture. As used herein, “swelling” refers to a clinical sign of observed swelling or congestion of a body part due to blood or fluid and associated with visible redness (discoloration). If swelling did not occur, the dose level was increased to 10 µmol / kg; if swelling occurred, the bolus dose level was reduced to 3.0 µmol / kg. A one-week clearance period was allowed between the two bolus doses, and animals were humanely euthanized by CO2 asphyxiation after final observation. Example 2: With cationic amino acids containing a long R group (such as lysine or arginine) or their non-natural D-isomers Compared to alkylated natriuretic peptides, alkylated natriuretic peptides containing alkylated Dap / Dab produce clinically observable levels in rats. There were fewer adverse reactions or ataxias. Furthermore, alkylated peptides exhibited improved pharmacokinetic properties compared to unmodified peptides. Mechanical and / or pharmacodynamic properties (see Example 3, Table 3).

[0279] The data in Table 2 were generated according to the experimental protocol below. Animal care and treatment: Male Sprague-Dowley rats aged 8 to 10 weeks (Charles River Laboratory, Hollister, CA) were randomly divided into two groups (n = 3 / group) and housed in disposable polypropylene cages with rodent cob bedding at the PharmaIN Animal Facility. Animals had free access to food (LabDiet Pico Rodent Diet #5053; Animal Specialties, Woodburn, Oregon) and water. Temperature (68–74℉) and humidity (30–60%) were maintained within controlled ranges using a 12-hour light / dark cycle. Drug administration and observation: During these dose range studies, rats were treated with a lead buffer containing up to 10 mg / kg of cationic alkyl-modified CNP (SEQ ID NO: 23–31, listed in Table 2 below; PharmaIN, Bothell, WA) by subcutaneous bolus injection between the scapulae at a dose volume of 1.0 mL / kg. Animals were closely monitored for the first 4 hours after injection, and then hourly for the remainder of the day, for any signs of swelling or discoloration (expansion side effect), lethargy (side effect), or ataxia (a marker of action toxicity). A 1-week clearance period was allowed between bolus doses in rats. Animals were humanely euthanized by CO2 asphyxiation after final observation. Compared to CNPs modified with the cationic alkyl moiety of formula (I) [e.g., SEQ ID NO: 29-31 (see Table 2)], cationic alkyl groups containing longer R groups of cationic amino acids (both natural and non-natural) (such as lysine or arginine) (e.g., CH3(CH2)14(C=O)-KKGGGKK- [SEQ ID NO: 70], CH3(CH2)14(C=O)-GGGKKKK- [SEQ ID NO: 71], CH3(CH2)14(C=O)-kkkkGGG- [SEQ ID NO: 72], CH3(CH2)14(C=O)-KKGGGRR- [SEQ ID NO: 73], CH3(CH2)14(C=O)-KKGGG-Dab-Dab- [SEQ ID NO: 74]) produce highly toxic conjugates [e.g., SEQ ID NO: 29-31 (see Table 2)]. 23-28 (see Table 2)]. Example 3: Pharmacokinetics of several natriuretic peptides (NPs) modified with alkylated cationic Dap or Dab residues. Pharmacokinetic data. These modifications exhibited improved pharmacokinetics, such as higher 2-hour and 6-hour plasma cGMP. The levels demonstrated. Compared to the corresponding natural NP, when administered to mice at a bolus dose of 1.0 mg / kg (≤ 0.45 µmol / kg)... When administered subcutaneously, the pharmacokinetics are also improved, as evidenced by its significant presence in the blood (NP level).

[0281] The data in Table 3 were generated according to the experimental protocol below. Animal care and handling: Male CD-1 mice aged 6 to 9 weeks (Charles River Laboratory, Hollister, California) were housed in groups of 5–6 in disposable polypropylene cages with rodent cob bedding and free access to LabDiet Pico rodent #5053 food and water. Animals were housed in the PharmaIN animal facility under controlled temperature (68–74℉) and humidity (30–60%), with a 12-hour light / dark cycle. Drug administration and blood sampling: All animals were treated with one of the following natriuretic peptides: natural human ANP (SEQ ID NO: 44; Chempep Inc., Wellington, FL), BNP (SEQ ID NO: 48; Tocris, Minneapolis, MN), CNP (SEQ ID NO: 32; Chempep Inc., Wellington, FL), or cationic alkyl-modified NP (as listed in Table 3; PharmaIN, Bosell, Washington) via a bolus injection into the interscapular region. The test product was prepared or dissolved in sterile water for injection on the day of administration. No adverse reactions were observed in any animals at this bolus dose. Blood samples were collected via retroorbital hemorrhage at 2 and 6 hours post-injection, with two hemorrhages collected from each animal at two distinct time points. Samples were collected in K2EDTA tubes and centrifuged (2000 xg; 15 min, 4°C for 30 min after collection). The resulting plasma samples were stored at -80°C. Biochemical analysis: Plasma concentrations of the corresponding natriuretic peptides were analyzed using the following commercially available ELISA kits from Phoenix Pharmaceuticals (Burlingham, CA): ANP ELISA (Catalog No. EKE-005-06), BNP ELISA (Catalog No. EKE-011-03), and CNP ELISA (Catalog No. EKE-012-03). The ANP and BNP kits detect only the corresponding derivatives, while the CNP kit detects the cyclic structure of CNP and all CNP derivatives with the same reactivity level. Plasma cGMP was analyzed using a commercially available kit from Abogen Pharmaceuticals (ab133052, Waltham, MA). Example 4: Compared with natural CNP, subcutaneous administration of cationic alkyl-modified CNP prolongs plasma retention time and increases... Strong biological activity.

[0283] The data in Figure 1 were generated according to the experimental protocol below. Animal care and handling: Male CD-1 mice aged 6–9 weeks (Charles River Laboratory, Hollister, California) were housed in groups of 5–6 in disposable polypropylene cages with rodent cob bedding and free access to LabDiet Pico rodent #5053 food and water. Animals were housed in the PharmaIN animal facility under controlled temperature (68–74℉) and humidity (30–60%), with a 12-hour light / dark cycle. Drug administration: Male CD-1 mice received a single subcutaneous injection of 1.0 mg / kg of natural human CNP (SEQ ID NO: 32, sequence GLSKGCFGLKLDRIGSMSGLGC; Chempep Inc., Wellington, Florida) or a cationic alkyl-modified CNP derivative synthesized at PharmaIN (SEQ ID NO: 29, sequence CH3(CH2)). 14 (C=O)-Aeea-Aeea-Aeea-Dab-Dab-GLSKGCFGLKLDRIGSQSGLGC, or SEQ ID NO: 31, with the sequence CH3(CH2). 14 (C=O)-Aeea-Aeea-Aeea-Dap-Dap-GLSKGCFGLKLDRIGSQSGLGC). The lead buffer is used to prepare or dissolve all test products administered on the same day. Blood Sampling: Blood samples were collected via retro-orbital hemorrhage at several time points (0, 0.5, 1, 2, 4, 6, 8, and 24 hours) and processed in K2EDTA tubes (2000 xg; 15 min, 4°C for 30 minutes after collection) to obtain plasma. Plasma samples were aliquoted and stored at -80°C until analysis. Biochemical Analysis: For pharmacokinetic studies, plasma aliquots were thawed at 4°C and analyzed using the commercially available CNP ELISA kit (catalog number EKE-012-03) from Phoenix Pharmaceuticals, which detects the cyclic structure of CNP and all CNP derivatives at the same level of reactivity as the ELISA kit. CNP derivatives are modified human CNP molecules in which the methionine in the cyclic portion of the natural CNP (i.e., GLSKGCFGLKLDRIGSMSGLGC) is replaced by glutamine (Q) (i.e., GLSKGCFGLKLDRIGSQSGLGC), and for SEQ ID NO: 29 and 31, the N-terminus is replaced with CH3(CH2), respectively. 14(C=O)-Aeea-Aeea-Aeea-Dab-Dab [SEQ ID NO: 10] or CH3(CH2) 14 (C=O)-Aeea-Aeea-Aeea-Dap-Dap [SEQ ID NO: 14] extension. For pharmacodynamic or bioactivity studies of cGMP, plasma aliquots were thawed at 4°C and then analyzed using a commercially available cGMP kit from Abogen Laboratories (ab133052, Waltham, MA).

[0284] Example 5: Single and repeated administration of cationic alkyl-modified C-type natriuretic peptides increased LPS-induced sepsis and ALI. Survival probability of animal models.

[0285] When administered subcutaneously (SC) or intratracheally (IT) to mice, both SEQ ID NO: 31 and SEQ ID NO: 29 effectively improved ALI survival. The data in Figure 2 were generated according to the experimental protocol described below. Animal care and treatment: Male C57BL / 6J mice (6 weeks old) were purchased from Kyudo (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and a standard mouse diet (MF diet, Oriental Yeast Co., Ltd. Tokyo, Japan). Figure 2A Drug administration: Mice were injected intraperitoneally (IP) with LPS (15 mg / kg; Sigma-Aldrich) and treated with various test products, including cationic alkyl-modified CNPs (SEQ ID NO: 31 and 29; 0.3 mg / kg (0.1 µmol / kg) SC). Control groups received LPS treatment without any test product. Test products were administered immediately after LPS administration. Survival was monitored every 2 hours over 8–56 hours, after which mice were euthanized under isoflurane anesthesia. Statistical analysis was based on the Gihen-Breslow-Wilkerson test using GraphPad Prism (n = 10, 10, and 10; controls, SEQ ID NO: 31, and SEQ ID NO: 29). P<0.01, P<0.05, compared to the control group. Figure 2BDrug administration: Mice were administered LPS (20 mg / kg; Sigma-Aldrich) via intratracheal (IT) injection and treated with various test products, including cationic alkyl-modified CNPs (SEQ ID NO: 31 and 29; 0.3 mg / kg (0.1 µmol / kg) IT). Control groups received LPS treatment without any test product. Test products were administered immediately after LPS administration and repeated every 24 hours for a total of three bolus doses. Survival was monitored every 8 hours for 72 hours, after which mice were euthanized under isoflurane anesthesia. Statistical analysis was based on the Gihen-Breslow-Wilkerson test using GraphPadPrism (n = 6, 6, and 6; controls, SEQ ID NO: 31, and SEQ ID NO: 29). P<0.01, P<0.05, compared to the control group.

[0286] Example 6: Bulk administration of cationic alkyl-modified C-type natriuretic peptides inhibited lung injury and indicated the regression of ALI / ARDS.

[0287] The data in Figure 3 were generated according to the experimental protocol described below. Animal care and treatment: Male C57BL / 6J mice (6 weeks old) were purchased from Yukido Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and a standard mouse diet (MF diet, Oriental Yeast Co., Ltd., Tokyo, Japan). Drug administration: Mice were administered LPS (0.05 mg / kg IT; Sigma-Aldrich) followed by treatment with various test products, including cevelexatol (150 mg / kg; Nipro, Osaka, Japan), an inhibitor of human neutrophil elastase injected intraperitoneally (IP) as a positive control, and cationic alkyl-modified CNP (SEQ ID NO: 31, 29, and 30; 0.3 mg / kg (0.1 µmol / kg) SC). Test products were administered immediately after LPS injection. Additionally, a normal control (NC) group without LPS administration and a control group receiving only LPS without any test products were included. Twenty-four hours later, the mice were euthanized under isoflurane anesthesia, and their lungs were collected for analysis. Figure 3BBiochemical analysis of lung samples: Lungs were minced in Tri-Reagent (Cosmo Bio, Tokyo, Japan) and then CHCl3 was added. After incubation at room temperature for 3 minutes, the samples were centrifuged (12,000 × g, 4°C, 15 minutes). The aqueous layer was collected, and an equal volume of 2-propanol was added. After incubation at room temperature for 10 minutes, the samples were centrifuged (12,000 × g, 4°C, 15 minutes), and the supernatant was discarded. Then, 75% EtOH was added, and the samples were centrifuged (12,000 × g, 4°C, 5 minutes). The supernatant was discarded, and the pellets were dissolved in nuclease-free water (Ambion, MA, USA). The gene expression levels of S100A8 and S100A9 were determined by qRT-PCR analysis using a cDNA synthesis kit (Kagem, Venlo, Netherlands). The expression of myeloid cell-derived proteins (S100A8 / A9) was elevated in several types of inflammatory lung disease. Statistical analysis was based on the Dunnett test performed using GraphPad (n = 5, 8, 8, 8, 8, 8 and 8; NC, Control, PC, A [SEQ ID NO: 31], B [SEQ ID NO: 29], Control, D [SEQ ID NO: 30]). P<0.001, P<0.01, P<0.05, relative to each corresponding control group. Figure 3C Biochemical analysis of (D): Lung tissue was fixed with 4% paraformaldehyde. The fixed lung tissue was embedded in paraffin and sectioned. Immunohistochemical staining was performed on the sections using anti-MPO rabbit polyclonal antibody (Agilent Technologies, Santa Clara, CA) followed by horseradish peroxidase-labeled anti-rabbit IgG goat polyclonal antibody (Nichirei Bioscience Inc., Tokyo, Japan). Detection was performed using 3,3'-diaminobenzidine-4HCl (DAB) (Agilent Technologies, Santa Clara, CA). Myeloperoxidase positivity (MPO) was detected by field of view. + The number of cells was quantified. Increased neutrophil count is common in ALI and ARDS. MPO +Cells serve as a direct measure of neutrophil presence. Statistical analysis was based on the Dunnett test performed using GraphPad (n = 5, 8, 8, 8, 8, 8 and 8; NC, Control, PC, A [SEQ ID NO: 31], B [SEQ ID NO: 29]; Control, D [SEQ ID NO: 30]). P<0.001, relative to each corresponding control group.

[0288] Example 7: Cationic alkyl-modified CNP derivatives reduce neutrophil infiltration in the lungs, indicating ALI / ARDS faded.

[0289] The data in Figure 4 were generated according to the experimental protocol described below. Animal care and treatment: Male C57BL / 6J mice (6 weeks old) were purchased from Yukido Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and a standard mouse diet (MF diet, Oriental Yeast Co., Ltd., Tokyo, Japan). Drug administration: Mice were administered LPS (0.05 mg / kg IT; Sigma-Aldrich) followed by treatment with various test products, including cevelexatol (150 mg / kg; Nipro, Osaka, Japan), an inhibitor of human neutrophil elastase injected as a positive control (IP), and cationic alkyl-modified CNP (SEQ ID NO: 31, 29, and 30; 0.3 mg / kg (0.1 µmol / kg) SC). Test products were administered immediately after LPS injection. Additionally, a normal control (NC) group without LPS administration and a control group receiving only LPS without any test products were included. Twenty-four hours later, mice were euthanized under isoflurane anesthesia, and bronchoalveolar lavage fluid (BALF) was collected. Biochemical analysis: The total number of cells in the BALF was counted using a counting chamber. The total protein concentration in the BALF was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Statistical analysis was based on the Dunnett test performed using GraphPad (n = 5, 8, 8, 8, 8, 8 and 8; NC, Control, PC, A [SEQ ID NO: 31], B [SEQ ID NO: 29]; Control, D [SEQ ID NO: 30]). P < 0.001, relative to each corresponding control group. ALI and ARDS were associated with an increase in cells, particularly neutrophils, in BALF. To assess the regression of ALI / ARDS in animal models, it is common practice to measure cell count ( Figure 4B) and total protein level ( Figure 4C These markers act as markers of neutrophils. A decrease in these markers indicates regression of ALI / ARDS.

[0290] Example 8: The table shows that treatment with cationic alkyl-modified CNP derivatives weakened LPS-induced BALF. Upregulation of inflammatory cytokines.

[0291] The data in Table 4 were generated according to the experimental protocol described below. Animal care and treatment: Male C57BL / 6J mice (6 weeks old) were purchased from Yukido Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and a standard mouse diet (MF diet, Oriental Yeast Co., Ltd., Tokyo, Japan). Drug administration: Mice were administered LPS (0.05 mg / kg IT; Sigma-Aldrich) followed by treatment with various test products, including cevelex (150 mg / kg; Nipro, Osaka, Japan), an inhibitor of human neutrophil elastase injected as a positive control (IP), and cationic alkyl-modified CNP (SEQ ID NO: 31, 29, and 30; 0.3 mg / kg (0.1 µmol / kg) SC). Test products were administered immediately after LPS injection. Additionally, a normal control (NC) group without LPS administration and a control group receiving only LPS without any test products were included. Twenty-four hours later, mice were euthanized under isoflurane anesthesia, and BALF was collected. Biochemical analysis: ALI and ARDS are typically characterized by elevated levels of inflammatory cytokines in BALF, including macrophage chemokine-1 (MCP1), interleukin-6 (IL-6), and tissue necrosis factor-α (TNFα). Decreased concentrations of these cytokines can indicate the regression of ALI / ARDS. The concentrations of each cytokine (MCP1, IL-6, and TNFα) were measured using an ELISA kit (R&D Systems, Minneapolis, MN). Previous studies have highlighted the important role of TNFα (see, for example, PLoS One, July 22, 2014; 9(7): e102967) and demonstrated upregulation of TNFα and IL-6 in non-survivors (see, for example, Chest, 1997:111:1306-21), while MCP-1 was elevated in the group with ALI / ARDS (see, for example, International Journal of Molecular Sciences, 2019:20(9): 2218). Statistical analysis was based on the Dunnett test performed using GraphPad. (n = 5, 8, 8, 8 and 8; NC, Control, PC, SEQ ID NO: 31, SEQ ID NO: 29). P<0.001, P<0.01, P<0.05, relative to each corresponding control group. Example 9: The use of cationic alkyl-modified CNP derivatives in acute exacerbations (IPF-AE) of idiopathic pulmonary fibrosis in the lungs. Its role in the inflammatory state, indicating the resolution of ALI / ARDS.

[0293] The data in Figure 5 were generated according to the experimental protocol below. Animal care and treatment: Male C57BL / 6J mice (6 weeks old) were purchased from Yukido Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and a standard mouse diet (MF diet, Oriental Yeast Co., Ltd., Tokyo, Japan). Drug administration: Mice were administered bleomycin (Bleo; 1.0 mg / kg; Nippon Kayaku Tokyo, Japan) via IT, followed by LPS (0.025 mg / kg; Sigma-Aldrich, St. Louis, MO, USA) via IT 3 weeks later. Figure 5A As shown, subcutaneous treatment was administered at 0.3 mg / kg (0.1 µmol / kg) using SEQ ID NO:29 or SEQ ID NO:31. Additionally, a normal control (NC) group without LPS / Bleo treatment, a Bleo group without LPS treatment, and a control group without test product treatment were also included. On the last day, mice were euthanized under isoflurane anesthesia, and their lung tissue was collected and weighed. Figure 5B An increased lung weight to body weight ratio is a commonly measured parameter indicating lung injury. Figure 5C Biochemical analysis: Lung tissue was collected and fixed with 4% paraformaldehyde. Immunohistochemical staining was performed on paraffin sections of fixed lung tissue using anti-MPO rabbit polyclonal antibody (Agilent Technologies, Santa Clara, California), horseradish peroxidase (HRP)-labeled anti-rabbit IgG goat polyclonal antibody (Nichirei Biosciences, Tokyo, Japan), and 3,3'-diaminobenzidine-4HCl (DAB) (Agilent Technologies, Santa Clara, California). MPO expression was assessed by Image J (NIH, Bethesda, MD, USA). Figure 5D Biochemical analysis of lung tissue: Lung tissue was minced in lysis buffer and diluted with PBS (Fujifilm, Tokyo, Japan). Macrophage chemokine-1 (MCP1) was measured using an ELISA kit (R&D Systems, Minneapolis, Minnesota). Figure 5EBiochemical analysis of lung tissue: Lung tissue was minced in Tri-Reagent (Cosmo Biotech, Tokyo, Japan) and then CHCl3 was added. After incubation at room temperature for 3 minutes, the sample was centrifuged (12,000 × g, 4°C, 15 minutes). The aqueous layer was collected, and an equal volume of 2-propanol was added. After incubation at room temperature for 10 minutes, the sample was centrifuged (12,000 × g, 4°C, 15 minutes), and the supernatant was discarded. Then, 75% EtOH was added, and the sample was centrifuged (12,000 × g, 4°C, 5 minutes). The supernatant was discarded, and the pellets were dissolved in nuclease-free water (Ambion, Massachusetts, USA). The gene expression level of IL-6 was determined by qRT-PCR analysis using a cDNA synthesis kit (Kagem, Venlo, Netherlands). Previous reports have demonstrated upregulation of neutrophil (see, for example, Kona M. et al., *Respir. Med.*, 2021, Vol. 186.), MCP1 (see, for example, Arai T. et al., *BMJ Open Respir. Res.*, 2021, Vol. 8, No. 1.), and IL-6 (see, for example, Lee J. et al., *PLOS ONE*, 2021, Vol. 16, No. 7.) cell numbers in non-survivors. Statistical analysis was based on the Stourden's t-test performed using GraphPad (n = 5, 5, 8, 8, 8; NC, Bleo, Control, A [SEQ ID NO: 29], B [SEQ ID NO: 31]). ## P<0.01 and # P<0.05.

[0294] Example 10: Repeated subcutaneous application of cationic alkyl-modified CNP derivatives to in situ mammary glands using E0771 cells Significant antitumor activity was observed in a mouse model of cancer.

[0295] At the end of the study, the groups treated with cationic alkyl-modified CNPs (SEQ ID NO: 29, 30, and 31) showed a significant reduction in tumor volume compared to the control group. The cationic alkyl sequence without CNPs (SEQ ID NO: 14) was also tested in this model, but did not show a significant reduction in tumor volume. Therefore, it can be concluded that the conjugation of CNPs is crucial for antitumor activity. The data in Figure 6 were generated according to the experimental protocol below. Animal care and treatment: Female C57BL / 6J mice (6 weeks old) were purchased from Yukido Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and a standard mouse diet (MF diet, Oriental Yeast Co., Ltd., Tokyo, Japan, or PicoLab rodent diet 20, LabDiet Corp., St. Louis, Missouri). Implantation and Drug Administration: E0771 breast cancer cells (250,000 cells / mouse; Cosmo Bio, Tokyo, Japan) were implanted into the left mammary glands of mice and randomly assigned to groups of n = 10. Starting on day 4 post-inoculation, sterile water for injection containing cationic alkyl-modified CNP derivatives (SEQ ID NO: 29, 30, and 31) (Otsuka Pharmaceutical, Tokushima, Japan) was administered subcutaneously at a bolus dose of 0.3 mg / kg (0.1 µmol / kg; dose volume 10 mL / kg) once daily for 5 days (5 days of administration, 2 days of rest), for 3 cycles. The same procedure was used to administer the drugs to a control group that received only buffer solution to establish baseline tumor growth kinetics. Tumor size was measured using calipers. Statistical analysis was based on the Dunnett test performed using GraphPad (n = 10, 10, 10, and 10; Control, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31). P<0.0001, compared to the control group.

[0296] Example 11: Fluorescence polarization (FP) assay of natriuretic peptide receptor B (NPRB) and natriuretic peptide receptor C (NPRC) Based solely on 5(6)-carboxyfluorescein (F A probe (5 nM CNP-F) that binds to C-type natriuretic peptide (CNP) It possesses rapid rotation alone and induces a low fluorescence polarization (FP) signal. For example... Figure 7As shown, when human NPRB or NPRC (50 nM) is added, the CNP probe binds to these receptors and induces slow rotation and a high FP signal. In the presence of NPRA, no change in FP signal was detected, indicating that the CNP probe did not bind to NPRA. In the presence of [SEQ ID. No. 31], a low FP signal demonstrates that the CNP probe binds to both NPRB and NPRC.

[0297] Fluorescence polarization (FP) measurement CNP-F was incubated at a final concentration of 5 nM in an assay buffer containing PBS, pH 7.4, and 0.01% Triton X-100, in the presence of 50 nM human NPRB or NPRC. Probe. Before FP measurement, place a 100 μL volume of CNP-F... The probe and NPR were dispensed into black 96-well Costa flat-bottomed polystyrene plates. Next, 1 μL of SEQ ID. No. 31 (final concentration 150 nM) was added to the premixed probe and NPR. The plates were incubated at room temperature for 10 minutes. Then, fluorescence polarization was measured on a Flexstation 3 (excitation wavelength: 480 nm; emission wavelength: 525 nm, with a cutoff at 515 nm).

[0298] CNP-F Probe synthesis.

[0299] CNP-F was prepared using solid-phase peptide synthesis. In short, a linear peptide (sequence: F) -GLSKGCFGLKLDRIGSMSGLGC,F 5(6)-Carboxyfluorescein was synthesized in an automated microwave synthesizer (CEM, Matthews, NC). Next, the crude linear peptide was cleaved with 95% TFA in the presence of a carbocation scavenger and diethyl ether precipitation. Disulfide bond formation of the peptide was performed at a high dilution in 10% DMSO. Finally, the product was purified and characterized by reversed-phase HPLC (1260 Infinity II preparative LC system, Agilent Technologies, Santa Clara, CA), and its quality was confirmed using an LCMS system (6100 series single quadrupole LC / MS, Agilent Technologies, Santa Clara, CA).

[0300] Recombinant NPR-Fc fusion protein The extracellular domains (ECDs) of recombinant human NPRA, NPRB and NPRC are expressed in mammalian cells as soluble human IgG1 Fc fusions, which are essentially the same as previously described (Bennet et al., Journal of Biochemistry (JBC) 266(34) 23060-23067, 1991). In summary, the mature human NPR ECD peptide sequence, NPRA N1-L439 (GenBank accession number XP_005245275.1), NPRB R1-T433 (GenBank accession number NP_003986.2), and NPRC Q1-S434 (GenBank accession number NP_001191304.1) were synthetically linked with amino acid E216-G446 of the human IgG1 heavy chain (accession number P01857.2) (IMGT.org, EU number) (Azenta, Burlington, MA) and cloned into pCMV6-a-puro (Invitrogen, Carlsbad, CA) for mammalian expression. A single C>S substitution, an unpaired cysteine ​​residue, was introduced at position C232 of the NPRA to eliminate disulfide bond aggregation (Olympic Protein Technologies, PA1 report, unpublished). All NPR-Fc plasmid sequences were validated, and the plasmids were generated using endotoxin-free reagents (Azenta, Burlington, MA).

[0301] Each NPR-Fc was transfected in Expi293 cells (A-14635, Thermo-Fisher Scientific) at a scale of 200 ml. After 4 days of growth, the transfection supernatant was collected, filtered, and protein A (MabSelect SuRe, Cytiva) was captured and eluted in 0.1 M citrate buffer pH 6.0 / 3.5 mM MgCl2 / 2% glycerol for use in NPRA-Fc and NPRC-Fc. NPRB-Fc was eluted at pH 6.6 (Pierce Mild Elution Buffer, Thermo-Fisher Catalog No. 21027). SDS-PAGE analysis showed that, under reducing conditions, the 80–85 kDa band was larger than the expected 75 kDa band, likely due to glycosylation in the ECD of all three NPRs (Potter LR et al., Handb Exp Pharmacol. 2009;(191):341–366). SEC analysis confirmed that the main peak ran greater than 153 kDa MW markers. Preparative SEC removed HMW proteins from NPRA and NPRC-Fc and purified both to 93% monomericity, subsequently exchanged to PBS via buffer. NPRB-Fc, at 98% monomericity, required no further purification. (Olympic Protein Technologies, PA1 report, unpublished).

[0302] Example 12: Effects of cationic alkyl-modified CNP derivatives, alone or in combination with pirfenidone, as indicated in... Reduced pulmonary fibrosis in a mouse model of idiopathic pulmonary fibrosis (IPF) (inversely proportional to alveolar area).

[0303] Generate according to the following experimental scheme Figure 8A and 8B Data. Animal care and treatment: Male C57BL / 6J mice (6 weeks old) were purchased from Yukido Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and a standard mouse diet (MF diet, Oriental Yeast Co., Ltd., Tokyo, Japan). To induce pulmonary fibrosis, mice were administered bleomycin (Bleo; 1.0 mg / kg; Nippon Kayaku Co., Ltd., Tokyo, Japan) via IT. Drug administration began 7 days later: administration of SEQ ID NO: 31 (SC, at 0.3 mg / kg (0.1 µmol / kg)), pirfenidone (PO, at 100 mg / kg), or a combination of pirfenidone (Pir; PO, at 100 mg / kg) and SEQ ID NO: 31 (SC, at 0.3 mg / kg (0.1 µmol / kg)), as shown. Figure 8AAs shown. Additionally, this study included a normal control (NC) group without Bleo treatment and a Bleo control group without test product treatment. Mice were euthanized under isoflurane anesthesia on day 21, and their lung tissue was collected. Figure 8B Biochemical analysis: Lung tissue was collected and fixed with 4% paraformaldehyde, and Azan staining was performed by Kyushu University (Fukuoka, Japan). Alveolar area was measured in a blinded manner using Image J (NIH, Bethesda, Maryland, USA), and was inversely proportional to fibrosis area. Statistical analysis was based on Stourden's t-test performed using GraphPad (n = 3, 7, 7, 7, and 7; NC, Bleo controls, SEQ ID NO: 31, Pir, and combinations thereof (SEQ ID NO: 31 and Pir). P < 0.001, ns = not significant relative to NC. Groups treated with SEQ ID NO: 31 alone or in combination with pirfenidone showed significantly larger alveolar areas, demonstrating the presence of healthy tissue and reduced fibrosis.

[0304] Example 13: Repeated subcutaneous administration of cationic alkyl-modified CNP derivatives as monotherapy or in combination with immune testing The combination of spot inhibitors showed significant antitumor activity in an orthotopic breast cancer mouse model using E0771 cells.

[0305] Generate according to the following experimental scheme Figure 9Data. Animal care and treatment: Female C57BL / 6J mice (6 weeks old) were purchased from Yukido Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and a standard mouse diet (CRF diet; Oriental Yeast Co., Ltd., Tokyo, Japan). Implantation and drug administration: E0771 breast cancer cells (250,000 cells / mouse; Cosmo Bio Co., Ltd., Tokyo, Japan) were in situ implanted into the left mammary glands of mice and randomly assigned to groups of n = 7–8. Drug administration began on day 4 post-vaccination with anti-PD1 antibody (aPD1; BioX cell, West Lebanon, NH, #RMP1-14) administered intraperitoneally at 5 mg / kg twice weekly for 2 cycles; or subcutaneously administered 0.3 mg / kg (0.1 µmol / kg) of sterile water for injection containing SEQ ID NO:31 (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) once daily for 5 days (5 days of administration, 2 days of rest) for 3 cycles; or a combination of aPD1 (5 mg / kg IP, twice weekly; 2 cycles) and SEQ ID NO:31 (0.3 mg / kg SC, once daily, 5 days of administration and 2 days of rest; 3 cycles). A control group receiving only sterile water for injection was administered the same way as with SEQ ID NO:31 to establish baseline tumor growth kinetics. Tumor size was measured using calipers. Statistical analysis was based on the Dunnett test performed using GraphPad (n = 8, 7, 8, and 7; control, aPD1, SEQ ID NO: 31, and combination (aPD1 and SEQ ID NO: 31)). P<0.0001, P<0.05, relative to control. At the end of the study, the group treated with cationic alkyl-modified CNP (SEQ ID NO: 31) showed a significant reduction in tumor volume compared with the control group and the group treated with the immune checkpoint inhibitor PD1 alone.

[0306] Example 14: Radiation, immune checkpoint inhibitors, and repeated subcutaneous administration of cationic alkyl-modified CNP derivatives The combination showed reduced bone metastasis and significantly improved overall survival in a mouse model of orthotopic bone metastasis using E0771 breast cancer cells. Survival rate.

[0307] The data in Figure 10 were generated according to the experimental protocol below. Animal care and treatment: Female C57BL / 6J mice (6 weeks old) were purchased from Oriental Bioservices (Kobe, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and a standard mouse diet (CRF diet; Oriental Yeast Co., Ltd., Tokyo, Japan). Implantation and drug administration: E0771 breast cancer cells (Cosmo Bio Inc., Tokyo, Japan; 250,000 cells / mouse, in RPMI 1640 medium (Fujifilm Inc., Tokyo, Japan)) were implanted in situ into the left mammary glands of mice, and E0771 mouse breast cancer cells (500,000 cells / mouse, 50% matrix gel (Corning, NY, USA, #354234)) were implanted into the femurs. Then, the mice were randomly divided into several treatment groups: 1) control (n = 5), 2) SEQ ID NO: 31 (n = 5), 3) SEQ ID NO: 31 and aPD1 (n = 5), 4) radiation (n = 6), 5) radiation and aPD1 (n = 5), 6) SEQ ID NO: 31 and radiation (n = 6), 7) SEQ ID NO: 31, radiation and aPD1 (n = 6). Figure 10AThe drug administration regimen was demonstrated as follows: starting on day 5 post-vaccination, a bolus dose of 0.3 mg / kg (0.1 µmol / kg) of a buffer solution containing SEQ ID NO:31 (15 mM succinic acid (TCI, Tokyo, Japan, #S0100), 4% (w / v) D-mannitol (TCI, Tokyo, Japan, #M0044), 10 mM hydroxypropyl-β-cyclodextrin (TCI, Tokyo, Japan, #H0979, pH 4.4) was administered subcutaneously once daily for 5 days (5 days of administration, 2 days of rest), for 4 cycles; aPD1 (BioX Cell, West Lebanon, New Hampshire, #RMP1-14) was administered intraperitoneally at 5 mg / kg twice weekly for 2 cycles; on days 5, 8, and 12, a mixture of three types of anesthetic (0.3 µmol / kg) was administered. Mice were anesthetized with three groups of 5 Gy irradiations to bone via an X-ray radiation system (mediXtec Japan Corporation, Chiba, Japan, MX-160Labo) under X-ray radiation system (mediXtec Japan Corporation, Chiba, Japan). The control group, receiving only buffer, was administered the same regimen as in SEQ ID NO: 31 to establish baseline tumor growth kinetics. Cell survival was monitored up to day 33 post-seeding. Figure 10B After the remaining mice were euthanized as planned, tumors were collected and their size was measured using calipers. Statistical analysis was based on the log-rank (Mantel-Cox) test performed using GraphPad (for each group: 1) Control (n = 5), 2) SEQ ID NO: 31 (n = 5), 3) SEQ ID NO: 31 and aPD1 (n = 5), 4) Radiation (n = 6), 5) Radiation and aPD1 (n = 5), 6) SEQ ID NO: 31 and radiation (n = 6), 7) SEQ ID NO: 31, radiation and aPD1 (n = 6) P < 0.01, relative to group 5. At the end of this study, the combination of SEQ ID NO: 31, radiation, and aPD1 (an immune checkpoint inhibitor) showed a significant improvement in survival. Furthermore, the addition of SEQ ID NO: 31 reduced the incidence of bone metastases in all corresponding groups.

[0308] Example 15: Amputation and repeated subcutaneous administration of cationic alkyl-modified CNP derivatives combined with isolated amputation phases Compared with the mouse model of orthotopic lung metastasis using osteosarcoma LM8 cells, it showed a significant reduction in lung metastasis.

[0309] Generate according to the following experimental scheme Figure 11A-11C Data. Animal care and treatment: Male CH3 / He mice (7 weeks old) were purchased from Yukido Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and a standard mouse diet (MF diet; Oriental Yeast Co., Ltd., Tokyo, Japan). Implantation and drug administration: LM8 osteosarcoma cells (RCB, Tsukuba, Japan, #RCB1450) were implanted in situ into the femurs of mice (1,000,000 cells / mouse). Mice were then randomly assigned to groups of n = 7. Figure 11A The drug administration regimen was demonstrated. Starting on day 4 post-inoculation, mice were subcutaneously administered a bolus dose of 0.3 mg / kg (0.1 µmol / kg) of a buffer solution containing SEQ ID NO:31 (15 mM succinic acid (TCI Ltd., Tokyo, Japan, #S0100), 4% (w / v) D-mannitol (TCI Ltd., Tokyo, Japan, #M0044), 10 mM hydroxypropyl-β-cyclodextrin (TCI Ltd., Tokyo, Japan, #H0979, pH 4.4) once daily for 5 days (5 days of administration, 2 days of rest), for slightly more than 3 cycles. On day 7 post-inoculation, all mice underwent amputation under isoflurane anesthesia to remove the primary tumor, and the sutures were closed with sutures (AlfresaPharma Corporation, Osaka, Japan, #HR0806NW45-KF2). In the alternative treatment, the amputation control group also received buffer solution once a day for 5 days (5 days of treatment followed by 2 days of rest), for a little over 3 cycles. On day 34, the mice were sacrificed and lung tissue was collected. Figure 11B (Biochemical analysis) The collected lung tissue was immersed in 4% paraformaldehyde (Fujifilm Corporation, Tokyo, Japan, #163-20145). The tissue was then paraffin-embedded, and the obtained sections were stained with hematoxylin and eosin (H&E) by Kyodo Byori (Kobe, Japan). Lung images were observed using a high-resolution microscope (Keyence Corporation, Tokyo, Japan, #BZ-X700), and lung metastases were assessed by direct counting of present metastatic nodules. Figure 11C Outliers were identified using the ROUT test (Q = 1%). All statistical analyses were based on the Dunnett test performed using GraphPad (n = 7 and 7; control, combination (amputation and SEQ ID NO: 31)). P<0.01, relative to the control group. Surgical interventions such as amputation, in addition to chemotherapy, can also be used to prevent lung metastases. At the end of the study, the combination of repeated SC doses of SEQ ID NO: 31 and amputation reduced the incidence of lung metastases compared to amputation alone.

[0310] Example 16: Repeated subcutaneous administration of cationic alkyl-modified CNP derivatives as monotherapy and in combination with immunoassay The combination of checkpoint inhibitors showed a significant reduction in tumor volume in a mouse model of subcutaneous colon cancer using MC38 cells.

[0311] The data in Figure 12 were generated according to the experimental protocol below. Animal care and treatment: Male C57BL / 6J mice (6 weeks old) were purchased from Yukido Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and a standard mouse diet (MF diet; Oriental Yeast Co., Ltd., Tokyo, Japan). Implantation and drug administration: MC38 colon cancer cells were subcutaneously implanted into the right flank of mice (1,000,000 cells / mouse), and the mice were randomly assigned to groups of n = 8-9. Drug administration began on day 4 post-vaccination, with anti-tigit antibody (Absolute antibody, Shirley, MA, USA, #Ab01258-1.1-VXX, clone 1B4) administered intraperitoneally at a dose of 5 mg / kg, twice weekly for 2 cycles; and a bolus dose of 0.3 mg / kg (0.1 µmol / kg) subcutaneously administered a buffer solution containing SEQ ID NO: 31 (15 mM succinic acid (TCI, Tokyo, Japan, #S0100), 4% (w / v) D-mannitol (TCI, Tokyo, Japan, #M0044), 10 mM hydroxypropyl-β-cyclodextrin (TCI, Tokyo, Japan, #H0979, pH 4.5) once daily for 5 days (5 days of administration, 2 days of rest) for 3 cycles; or anti-tigit antibody (5 mg / kg IP, twice weekly; 2 cycles) and SEQ ID NO: 31 (0.3 µmol / kg) at a dose of 0.1 µmol / kg, pH 4.5. The combination of (mg / kg SC, once daily, for 5 days with 2 days of rest; 3 cycles) was used. A control group receiving only buffer was administered the same way as in SEQ ID NO: 31 to establish baseline tumor growth kinetics. Tumor size was measured using calipers on days 4, 7, 14, and 22 post-inoculation. Mice were humanely euthanized after final tumor measurement on day 22. Statistical analysis was based on the Dunnett test performed using GraphPad (n = 9, 8, 8, and 8; control, anti-tigit antibody, SEQ ID NO: 31, combination (anti-tigit antibody and SEQ ID NO: 31)). P<0.05, relative to the anti-tigit antibody monotherapy group. At the end of the study, the group treated with cationic alkyl-modified CNP (SEQ ID NO: 31) showed a significant reduction in tumor volume compared with the control group and the group treated with immune checkpoint anti-tigit antibody alone.

[0312] Example 17: HeLa cells treated with cationic alkyl-modified CNP derivatives showed resistance to baseline cyclic monophosphate. Significant inhibition of adenosine levels.

[0313] Generate according to the following experimental scheme Figure 13 Data. In vivo protocol: HeLa cells were purchased from ATCC (Manassas, Virginia, USA). Cells were cultured at 37°C, 100% humidity, and 5% CO2 in DuPont modified Eagle medium (DMEM) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, Missouri, USA, #F2442) (Fujifilm Inc., Tokyo, Japan, #044-29765). Cells were harvested and cultured at 10... 7 Cells were suspended at a concentration of 10 cells / mL in ENGS (Lifeline, San Diego, CA, USA, #LEC-LL0002), and 5 μL of cell suspension was added to each well of a 96-well low-adhesion plate (PerkinElmer, Waltham, MA, USA, #66PL96005). Cells were then treated (n = 4 wells) with 5 μL of SEQ ID NO: 31 (final concentration 10 µg / mL) in ENGS containing 1 mM IBMX (Fujifilm Corporation, Tokyo, Japan, #099-03411) for 10 minutes. cAMP levels were assessed using a cAMP assay kit (PerkinElmer, Waltham, MA, USA, #62AM4PEB) and a plate reader (PerkinElmer, Waltham, MA, #Nivo) according to the manufacturer's protocol. Statistical analysis of untreated control wells (n = 4 wells) was performed using GraphPad Prism. P<0.05. The NPR-C receptor is involved in inhibiting adenylate cyclase activity, leading to a reduction in cyclic adenosine monophosphate (cAMP) production. This study concludes that baseline cAMP levels were altered (inhibited) in HeLa cells (known to express NPR-C) treated with SEQ ID NO: 31, indicating binding to NPR-C.

[0314] The teachings of all patents, published applications and references cited in this article are incorporated herein by reference in their entirety.

[0315] Although exemplary embodiments have been specifically shown and described, those skilled in the art will understand that various changes in form and detail may be made thereto without departing from the scope of the embodiments as covered by the appended claims.

Claims

1. A compound comprising a cationic alkyl moiety of formula (I): J-(CH2)x(CO)-(A)y-(B)z- （I）， in: J is either HOOC or CH3; x is 10⁻¹⁶; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-aminobutyric acid (γAbu), γ-linked glutamic acid (γE) and α-linked glutamic acid (E); y is 2-4; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2-4; Wherein -(B)z- contains no more than 2 Dab residues, and wherein the Dap or Dab residues are linked by an α-amino group.

2. The compound according to claim 1, wherein: J is CH3; x is 10, 12, 14, or 16; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

3. The compound according to claim 1, wherein: J is CH3; x is 10, 12, 14, or 16; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-linked glutamic acid (γE); y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

4. The compound according to claim 1, wherein: J is CH3; x is 10, 12, 14, or 16; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

5. The compound according to claim 1, wherein: J is CH3; x is 10, 12, 14, or 16; A is γ-aminobutyric acid (γAbu). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

6. The compound according to claim 1, wherein: J stands for HOOC; x is 10, 12, 14, or 16; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-aminobutyric acid (γAbu). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

7. The compound according to claim 1, wherein: J stands for HOOC; x is 10, 12, 14, or 16; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and γ-linked glutamic acid (γE). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

8. The compound according to claim 1, wherein: J stands for HOOC; x is 10, 12, 14, or 16; A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

9. The compound according to claim 1, wherein: J stands for HOOC; x is 10, 12, 14, or 16; A is γ-linked glutamate (γE). y is 3; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2 or 3.

10. The compound according to claim 1, wherein: J is CH3; x is 14; (A)y is Aeea-Aeea-Aeea 、gAbu - Abu - cAbu、cAbu -Aeea-Aeea、 cE -Aeea-Aeea、 E - Aeea-Aeea;Aeea-Aeea 、gAbu - cAbu、cAbu -Aeea ,γE -Aeea or E -Aeea; and also (B)z is Dap-Dap, Dab-Dab, Dab-Dap or Dap-Dab.

11. The compound according to claim 1, wherein: J is CH3; x is 14; (A)y is Aeea-Aeea-Aeea 、gAbu - Abu - cAbu、cAbu -Aeea-Aeea ,γE -Aeea-Aeea or E - Aeea-Aeea; and also (B)z is Dap-Dap, Dab-Dab, Dab-Dap or Dap-Dab.

12. The compound of claim 1, wherein the cationic alkyl moiety is selected from SEQ ID NO: 10 to 22 and 51 to 69.

13. The compound of claim 1, wherein the cationic alkyl moiety is selected from SEQ ID NO: 10 to 22.

14. The compound according to any one of claims 1 to 13, wherein the compound, when conjugated with a peptide, does not cause clinically observable ataxia after parenteral bolus administration in rats at doses of 10 µmol / kg and lower.

15. The compound according to any one of claims 1 to 14, used for modifying a peptide by covalent conjugation.

16. A conjugated peptide of formula (II): CH3(CH2)x(CO)-(A)y-(B)z-peptide (II) in: x is 10⁻¹⁶; A is independently selected from the group consisting of: 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), γ-aminobutyric acid (γAbu), γ-linked glutamic acid (γE) and α-linked glutamic acid (E); y is 2-4; B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab); and z is 2-4; in: -(B)z- contains no more than 2 Dab residues, wherein the Dap or Dab residues are linked by an α-amino group; CH3(CH2)x(CO)-(A)y-(B)z- is covalently linked to the N-terminus of the peptide or to one of the amino groups on the side chain of the peptide; At equivalent bolus doses, the bioactivity of the conjugated peptide is equal to or greater than that of the unmodified peptide; and / or At the same time point following bolus administration of an equivalent dose, the blood level of the conjugated peptide was equal to or higher than that of the unconjugated peptide.

17. The conjugated peptide of claim 16, wherein the conjugated peptide binds to a natriuretic peptide receptor and has no adverse effects or ataxia in rats at 3.0 µmol / kg and lower bolus doses.

18. The conjugated peptide according to claim 16 or 17, wherein the CH3(CH2)x(CO)-(A)y-(B)z- moiety is covalently linked to the N-terminus of the peptide.

19. The conjugated peptide according to any one of claims 16 to 18, wherein the peptide is a natriuretic peptide or a natriuretic peptide derivative of SEQ ID NO: 32, 44, 48 or 75.

20. The conjugated peptide according to any one of claims 16 to 19, wherein the CH3(CH2)x(CO)-(A)y-(B)z- portion is selected from SEQ ID NO: 10 to 22 and 51 to 69.

21. The conjugated peptide according to claim 20, wherein the CH3(CH2)x(CO)-(A)y-(B)z- portion is selected from SEQ ID NO: 10 to 22.

22. The conjugated peptide according to any one of claims 16 to 20, wherein the peptide is a natriuretic peptide derivative, and one or more methionine residues of the natriuretic peptide derivative are replaced by glutamine (Q), leucine (L), oroleucine (Nle) or methoxinine (Mox).

23. The conjugated peptide according to any one of claims 16 to 22, wherein the peptide is a natriuretic peptide or a derivative thereof according to SEQ ID NO: 32, wherein one or more methionine residues are replaced by glutamine (Q), and the CH3(CH2)x(CO)-(A)y-(B)z- portion is selected from SEQ ID NO: 10 to 22.

24. The conjugated peptide according to any one of claims 16 to 23, wherein the conjugated peptide is selected from SEQ ID NO:29-31, 33-43, 45-47, 49-51.

25. The conjugated peptide according to claim 24, wherein the conjugated peptide is selected from SEQ ID NO: 29-31 and 33-43.

26. The conjugated peptide according to claim 25, wherein the conjugated peptide is selected from SEQ ID NO: 29-31.

27. The conjugated peptide according to claim 26, wherein the conjugated peptide is SEQ ID NO:

29.

28. The conjugated peptide according to claim 26, wherein the conjugated peptide is SEQ ID NO:

30.

29. The conjugated peptide according to claim 26, wherein the conjugated peptide of formula (II) is SEQ ID NO:

31.

30. The conjugated peptide according to any one of claims 16 to 29, wherein the conjugated peptide binds to natriuretic peptide receptor B (NPRB), natriuretic peptide receptor C (NPRC), or a combination thereof.

31. The conjugated peptide according to any one of claims 16 to 30, wherein the conjugated peptide is an NPRB agonist.

32. The conjugated peptide according to any one of claims 16 to 31, wherein the conjugated peptide is an NPRC agonist.

33. The conjugated peptide according to any one of claims 16 to 32, wherein the conjugated peptide produces a physiological effect selected from the following: sustained increase in blood cGMP, alteration of cAMP, alteration of blood pressure, increase in sepsis survival, increase in acute lung injury survival, increase in acute respiratory distress syndrome survival, reduction of MPO-positive cells, reduction of the number of cells in alveolar fluid or bronchoalveolar lavage fluid, reduction of the amount of protein in alveolar fluid or bronchoalveolar lavage fluid, reduction of endothelial permeability, reduction of lung weight by body weight, reduction of monocyte chemoattractant protein-1, reduction of IL-6, reduction of TNF-α, reduction of A1008 / A9, reduction of fibrosis, reduction of tumor volume, reduction of metastasis, reduction of inflammation, antiproliferative effect, reduction of cancer burden, inhibition of cyclooxygenase 2 (COX-2) expression, antagonism of the renin-angiotensin-aldosterone system, inhibition of cardiac hypertrophy or combinations thereof.

34. The compound according to any one of claims 1 to 15 or the conjugated peptide according to any one of claims 16 to 33, for use in the preparation of a pharmaceutical composition.

35. The compound or conjugated peptide for use according to claim 34, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers or excipients.

36. The compound or conjugated peptide for use according to claim 35, wherein one or more pharmaceutically acceptable carriers or excipients comprise a bulking agent, a buffer, a stabilizer, a preservative, or a combination thereof.

37. The compound according to any one of claims 1 to 15 or the conjugated peptide according to any one of claims 16 to 33, for use in treating a disease or condition of a subject in need.

38. The compound or conjugated peptide for use according to claim 37, wherein the compound or the conjugated peptide is selected from: a) Any one of SEQ ID NO: 29-31, 33-43, 45-47, 49-51, or b) Any one of SEQ ID NO: 29-31, 33-43, 45-47, or c) Any one of SEQ ID NO: 29-31, 33-43, or d) Any one of SEQ ID NO: 29-31, or e) SEQ ID NO: 29 or f) SEQ ID NO: 30, or g) SEQ ID NO:

31.

39. The compound for use according to any one of claims 37 or 38, wherein the compound or the conjugated peptide comprises SEQ ID NO:

31.

40. The compound or conjugated peptide for use according to any one of claims 37 to 39, wherein the disease or condition affects the lungs (e.g., ALI, ARDS, COVID, inflammation, sepsis, fibrosis, or cancer), liver (e.g., non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), inflammation, fibrosis, or cancer), heart (e.g., heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), acute heart failure, or congestive heart failure), bones / joints (e.g., osteoporosis, osteoarthritis, rheumatoid arthritis, inflammation, cancer, or dwarfism), kidneys (e.g., chronic kidney disease (CKD), acute kidney injury (AKI), drug-induced kidney injury, inflammation / nephritis, renal fibrosis, glomerulosclerosis, or renal cancer), prostate (e.g., benign prostatic hyperplasia or prostate cancer), brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, testes, ovaries, uterus, and / or blood vessels).

41. The compound or conjugated peptide for use according to any one of claims 37 to 39, wherein the disease or symptom is pre-metastatic cancer or post-metastatic cancer.

42. The compound or conjugated peptide for use according to claim 41, wherein the cancer is a cancer selected from any one or more of the following organs: lung, pleura, liver, heart, bone / joint, kidney, prostate, breast, brain, eye, skin, muscle, blood, blood vessels, gastrointestinal tract, bladder, testis, ovary and / or uterus.

43. The compound or conjugated peptide for use according to any one of claims 37 to 39, wherein the disease or symptom is pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), or COVID in the subject of need.

44. The compound or conjugated peptide for use according to any one of claims 37 to 39, wherein the disease or symptom is fibrosis.

45. The compound or conjugated peptide for use according to any one of claims 37 to 44, wherein the treatment comprises administering to the subject a therapeutically effective bolus dose of 10.0 µmol / kg or less and / or 10.0 µmol / kg to 0.0001 µmol / kg, including the end value.

46. ​​The compound or conjugated peptide for use according to any one of claims 37 to 45, wherein the compound is administered to the subject as a single therapy or in combination with one or more other agents or treatments.

47. The compound or conjugated peptide for use according to claim 46, wherein the one or more additional agents or treatments are selected from immune checkpoint inhibitors, surgery / amputation, radiation, chemotherapy, or combinations thereof.

48. The compound for use according to any one of claims 37 to 46, wherein the compound is applied subcutaneously, by infusion, by inhalation, by nasal spray, orally, by eye drops, and / or by topical application.

49. The compound or conjugated peptide for use according to any one of claims 37 to 42, wherein the compound or the conjugated peptide is applied to the subject subcutaneously, by infusion, by inhalation, by nasal spray, orally, by eye drops, and / or by topical application.

50. A composition comprising a compound according to any one of claims 1 to 15 or a conjugated peptide according to any one of claims 16 to 33, and one or more pharmaceutically acceptable carriers or excipients.

51. The composition of claim 49, wherein the one or more pharmaceutically acceptable carriers or excipients comprise a leavening agent, a buffer, a stabilizer, a preservative, or a combination thereof.

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