Cationic lipids for covalent modification of peptides

Abstract

Provided herein are compounds (e.g., peptides) comprising a cationic alkyl moiety, compositions comprising such compounds, and methods of using such compounds to treat, for example, a disease, disorder, or pathological condition. In some embodiments, the conjugated peptide has equivalent or greater biological activity than the unmodified peptide at an equivalent bolus dose, equivalent or greater blood levels than the unconjugated peptide at the same time point after bolus administration at an equivalent dose, or a combination thereof. Provided herein are compositions comprising the compounds or conjugated peptides of this disclosure for use in the manufacture of medical compositions.

Description

[Background technology]

[0001] Related applications This application claims the benefits of U.S. Provisional Application No. 63 / 493,290, filed on March 30, 2023. The entire teachings of the above application are incorporated herein by reference. Embedding by referencing data in XML

[0002] This application incorporates, by reference, the sequence listings contained in the following eXtensible Markup Language (XML) files filed concurrently with this specification. a) File name: 6165.1024-001_SL.xml, creation date: March 29, 2024, size: 67,928 bytes. Government support

[0003] This invention was made with government support under grant number HL156295 granted by the National Institutes of Health. The government has certain rights to this invention.

[0004] Alkyl chain modification of natural peptides is one approach used to enhance the pharmacokinetic properties and / or extend the in vivo half-life of peptides that are inherently unstable in vivo (see, e.g., Menacho-Melgar et al, J Control Release. 2019. Feb. 10;295:1-12). To date, most modified peptides utilize fatty acids or alkyl chains that do not contain cationic moieties, because the inherent toxicity of cationic alkyl compounds and cationic lipid compounds has been well demonstrated (see, e.g., Cui et al, The Royal Society of Chemistry. 2018. pp 473-479). Modifying peptide drugs with cationic lipids is known to increase their toxicity, often resulting in a narrow therapeutic range where the toxic dose is very close to the dose desired for biological activity. Consequently, this approach is generally considered undesirable. Furthermore, peptide modification can lead to loss of biological activity or decreased solubility of the peptide. Despite these drawbacks, cationic alkyl and cationic lipid modifications can improve in vivo half-life by potentially interacting with or binding to cell membranes and proteins. However, this same property also leads to increased toxicity, as the very strong binding to cell membrane components is thought to cause hemolysis, caspase-induced apoptosis, mitochondrial dysfunction due to decreased membrane potential, elevated reactive oxygen species (ROS) levels, cell arrest in S phase, and / or other unknown mechanisms leading to toxicity. Therefore, the use of cationic alkyl or cationic lipid moieties in peptide modifications has been restricted due to toxicity concerns that may reduce the therapeutic index. The toxicity of cationic lipids is strongly associated with the presence of multiple amino group heads, which have been shown to kill 50% of cells at concentrations above μM in culture. As a result, the application of cationic lipids has been limited to killing cells with abnormally high negative charge membranes compared to normal mammalian cells, such as cancer cells (see, for example, Cui et al, The Royal Society of Chemistry 2018, pp. 473-479) and bacterial cells (see, for example, Mawuch et al, Int. J. Mol. Sci. 2020, 21, 8944). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Menacho-Melgar et al,J Control Release.2019.Feb.10;295:1-12 [Non-Patent Document 2] Cui et al,The Royal Society of Chemistry.2018.pp 473-479 [Non-Patent Document 3] Mawuch et al,Int.J.Mol.Sci.2020,21,8944 [Overview of the Initiative] [Means for solving the problem]

[0006] This specification includes formula (I): J-(CH2)x(CO)-(A)y-(B)z- (I) A compound comprising a cationic alkyl moiety represented by, J is either HOOC or CH3. x is between 10 and 16. A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), gamma-aminobutyric acid (γAbu), gamma-linked glutamic acid (γE), and alpha-linked glutamic acid (E). y is between 2 and 4, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is between 2 and 4, -(B)z- contains two or fewer Dab residues, and the Dap residues or Dab residues are linked via alpha-amino acids. A compound is provided.

[0007] Furthermore, this specification includes formula (II): CH3(CH2)x(CO)-(A)y-(B)z-peptide moiety (II) A conjugated peptide represented by, x is between 10 and 16. A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), gamma-aminobutyric acid (γAbu), gamma-linked glutamic acid (γE), and alpha-linked glutamic acid (E). y is between 2 and 4, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is between 2 and 4, -(B)z- contains two or fewer Dab residues, and the Dap or Dab residues are linked via alpha-amino compounds. CH3(CH2)x(CO)-(A)y-(B)z- is covalently attached to the N-terminus of the peptide moiety or linked to one of the side-chain amino groups of the peptide moiety. Conjugated peptides are also provided.

[0008] In some embodiments, the conjugated peptide has biological activity equal to or greater than that of an unmodified peptide at an equivalent bolus dose, blood levels equal to or greater than those of an unconjugated peptide at the same time point after bolus administration at an equivalent dose, or a combination thereof.

[0009] Compositions comprising a compound or conjugated peptide of the present disclosure for use in the manufacture of a medicament are provided herein.

[0010] In addition, compounds, conjugated peptides, and compositions of the present disclosure are provided for use in treating a disease or condition in a subject in need thereof.

[0011] Also provided herein are compositions comprising a compound or conjugated peptide of the present disclosure and one or more pharmaceutically acceptable carriers or excipients.

[0012] The foregoing will become apparent from the following more detailed description of the exemplary embodiments. As illustrated in the accompanying drawings, like reference numerals refer to the same parts in different figures. The drawings are not necessarily to scale; instead, emphasis is placed on illustrating embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] [Figure 1]A and B show the PK / PD profiles after a single subcutaneous (SC) injection of 1.0 mg / kg (0.45, 0.33, and 0.33 μmol / kg, respectively) of natural CNP (SEQ ID 32), SEQ ID 31, and SEQ ID 29 into mice. Error bars represent the standard error of the mean. A) Plasma CNP [mean (SEM), n=5] after subcutaneous administration of 1.0 mg / kg of natural CNP (SEQ ID 32), SEQ ID 31, and SEQ ID 29 (0.45, 0.33, and 0.33 μmol / kg, respectively) in buffer to CD-1 mice. In all groups, baseline CNP levels before administration were 4.5 (1.4) ng / mL [mean (SD), n=15]. Sequences ID 31 and 29 not only increase baseline plasma CNP levels by more than 5-fold and more than 10-fold, respectively, but also show a sustained increase in CNP levels compared to natural CNP. Plasma CNP in CD-1 mice was measured using the CNP ELISA kit from Phoenix Pharmaceuticals (EKE-012-03, Burlingame, CA). B) Plasma cGMP after subcutaneous administration of 1.0 mg / kg of natural CNP (SEQ ID NO: 32), SEQ ID NO: 31, and SEQ ID NO: 29 to CD-1 mice. In all groups, baseline plasma cGMP levels were 13.9 (5.7) pmol / mL [mean (SD), n=15]. SEQ ID NOs: 31 and 29 not only increased baseline plasma cGMP levels by more than 10 times, but also showed a sustained increase in cGMP levels compared to natural CNP. Plasma cGMP was measured using the cGMP ELISA kit from Abcam (ab133052, Waltham, MA). [Figure 2]A and B demonstrate that single and repeated administration of cationic alkyl-modified C-type natriuretic peptides enhances survival in LPS-induced sepsis and acute lung injury (ALI) animal models. A) Sepsis induction: C57BL / 6J male mice (n=10 in each of the three groups) were intraperitoneally (IP) injected with LPS (15 mg / kg) and treated with various test substances, including cationic alkyl-modified CNP (SEQ ID NOs. 31 and 29, 0.3 mg / kg (0.1 μmol / kg) SC). The control group received LPS treatment without the test substances. The test substances were administered immediately after LPS administration (indicated by vertical dotted lines). Survival rates were monitored every two hours from 8 to 56 hours, after which surviving mice were euthanized under isoflurane anesthesia. Statistical analysis was performed using the Gehan-Breslow-Wilcoxon test with GraphPad Prism (n=10, 10, and 10; control, SEQ ID NO: 31, and SEQ ID NO: 29). **P<0.01, *P<0.05 (compared to the control group). B) ALI induction: C57BL / 6J male mice (n=6 in each group, 3 groups) were intratracheally (IT) infused with LPS (20 mg / kg) and treated with various test substances, including cationic alkyl-modified CNP (SEQ ID NO: 31 and 29, 0.3 mg / kg (0.1 μmol / kg) IT). The control group received LPS treatment without test substances. Test substances were administered every 24 hours starting immediately after LPS administration, for a total of three bolus doses (shown by vertical dotted lines). Survival rates were monitored every 8 hours until 72 hours, after which surviving mice were euthanized under isoflurane anesthesia. Statistical analysis was performed using the Gehan-Breslow-Wilcoxon test with 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). [Figure 3-1]Figures A-D show that bolus administration of cationic alkyl-modified C-type natriuretic peptide suppresses lung injury and resolves acute lung injury (ALI) / acute respiratory distress syndrome (ARDS). In several types of inflammatory lung diseases, neutrophil counts increase and the expression of myeloid-derived proteins (S100A8 / A9) rises. MPO+ cells serve as an indicator that directly measures the presence of neutrophils. Therefore, in an animal model of ALI (Figure 3A), the expression of S100A8 and S100A9 (Figure 3B) and the presence of MPO+ cells in the lungs (Figures 3C, D) are measured as common markers of inflammation. This decrease indicates the resolution of ALI / ARDS and is consistent with the observed increase in survival rate (see Figure 2). A) shows a diagram illustrating the procedure in which C57BL / 6J mice were administered LPS (0.05 mg / kg IT) and then treated with various test substances, including the human neutrophil elastase inhibitor sivelestat (150 mg / kg) administered via IP injection as a positive control, and cationic alkyl-modified CNP (SEQ ID NOs. 31, 29, and 30; 0.3 mg / kg (0.1 μmol / kg) SC). The test substances were administered immediately after LPS injection. In addition, a normal control (NC) group that did not receive LPS and a control group that received only LPS without any test substances were also included. After 24 hours, the mice were euthanized under isoflurane anesthesia, and their lungs were collected for analysis. This procedure had to be repeated because the lung processing technique differed for each experiment. B) The lungs were finely chopped in Tri-Reagent and processed to measure the gene expression levels of S100A8 and S100A9 by qRT-PCR analysis using 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 (negative control), Control, PC (positive control), 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 (for each corresponding control group). C, D) Lung tissue was fixed, embedded in paraffin, and sectioned. Immunohistochemical staining was performed on the sections, and the number of myeloperoxidase-positive (MPO+) cells was quantified in each field of view.Statistical analysis was based on Dunnett's test 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 (vs. each corresponding control group). [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1] A - C indicate that cationic alkyl - modified CNP derivatives reduced pulmonary neutrophil infiltration. ALI and ARDS are associated with an increase in cells, particularly neutrophils, in bronchoalveolar lavage fluid (BALF). To evaluate the resolution of ALI / ARDS in an animal model, the cell count (B) and total protein amount (C) that function as neutrophil markers were measured. A decrease in these markers indicates the resolution of ALI / ARDS. A) shows a diagram of the procedure of treating mice with LPS (0.05 mg / kg IT) followed by various test substances such as the human neutrophil elastase inhibitor sivelestat (150 mg / kg) injected IP as a positive control and cationic alkyl - modified CNP (SEQ ID NOs: 31, 29, and 30; 0.3 mg / kg (0.1 μmol / kg) SC). The test substances were administered immediately after LPS injection. Additionally, a normal control (NC) group not treated with LPS and a control group treated with only LPS without the test substance were included. After 24 hours, the mice were euthanized under isoflurane anesthesia and BALF was collected. Statistical analysis was based on Dunnett's test 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 (vs. each corresponding control group). [Figure 4-2] Same as above. [Figure 5-1]Figures A-E show the effect of cationic alkyl-modified CNP derivatives on the inflammatory state during acute exacerbation of idiopathic pulmonary fibrosis (IPF-AE). Error bars indicate the standard error of the mean. In IPF-AE, increased pulmonary inflammation is observed, particularly in neutrophils, monocyte chemotactic protein 1 (MCP1), and interleukin-6 (IL-6). A decrease in these levels indicates resolution of IPF-AE. Figure A shows the procedure for treating C57BL / 6J mice with bleomycin (Bleo, 1.0 mg / kg IT administration). Three weeks later, the mice were treated with LPS (0.025 mg / kg IT), and as shown in the figure, they were SC-treated with SEQ ID NO: 29 or 31 at 0.3 mg / kg (0.1 μmol / kg) the day before LPS treatment, on the day of treatment, and after treatment. Furthermore, the study included a normal control (NC) group that did not receive LPS / Bleo treatment, a Bleo group that did not receive LPS treatment, and a control group that did not receive the test substance. On the final day, mice were euthanized under isoflurane anesthesia, and lung tissue was collected and weighed. B) An increase in lung weight / body weight ratio is a parameter of lung injury. C) Upregulation of MPO, a neutrophil marker, was significantly suppressed in the group treated with cationic alkyl-modified CNP derivatives. D) In ​​particular, SEQ ID NO: 31 showed a significant decrease in MCP1, another common indicator of inflammation. E) IL-6 levels did not show a significant difference compared to the NC group, indicating reduced 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 (compared to the control group). [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 6]Figures A and B show that repeated subcutaneous administration of cationic alkyl-modified CNP derivatives exhibited significant antitumor activity in a mouse model of orthotopic breast cancer using E0771 cells. Error bars represent the standard error of the mean. A) Tumor growth dynamics of 6-week-old C57BL / 6J female mice (n=10 in each group) inoculated with E0771 breast cancer cells (250,000 cells / mouse) into the mammary gland. Starting 4 days after inoculation, cationic alkyl-modified CNP derivatives (SEQ ID NOs. 29, 30, and 31) were administered subcutaneously once daily at a bolus dose of 0.3 mg / kg (0.1 μmol / kg) for 5 days (5 days administration, 2 days rest) for 3 cycles. Administration days are indicated by grid lines. The control group (the group administered only buffer) was administered in the same manner as the other groups to establish baseline tumor growth dynamics. B) The groups treated with cationic alkyl-modified CNP showed a significant reduction in tumor volume compared to the control group at the end of the study. However, cationic alkyl sequences without CNP (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 CNP binding is important for the observed antitumor activity. Statistical analysis was based on Dunnett's 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). [Figure 7] 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. With the CNP-based probe alone (5nM CNP-F*), the rotation is fast, resulting in a low fluorescence polarization (FP) signal. When human NPRB or NPRC (50nM) is added, the CNP probe binds to these receptors, resulting in slower rotation and a higher FP signal. In the presence of NPRA, no change in the FP signal is detected, suggesting that the CNP probe does not bind to NPRA. In the presence of SEQ ID NO: 31, the low FP signal indicates binding to both NPRB and NPRC. [Figure 8-1]A-B demonstrates that cationic alkyl-modified CNP derivatives, either alone or in combination with pirfenidone, reduced fibrosis in a mouse model of idiopathic pulmonary fibrosis (IPF). A) A diagram of the procedure for inducing pulmonary fibrosis is shown. Male C57BL / 6J mice (6 weeks old) were administered bleomycin (Bleo, 1.0 mg / kg) via intra-articular (IT) administration, and 7 days later, SEQ ID NO: 31 was administered once daily at 0.3 mg / kg on weekdays via SC administration, and / or pirfenidone was administered once daily (orally at 100 mg / kg). In addition, a normal control group without Bleo treatment (NC, n=3) and a Bleo control group without treatment with the test substance (n=7) were also included in this study. On day 21 after Bleo administration, the mice were euthanized under isoflurane anesthesia, and their lung tissue was collected. B) A portion of lung tissue was fixed with 4% PFA, stained with Azan, and alveolar area (inversely proportional to the degree of fibrosis) was measured in Image J in a blinded setting. Error bars indicate the standard error of the mean. Statistical analysis was performed using Student's t-test with GraphPad Prism (n=3, 7, 7, 7, and 7); NC, Bleo control, SEQ ID NO: 31, pirfenidone (Pir), and combination (SEQ ID NO: 31 & pirfenidone). ***P<0.001, ns=not significant (no significant difference) (compared to the control group). The groups treated with SEQ ID NO: 31 alone or in combination with pirfenidone had significantly higher alveolar area, indicating the presence of healthy tissue and reduced fibrosis. [Figure 8-2] Same as above. [Figure 9]Repeated subcutaneous administration of cationic alkyl-modified CNP derivatives, either as monotherapy or in combination with immune checkpoint inhibitors, significantly reduced tumor volume in an orthotopic mammary cancer mouse model using E0771 cells. Error bars represent the standard error of the mean. Tumor growth dynamics of 6-week-old C57BL / 6J female mice (n=7-8 in each group) orthotopically inoculated with E0771 mammary cancer cells (250,000 cells / mouse) into the mammary gland. Anti-PD1 antibodies (anti-PD1 Ab, aPD1) were administered intraperitoneally at 5 mg / kg twice a week for two cycles starting 4 days after inoculation. 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, 2 days of rest) for 3 cycles, or in combination with aPD1 (5 mg / kg IP administration twice weekly for 2 cycles) and SEQ ID NO: 31 (0.3 mg / kg SC administration once daily for 5 days, 2 days of rest, 3 cycles). The administration days for SEQ ID NO: 31 are indicated by dotted or grid lines. The control group (received buffer only) was administered in the same manner as SEQ ID NO: 31 to establish baseline tumor growth dynamics. At the end of the study, the group treated with SEQ ID NO: 31 showed a significant reduction in antiproliferative activity or tumor volume compared to the control group and the group treated with the immune checkpoint inhibitor aPD1 alone. Statistical analysis was based on Dunnett's test performed using GraphPad (n=8, 7, 8, and 7; control, aPD1, SEQ ID NO: 31, combination (combo) (aPD1 and SEQ ID NO: 31). *P<0.05, ****P<0.0001 (compared to the control group). [Figure 10-1]Figures A and B show that in a mouse model of orthotopic bone metastasis using E0771 breast cancer cells, the incidence of metastasis was reduced and overall survival was significantly improved by the combined use of radiation, immune checkpoint inhibitors, and repeated subcutaneous administration of cationic alkyl-modified CNP derivatives. Error bars indicate the standard error of the mean. A) This figure shows the procedure for orthotopic transplantation of E0771 breast cancer cells (250,000 cells / mouse in RPMI1640 medium) into the left mammary gland of a 6-week-old female C57BL / 6J mouse, and orthotopic transplantation of E0771 mouse breast cancer cells (500,000 cells / mouse in 50% Matrigel) into the femur. This figure also shows the schedule corresponding to each procedure performed throughout this study. After tumor transplantation, the SEQ ID NO: 31 treatment groups (Groups 2, 3, 6, and 7) received 0.3 mg / kg / 0.1 μmol / kg (in a buffer solution containing 15 mM succinate, 4% (w / v) D-mannitol, and 10 mM hydroxypropyl-β-cyclodextrin at pH 4.4) via intracellular (SC) administration. Administration was performed on days 5-9, 12-16, 19-23, and 26-29. The aPD1 treatment groups (Groups 3, 5, and 7) received 5 mg / kg via intracellular (IP) administration on days 5, 7, 12, and 14, while the radiation therapy groups (Groups 4, 5, 6, and 7) received three sets each of buffer solution and 5 Gy of X-ray radiation to the bone region on days 5, 8, and 12. After the remaining mice were euthanized as planned, survival rates were observed until day 33. At this point, tumor size was measured with calipers. B) Survival probabilities for groups 1 through 7 are shown. Since all groups were significantly better than the control group, statistical analysis was performed using GraphPad Prism based on the log-rank (Mantel-Cox) test for group 5 (radiation + aPD1). The addition of Sequence ID No. 31 resulted in a significant improvement in survival rates with the combination of the three treatments (**P<0.01). Furthermore, 5 out of 6 mice in group 6 (Sequence ID No. 31 + radiation) and 6 out of 6 mice in group 7 (Sequence ID No. 31 + radiation + aPD1) had no bone tumors compared to 1 out of 5 mice in group 4 (radiation alone) and group 5 (radiation + aPD1). These findings indicate that the addition of Sequence ID No. 31 reduces the incidence of metastasis and tumor burden. [Figure 10-2] Same as above. [Figure 11-1] In a mouse model of orthotopic lung metastasis using osteosarcoma LM8 cells, mutilation combined with repeated subcutaneous administration of a cationic alkyl-modified CNP derivative significantly reduced the incidence of lung metastasis compared to mutilation alone. Error bars indicate the standard error of the mean. A) A diagram shows the procedure for orthotopic transplantation of LM8 osteosarcoma cells (1,000,000 cells / mouse) into the femur of CH3 / He male mice (7 weeks old). This diagram also shows the corresponding treatment schedule for Sequence ID No. 31 and mutilation. SC (0.3 mg / kg / 0.1 μmol / kg, in buffer) was administered to the Sequence ID No. 31 treatment group on days 4-8, 11-15, 18-22, and 25-26 after tumor transplantation. On day 7 after inoculation, all mice were mutilated to remove the primary tumor. Instead of treatment, the mutilation control group was also administered buffer on the same day as Sequence ID No. 31 was administered. Mice were euthanized on day 34 and lung tissue was collected. B and C) 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 directly counting the present metastatic lung nodules. In the group treated with SEQ ID NO: 31 (resection), the number of metastatic nodules in the lungs was significantly reduced compared to the group treated with resection without any intervention (**P<0.01). Outliers were identified using the ROUT test (Q=1%). All statistical analyses were based on Dunnett's test performed using GraphPad (n=7 and 7; control (resection), combination (resection and SEQ ID NO: 31))**P<0.01 (compared to the control group). [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 12-1]Repeated subcutaneous administration of cationic alkyl-modified CNP derivatives, either as monotherapy or in combination with immune checkpoint inhibitors, significantly reduced tumor volume in a subcutaneous mouse model of colon cancer using MC38 cells. Error bars represent the standard error of the mean. A) A diagram showing the administration schedule of the immune checkpoint inhibitor (anti-TIGIT antibody (anti-TIGIT Ab)) and SEQ ID NO: 31. B) Tumor growth dynamics of 6-week-old male C57BL / 6J mice (n=8-9 in each group) subcutaneously inoculated with MC38 colon cancer cells (1,000,000 cells / mouse) in the right flank. From day 4 after inoculation, anti-TIGIT antibody was administered intraperitoneally at 5 mg / kg twice a week for 2 cycles. SEQ ID NO: 31 was administered subcutaneously once daily at a bolus dose of 0.3 mg / kg (0.1 μmol / kg) for 5 days (5 days administration, 2 days rest) for 3 cycles. Alternatively, a combination of anti-TIGIT antibody (5 mg / kg administered intravenously twice weekly for 2 cycles) and SEQ ID NO: 31 (0.3 mg / kg administered once daily for 5 days, followed by a 2-day rest period and then intracytoplasmic rehydration therapy for 3 cycles) was administered. The control group (received buffer only) was administered in the same manner as SEQ ID NO: 31 to establish baseline tumor growth dynamics. The group treated with SEQ ID NO: 31 showed a significant reduction in tumor volume and cancer burden compared to the control group, and the combination group (anti-TIGIT antibody + SEQ ID NO: 3) demonstrated a superior antitumor effect compared to anti-TIGIT antibody monotherapy. Statistical analysis was based on Dunnett's test 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). [Figure 12-2] Same as above. [Figure 13]HeLa cells treated with cationic alkyl-modified CNP derivatives showed binding to NPR-C based on significant inhibition of baseline cyclic adenosine monophosphate levels. In this study, HeLa cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS at 100% humidity, 5% CO2 concentration, and 37°C. Cells were harvested and suspended in ENGS at a concentration of 10⁷ cells / mL. 5 μL of the cell suspension was added to each well of a 96-well low-adhesion plate. Next, cells (n=4 wells) were treated with 5 μL of SEQ ID NO: 31 (final concentration 10 μg / mL) in ENGS containing 1 mM IBMX for 10 minutes. cAMP levels were assessed using a PerkinElmer (Waltham, MA, USA, #62AM4PEB) cAMP assay kit, following the manufacturer's protocol and using a PerkinElmer plate reader. Statistical analysis was performed on untreated control wells (n=4 wells) using GraphPad Prism. *P<0.05. The NPR-C receptor is suggested to inhibit adenylyl cyclase activity, which reduces cyclic adenosine monophosphate (cAMP) production. From this study, we can conclude 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. [Modes for carrying out the invention]

[0014] This disclosure provides novel compositions comprising compounds containing a single cationic alkyl chain moiety, which are useful for modifying biologically active molecules such as peptides and exhibit remarkably significant toxicity reductions when administered in vivo (for example, certain modifiers disclosed herein did not cause toxicity or ataxia in rats at 10 μmol / kg). The novel compositions comprise compounds comprising a single alkyl chain linked by a non-cationic linker to a chain of 2-4 cationic amino acid residues selected from diaminopropionic acid (Dap) and diaminobutanoic acid (Dab), with two or fewer Dab residues present in the chain. The non-cationic linker comprises 2-4 residues independently selected from 2-aminoethoxy-2-ethoxyacetic acid (Aeea), gamma-aminobutyric acid (γAbu), gamma-bound glutamic acid (γE), and glutamic acid (E). The cationic alkyl chains disclosed herein can be linked to peptides to enhance the pharmacokinetic and / or pharmacodynamic activity of those peptides (see Figure 1). For example, the activity can be enhanced compared to naturally occurring unmodified peptides and without the risk of causing further toxicity (for example, even at bolus doses of 3.0 μmol / kg or less [Table 1]). As illustrated herein, cationic alkyl chain-modified peptides, such as type C natriuretic peptide (CNP) (or its derivatives), can be used to increase survival rates from 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). Furthermore, cationic alkyl chain-modified CNP (or its derivatives) can be used, for example, as a therapeutic agent for treating cancer, without reaching doses that cause toxicity / ataxia (see Figures 6 and 9-12, and Examples 10 and 13-16). Further examples herein are cationic alkyl chain-modified atrial natriuretic peptides, type B natriuretic peptides, and their derivatives.

[0015] 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 hypoxemia. ALI is characterized by a PaO2 / FiO2 ratio of less than 300 Torr, while more severe ARDS is characterized by a PaO2 / FiO2 ratio of less than 200 Torr. They are also characterized by bilateral radiographic infiltrates and the absence of findings of left atrial hypertension. (See, for example, Bernard, GR, et al., J. Crit. Care, 1994.9(1):p.72-81, Rubenfeld, GD, et al., N Engl J Med, 2005.353(16):p.1685-93, Brun-Buisson, C., et al., Intensive Care Med, 2004.30(1):p.51-61, and Phua, J., et al., Am J Respir Crit Care Med, 2009.179(3):p.220-7). As used herein, PaO2 refers to the partial pressure of oxygen in arterial blood, and FiO2 is the fraction of oxygen in the inhaled air (for reference, the FiO2 of air is approximately 0.21, and the normal PaO2 / FiO2 is approximately 500 Torr). Both ALI and ARDS can lead to death or pulmonary fibrosis. ARDS is an overwhelming inflammatory pneumonia response to certain primary and secondary adverse stimuli, such as pneumonia (aseptic, viral, or bacterial), sepsis, aspiration, inhalation injury, drowning, or lung resection surgery (see, e.g., Alam, N., et al, Ann Thorac Surg, 2007. 84(4): pp. 1085-91). ARDS is characterized by rapidly developing respiratory failure requiring hospitalization in the intensive care unit (ICU) and mechanical ventilation support. When patients survive ALI / ARDS, lung scarring often negatively impacts their long-term quality of life (see, e.g., Rubenfeld, GD, et al., N Engl J Med, 2005. 353(16): pp. 1685-93, Dowdy, DW, et al., Intensive Care Med, 2006. 32(8): pp. 1115-24). To date, no effective drugs have been identified to treat acute lung injury (ALI) or ARDS, and there is a great need for such drugs.All drugs tested in human clinical trials to date for the treatment of ALI, including glucocorticoids, surfactants, N-acetylcysteine, inhaled nitric oxide, liposomal PGE1, ketoconazole, lysophyllin, salbutamol, procysteine, activated protein C, and inhaled albuterol, have failed (see, for example, Johnson ER and Matthay MA, J Aerosol Med Pulm Drug Deliv. 2010, 23(4):243-52). No drug for the treatment of ALI or ARDS in humans has yet been found by those skilled in the art. Supportive care for ALI includes oxygen administration to maintain arterial oxygen partial pressure (PaO2) above 55 mmHg or oxygen saturation (SaO2) above 88%, and fluid management. However, care must be taken not to over-supply oxygen to avoid oxygen toxicity (i.e., oxygen supply should be less than 60%). Furthermore, this treatment does not address the underlying alveolar inflammatory edema (fluid in the alveoli filled with blood proteins and inflammatory cells). Alveolar inflammatory edema, which underlies ALI or ARDS, is the root cause of decreased blood oxygenation.

[0016] Pulmonary fibrosis Pulmonary fibrosis (PF) is a progressive scarring of lung tissue and can be caused by many conditions, including infections (i.e., sepsis or pneumonia that may be sterile, viral, or bacterial), environmental factors (e.g., exposure to asbestos, silica, or certain gases), exposure to ionizing radiation (e.g., radiotherapy to treat thoracic tumors), chronic autoimmune diseases (e.g., lupus, rheumatoid arthritis), chronic inflammatory processes (e.g., sarcoidosis, Wegener's granulomatosis), or certain medications. Interstitial lung disease (ILD) is another broad term referring to PF and is treated as a synonym herein. Idiopathic pulmonary fibrosis (IPF) is PF of unknown cause. PF, or IPF, is a chronic, scarring lung disease characterized by progressive and irreversible decline in lung function, gradually leading to shortness of breath and a dry cough. It affects 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 inhalation of chemicals such as smoking, viral infections, or a family history of the disease. Other symptoms may include fatigue and abnormally large, dome-shaped fingernails and toenails (nail clubbing). See, for example, the National Institutes of Health's health topics page on idiopathic pulmonary fibrosis or the Wikipedia page on idiopathic pulmonary fibrosis. Complications may include pulmonary hypertension, heart failure, pneumonia, and pulmonary embolism.

[0017] Therefore, there is a need for safer and more effective compositions for the treatment of ALI and / or ARDS and PF. Such compositions can maintain or enhance plasma levels of CNP therapeutic agents while avoiding cardiovascular side effects such as hypotension. There is a need for long-half-life natriuretic peptide derivatives for use in the manufacture of pharmaceuticals for the treatment of ALI and / or ARDS. This disclosure seeks to meet such needs and provides further relevant advantages.

[0018] Natriuretic peptides (NPs) and natriuretic peptide receptors (NPRs)

[0019] Natriuretic peptides (NPs) are peptides that induce sodium excretion in the kidneys and lower blood pressure by binding to natriuretic transmembrane receptors that have a guanylate cyclase domain within cells, thereby lowering blood pressure through vasodilation. Binding activates guanylate cyclase activity, increasing blood and intracellular cGMP levels and leading to various physiological activities. Several natriuretic peptides are well known in this field. Type C natriuretic peptide (CNP, GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 32]) acts via natriuretic peptide receptor B (NPRB) and natriuretic peptide receptor C (NPRC) (e.g., 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). (See TJBiol.Chem.2001;276:6057-6060). On the other hand, 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 ID NO: 48]) either bind to or act via natriuretic peptide receptor A (NPRA). CNP, BNP, ANP, and URO are peptides with a cyclic structure necessary for their activity, made possible by the presence of disulfide bonds. NPs have various physiological activities, including vasodilatory, blood pressure-raising, and vascular fluid regulation through diuretic effects.For example, the inhibitory effect of ANP on inflammation induced by bacterial infection and the associated breakdown of endothelial barrier function has been reported (see, for example, Xing J., et al., J.appl.Physiol., 2011, 110(1), 213-224). Furthermore, CNP, BNP, ANP, and URO all bind to NPRCs (which lack guanylyl cyclase activity) and are removed and degraded (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 TJBiol.Chem. 2001; 276: 6057-6060).

[0020] Traditionally, natriuretic peptides have a very short half-life, and large bolus doses result in extremely high plasma peak concentrations (Cmax), causing dangerously low blood pressure, lethargy, and ataxia, and are the main cause of toxicity; therefore, they need to be administered continuously at low doses. To mitigate these adverse effects, natriuretic peptides are usually delivered by slow infusion. See, for example, Kimura et al., J Surg Res. 2015, 194(2); 631-637. One aspect of the disclosed invention mitigates the toxicity of natriuretic peptides by bolus administration while enhancing the cyclic GMP response and further increasing efficacy in treating the disease.

[0021] This disclosure relates to the surprising and unexpected discovery of compounds containing cationic alkyl moieties that are less toxic (e.g., requiring high doses to observe ataxia). The cationic alkyl moieties of this disclosure can be covalently bonded to molecules (e.g., peptides) to reduce in vivo degradation and / or extend blood presence or half-life without causing toxicity (ataxia). These benefits can occur at doses equivalent to or higher than therapeutically effective doses, and at higher doses than the same peptide modified with other cationic alkyl moieties (see Table 3 in Example 3). Furthermore, the disclosed compositions can enhance biological activity (pharmacological effect) compared to unmodified peptides.

[0022] This disclosure relates to a composition comprising a compound comprising a single positively charged alkyl chain having at least two derived from diaminopropionic acid (Dap) and / or diaminobutanoic acid (Dab) (defined herein as a cationic alkyl), which has the surprising and non-obvious characteristic of significantly reducing toxicity (tested by ataxia in rats) compared to compositions modified with cationic amino acids having larger or longer chains of R groups, including natural or non-natural D-type amino acids such as lysine or arginine. The novel composition comprises a single alkyl chain linked by a non-cationic linker to 2 to 4 cationic amino acid residues independently selected from diaminopropionic acid (Dap) and / or diaminobutanoic acid (Dab), wherein the cationic chain has 2 or fewer Dab residues. The non-cationic linker is 2-4 residues independently selected from 2-aminoethoxy-2-ethoxyacetic acid (Aeea), gamma-aminobutyric acid (γAbu), gamma-bound glutamic acid (γE), and glutamic acid (E). 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 exemplified herein, cationic alkyl-modified peptides such as C-type natriuretic peptide (CNP) (or its derivatives) can be used to increase survival rates from sepsis, acute respiratory distress syndrome, acute lung injury, and / or pulmonary fibrosis (see Examples 5-9 and 12, with relevant figures or tables). Furthermore, cationic alkyl-modified CNP (or its derivatives) can be used to treat or suppress cancer (see Examples 10 and 13-16). This disclosure relates to the remarkable discovery of safer cationic alkyl compositions that are safe at bolus doses of up to 10 μmol / kg (see Table 1 in Example 1). Furthermore, the cationic alkyl moieties of this disclosure can generally be covalently bonded to therapeutic peptides, thereby enhancing the peptides' in vivo stability, half-life (pharmacokinetics), and / or activity (pharmacological effects).In this case, even at bolus doses at least twice the therapeutically effective dose, and / or doses exhibiting significant (measurable) pharmacodynamic activity, no toxicity or ataxia is induced. In other words, peptide modifications using cationic alkyl modifications of this disclosure do not cause toxicity to the peptide at doses that provide significant pharmacological and / or therapeutic activity compared to unmodified peptides. This disclosure also relates to cationic alkyl-conjugated natriuretic peptides and their derivatives or compositions thereof that exhibit unexpectedly superior ability to increase blood cGMP compared to natural natriuretic peptides and lower toxicity compared to other cationic alkyl-conjugated natriuretic peptides. Examples of cationic alkyl-conjugated peptides in this disclosure include C-type natriuretic peptide (CNP), atrial natriuretic peptide (ANP), brain-type natriuretic peptide (BNP), and their corresponding derivatives, which exhibit unexpectedly superior biological activity and / or lower toxicity compared to the corresponding natural peptides or other modified natriuretic peptides known in the art.

[0023] For peptides to be considered for parenteral drug development, they must exhibit non-toxic therapeutic efficacy at practical or reasonable high doses via parenteral administration (e.g., subcutaneous, intramuscular, inhalation, or intravenous administration). For subcutaneous, intramuscular, and inhalation administration of peptides, desirable and practical high bolus doses in humans should not exceed 12 mg per person, so as not to result in excessive volume of infusion and / or peptide concentrations exceeding the solubility of the peptide in the infusion. A human bolus dose of 12 mg / 70 kg (or 0.17 mg / kg) is converted to rat and mouse doses of 1.0 mg / kg and 2.0 mg / kg, respectively, after allometric scaling. 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 a cationic lipid or cationic alkyl molar linked to a peptide in a 1:1 (mol:mol) ratio does not add toxicity / undesirable effects at practical or reasonable therapeutic doses, the dose at which no MTD or undesirable effects are observed for the cationic alkyl molar must be at least twice as large as the preferred and / or practical therapeutic molar dose of the peptide in the same species.

[0024] For the purposes of this disclosure, preferred compositions of the present invention include compounds comprising the cationic alkyl chain of the present disclosure, which, in some embodiments, have the ability to improve the in vivo pharmacokinetics, pharmacological effects, and / or bioavailability of a modified compound (e.g., a peptide) with minimal or no toxicity (ataxia) observed in rats at bolus doses up to 10 μmol / kg (see Table 1 of Example 1). For cationic alkyl moieties capable of improving the in vivo pharmacokinetics and / or pharmacological effects of peptides, preferred cationic alkyl moieties are those 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 (see Table 1 of Example 1). This preferred configuration ensures that the cationic lipid or cationic alkyl moiety, linked to the peptide in a 1:1 molar ratio for half-life extension and / or potency enhancement, does not impart toxic / undesirable effects to the peptide at doses of 3.0 μmol / kg or less. This disclosure provides compositions comprising an alkyl cationic moiety that can be linked or covalently linked to a peptide in a 1:1 ratio without the possibility of causing further toxicity to the peptide when administered to rats at preferred practical bolus doses of 3.0 μmol / kg or less. As an example, covalent linking of the cationic alkyl moiety of this disclosure to a natriuretic peptide did not induce ataxia alone at doses of 10 μmol / kg or less in rats, but the resulting conjugate was unexpectedly superior in its ability to increase blood cGMP and / or intracellular cGMP in vivo compared to natural peptides such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), or C-type natriuretic peptide (CNP), and their derivatives (see Table 3 in Example 3).

[0025] This disclosure describes modifications of peptides or derivatives to extend the half-life of peptides or derivatives without adding the risk of toxicity at therapeutic doses as a result of cationic alkyl modification. The modifiers of this disclosure are cationic lipids, which are generally known to be toxic in the art. However, surprisingly, when two or more unnatural amino acids of diaminopropionic acid (Dap) or diaminobutyric acid (Dab) form the cationic moiety of the cationic alkyl modifier, and these cationic amino acids are separated from the alkyl chain by non-cationic spacers such as Aeea, γAbu, γE, and / or 2-4 residues of E, it has been found that the toxicity is low (i.e., no ataxia and / or death) in rats. This is in contrast to alkyl modifications involving cationic amino acids with larger R groups (natural or unnatural D-type), such as lysine, and without non-cationic spacers between the alkyl chain and the cationic amino acids. The present invention enables the modification of peptides by cationic alkyl / lipid modification and limits the addition of toxicity to peptides. This ensures a larger safety margin at doses above the dose required for biological activity (pharmacological effect), or at practical and reasonable bolus doses (less than 3.0 μmol / kg for parenteral administration), which is extremely important in commercial drug development.

[0026] Most modified peptides to date use alkyl chains without a cationic moiety, as cationic lipids and cationic alkyls have been well demonstrated to be inherently toxic [Cui et al, 2018]. This disclosure provides cationic alkyl compositions that exhibit remarkably limited biological toxicity compared to other cationic alkyl compositions. Furthermore, the cationic alkyl compositions of this disclosure can covalently bond to peptides at residues not essential to the peptide's biological activity, resulting in peptide compositions with extended in vivo half-life or blood presence after bolus administration compared to unmodified peptides. In addition, the cationic alkyl moiety on the modified peptide can interact with the cellular anionic lipid membrane in combination with the peptide's specific interaction with its receptor, thereby increasing the biological activity or pharmacological potency of the peptide. The cationic alkyl compositions of this disclosure can be attached to or covalently bonded to peptides, such as natriuretic peptides. The compositions and methods of use of this disclosure are described herein.

[0027] The following describes an exemplary embodiment. composition Equation (I) This disclosure is based on formula (I): J-(CH2)x(CO)-(A)y-(B)z (I) A cationic alkyl compound or cationic lipid compound (for example, a compound for covalently modifying a peptide to improve its effectiveness) comprising a cationic moiety represented by, J is either HOOC or CH3. x is between 10 and 16. A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), gamma-aminobutyric acid (γAbu), gamma-bound glutamic acid (γE), and glutamic acid (E). y is between 2 and 4, B is independently selected from diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is between 2 and 4, -(B)z- has two or fewer Dab residues in its sequence, and the bonds between residues are amide bonds between Dab and / or Dap alpha-amino acids. Furthermore, the composition does not produce clinically observable adverse effects or ataxia after subcutaneous bolus administration at doses of 10 μmol / kg or less in rats. The present invention provides cationic alkyl compounds or cationic lipid compounds.

[0028] In assessing toxicity (using ataxia as a marker of toxicity), the dose was increased until ataxia (i.e., toxicity) was observed, up to 10 μmol / kg (the practical maximum bolus dose of a liquid parenterally administered peptide administered in a small amount of injection solution). As used in this disclosure, “ataxia” is a clinical sign observed as poor muscle control resulting in clumsy voluntary movements. Ataxia can result in difficulties with motor function, coordination, and / or eye movements.

[0029] One embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3 and 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 gamma-aminobutyric acid (γAbu), and z is 2.

[0030] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-aminobutyric acid (γAbu), and z is 2.

[0031] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-aminobutyric acid (γAbu), y is 3, and z is 2.

[0032] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-aminobutyric acid (γAbu), y is 3, and z is 2.

[0033] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-linked glutamic acid (γE), and z is 2.

[0034] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-linked glutamic acid (γE), and z is 2.

[0035] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-linked glutamic acid (γE), y is 3, and z is 2.

[0036] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-linked glutamic acid (γE), y is 3, and z is 2.

[0037] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 10, 12, 14, or 16, A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), and z is 2.

[0038] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), and z is 2.

[0039] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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.

[0040] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), y is 3, and z is 2.

[0041] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 10, 12, 14, or 16, A is gamma-aminobutyric acid (γAbu), and z is 2.

[0042] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is gamma-aminobutyric acid (γAbu), and z is 2.

[0043] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 10, 12, 14, or 16, A is gamma-aminobutyric acid (γAbu), y is 3, and z is 2.

[0044] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is gamma-aminobutyric acid (γAbu), y is 3, and z is 2.

[0045] One embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3 and 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 gamma-aminobutyric acid (γAbu), and z is 3.

[0046] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-aminobutyric acid (γAbu), and z is 3.

[0047] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-aminobutyric acid (γAbu), y is 3, and z is 3.

[0048] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-aminobutyric acid (γAbu), y is 3, and z is 3.

[0049] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-linked glutamic acid (γE), and z is 3.

[0050] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-linked glutamic acid (γE), and z is 3.

[0051] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-linked glutamic acid (γE), y is 3, and z is 3.

[0052] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-linked glutamic acid (γE), y is 3, and z is 3.

[0053] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 10, 12, 14, or 16, A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), and z is 3.

[0054] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), and z is 3.

[0055] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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.

[0056] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), y is 3, and z is 3.

[0057] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 10, 12, 14, or 16, A is gamma-aminobutyric acid (γAbu), and z is 3.

[0058] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is gamma-aminobutyric acid (γAbu), and z is 3.

[0059] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 10, 12, 14, or 16, A is gamma-aminobutyric acid (γAbu), y is 3, and z is 3.

[0060] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, A is gamma-aminobutyric acid (γAbu), y is 3, and z is 3.

[0061] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-aminobutyric acid (γAbu), and z is 2.

[0062] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-aminobutyric acid (γAbu), and z is 2.

[0063] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-aminobutyric acid (γAbu), y is 3, and z is 2.

[0064] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-aminobutyric acid (γAbu), y is 3, and z is 2.

[0065] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-linked glutamic acid (γE), and z is 2.

[0066] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-linked glutamic acid (γE), and z is 2.

[0067] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-linked glutamic acid (γE), y is 3, and z is 2.

[0068] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-linked glutamic acid (γE), y is 3, and z is 2.

[0069] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 10, 12, 14, or 16, A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), and z is 2.

[0070] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), and z is 2.

[0071] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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.

[0072] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), y is 3, and z is 2.

[0073] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 10, 12, 14, or 16, A is gamma-aminobutyric acid (γAbu), and z is 2.

[0074] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is gamma-aminobutyric acid (γAbu), and z is 2.

[0075] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 10, 12, 14, or 16, A is gamma-aminobutyric acid (γAbu), y is 3, and z is 2.

[0076] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is gamma-aminobutyric acid (γAbu), y is 3, and z is 2.

[0077] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-aminobutyric acid (γAbu), and z is 3.

[0078] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-aminobutyric acid (γAbu), and z is 3.

[0079] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-aminobutyric acid (γAbu), y is 3, and z is 3.

[0080] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-aminobutyric acid (γAbu), y is 3, and z is 3.

[0081] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-linked glutamic acid (γE), and z is 3.

[0082] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-linked glutamic acid (γE), and z is 3.

[0083] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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 gamma-linked glutamic acid (γE), y is 3, and z is 3.

[0084] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea) and gamma-linked glutamic acid (γE), y is 3, and z is 3.

[0085] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 10, 12, 14, or 16, A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), and z is 3.

[0086] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), and z is 3.

[0087] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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.

[0088] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), y is 3, and z is 3.

[0089] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 10, 12, 14, or 16, A is gamma-aminobutyric acid (γAbu), and z is 3.

[0090] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is gamma-aminobutyric acid (γAbu), and z is 3.

[0091] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 10, 12, 14, or 16, A is gamma-aminobutyric acid (γAbu), y is 3, and z is 3.

[0092] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is HOOC, x is 14, A is gamma-aminobutyric acid (γAbu), y is 3, and z is 3.

[0093] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3 or HOOC, x is 10, 12, 14, or 16, and (A)y is Aeea-Aeea-Aeea, γAbu-γAbu-γAbu, γAbu-Aeea-Aeea, γE-Aeea-Aeea, E-Aeea-Aeea, Aeea-Aeea, γAbu-γAbu, γAbu-Aee a, γE-Aeea, 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.

[0094] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3 or HOOC, x is 14, and (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-A eea, or E-Aeea, and (B)z is Dap-Dap, Dab-Dab, Dab-Dap, Dap-Dab, Dap-Dap-Dap, Dab-Dab-Dap, Dab-Dab-Dap, Dap-Dab-Dap-Dap, Dap-Dab-Dap-Dap, Dab-Dab-Dap-Dap, or Dap-Dab-Dap-Dap.

[0095] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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-Dab, Dap-Dap-Dap, Dab-Dab-Dap, Dab-Dab-Dap-Dap, Dab-Dab-Dap-Dap, or Dap-Dab-Dap-Dap.

[0096] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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-Dab, Dap-Dap-Dap, Dab-Dab-Dap, Dab-Dab-Dap-Dap, Dab-Dab-Dap-Dap, or Dap-Dab-Dap-Dap.

[0097] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 10, 12, 14, or 16, and (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.

[0098] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where J is CH3, x is 14, and (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-Dap-Dap, Dap-Dab-Dap, Dap-Dab-Dap-Dap, Dab-Dab-Dap-Dap, Dab-Dab-Dap-Dap, or Dap-Dab-Dap-Dap.

[0099] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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-Dab, Dap-Dap-Dap, Dab-Dab-Dap, Dab-Dab-Dap-Dap, Dab-Dab-Dap-Dap, or Dap-Dab-Dap-Dap.

[0100] Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), where 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-Dab, Dap-Dap-Dap, Dab-Dab-Dap, Dab-Dab-Dap-Dap, Dab-Dab-Dap-Dap, or Dap-Dab-Dap-Dap.

[0101] In another embodiment of the present disclosure, the cationic moiety of formula (I) is [SEQ ID NO: 10 to 22 (see Table 1 of Example 1) or 51 to 69], where SEQ ID NO: 10 to 22 and 51 to 69 are 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-Dab [SEQ ID NO: 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-Dab[SEQ ID NO: 56], CH3(CH2)14(C=O)-γE-Aee a-Aeea-Dab-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-A eea-Dab-Dab [SEQ ID NO: 59], (HOOC)(CH2)14(C=O)-Aeea-Aeea-Aeea-Dap-Dab[SEQ ID NO: 60], (HOOC)(CH2)14(C=O)-Aeea-Aeea-Aeea- Dab-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 ID NO: 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 [Sequence ID 65], (HOOC)(CH2)14(C=O)-γE-Aeea-Aeea-Dap-Dap [Sequence ID 66], (HOOC)(CH2)14(C=O)-γE-Aeea-Aeea-Dab-Dab [Sequence ID 67], (HOOC)(CH2)14(C=O)-γE-Aeea-Aeea-Dap-Dab [Sequence ID 68], or (HOOC)(CH2)14(C=O)-γE-Aeea-Aeea-Dab-Dap [Sequence ID 69].

[0102] Another embodiment of the present disclosure is a composition of formula (I) for use in the manufacture of pharmaceuticals, such as medical compositions.

[0103] Another embodiment of the present disclosure is a composition (e.g., a pharmaceutical composition) comprising a compound of formula (I) containing an alkyl cationic moiety (e.g., conjugated to a peptide) and one or more pharmaceutically acceptable carriers and / or excipients.

[0104] Another embodiment of the present disclosure is a composition comprising a compound of formula (I) having a cationic site, further comprising an immunomodulator or anticancer agent.

[0105] Formula (II) Another embodiment of the present disclosure is a compound comprising a cationic moiety of formula (I), further comprising a covalently bonded peptide of formula (II): J-(CH2)x(CO)-(A)y-(B)z-peptide moiety The compound yields a conjugated peptide represented by formula (II), wherein the portion corresponding to formula (I) is covalently bonded to the peptide via an amide bond to Dap or Dab, and the variable elements within the portion corresponding to formula (I) are defined as being the same as those in formula (I). The conjugated peptide of formula (II) has the following characteristics: i) higher biological activity than the unmodified peptide at an equivalent bolus dose (mol / kg), ii) a longer in vivo half-life and / or higher blood concentration over time after bolus administration compared to the unmodified peptide while maintaining activity, and / or iii) no toxicity or ataxia in rats at bolus doses of 3.0 μmol / kg or less.

[0106] A compound of formula (II) wherein the compound binds to a natriuretic peptide receptor, and formula (II) does not cause undesirable effects or ataxia in rats at a bolus dose of 3.0 μmol / kg or less.

[0107] In another embodiment of formula (II), the cationic alkyl moiety of formula (I) is covalently bonded to the N-terminus of the peptide or to a side-chain amino group (pendant amino) on the peptide.

[0108] In another embodiment of formula (II), the cationic alkyl moiety of formula (I) is covalently linked to the N-terminus of the peptide.

[0109] In one embodiment of formula (II), the cationic moiety of formula (I) is selected from [SEQ ID NOs: 10-22 and 51-69].

[0110] In one embodiment of formula (II), the peptide portion is a natriuretic peptide or a natriuretic peptide derivative. In this specification, “derivative” as applied to a peptide means a modified natural peptide in which one or more amino acids are substituted, one or more amino acids are added, one or more amino acids are removed, or any combination thereof, while retaining the biological activity of the natural form of the peptide.

[0111] In one embodiment of formula (II), the peptide portion is a natriuretic peptide [SEQ ID NOs: 32, 44, 48, 75] or a natriuretic peptide derivative, wherein one or more methionine residues in the natriuretic peptide are substituted with glutamine (Q), norleucine (Nle), methoxynine (Mox), or lucin (L).

[0112] In one embodiment of formula (II), the peptide portion is a natriuretic peptide [SEQ ID NOs: 32, 44, 48, 75] or a natriuretic peptide derivative, wherein one or more methionine residues in the natriuretic peptide are substituted with glutamine (Q), norleucine (Nle), methoxynine (Mox), or lucin (L), and the cationic alkyl portion of formula (I) is selected from [SEQ ID NOs: 10-22 and 51-69].

[0113] In one embodiment of formula (II), the peptide is a natriuretic peptide derivative in which one or more methionine residues in the natriuretic peptide are substituted with glutamine (Q), norleucine (Nle), methoxynine (Mox), or lucin (L), and the portion of formula (I) is selected from [SEQ ID NOs: 10-22 and 51-69].

[0114] In one embodiment of formula (II), the peptide is a natriuretic peptide derivative in which one or more methionine residues in the natriuretic peptide are substituted with glutamine (Q), norleucine (Nle), methoxynine (Mox), or lucin (L), and the portion of formula (I) is selected from [SEQ ID NOs: 10-22].

[0115] 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.

[0116] 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).

[0117] In one embodiment of formula (II), the peptide is a natriuretic peptide receptor B (NPRB) agonist.

[0118] In one embodiment of formula (II), the peptide is a natriuretic peptide receptor C(NPRC) agonist.

[0119] In one embodiment of formula (II), the peptide is a natriuretic peptide receptor A (NPRA) agonist.

[0120] 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, suppression of cardiac hypertrophy, prolonged elevation of blood cGMP, changes in cAMP, improved survival from sepsis, improved survival from acute lung injury, improved survival from acute respiratory distress syndrome, decreased MPO-positive cells, decreased cell count in alveolar fluid or bronchoalveolar lavage fluid, decreased protein content in alveolar fluid or bronchoalveolar lavage fluid, decreased lung weight per unit body weight, decreased monocyte chemoattractant protein-1 (MCP-1), decreased IL-6, decreased TNF-alpha, decreased A1008 / A9, decreased fibrosis, decreased tumor volume, decreased inflammation, and / or decreased cancer burden.

[0121] In one embodiment of formula (II), the peptide is a natriuretic peptide derivative in which one or more methionine residues in the natriuretic peptide are substituted with glutamine (Q).

[0122] In one embodiment of formula (II), the conjugated peptides are defined by SEQ ID NOs: 29-31, 33-43, 45-47, 49, and 50.

[0123] In one embodiment of formula (II), the conjugated peptides are defined by SEQ ID NOs: 29-31, 33-43.

[0124] In one embodiment of formula (II), the conjugated peptides are defined by SEQ ID NOs: 29-31.

[0125] In one embodiment of formula (II), the conjugated peptide is defined by SEQ ID NO: 29.

[0126] In one embodiment of formula (II), the conjugated peptide is defined by SEQ ID NO: 30.

[0127] In one embodiment of formula (II), the conjugated peptide is defined by SEQ ID NO: 31.

[0128] A compound comprising a cationic moiety of formula (I) and / or a conjugated peptide of formula (II) for use in the manufacture of medical compositions and / or in the treatment of diseases.

[0129] As is evident from the presented examples, the use of the compounds or compositions of this disclosure is effective in resolving alveolar inflammatory edema, improving blood oxygenation, and / or improving survival rates. Furthermore, the compounds or compositions of this disclosure are effective in preventing and resolving pulmonary fibrosis.

[0130] Another embodiment of the present invention is a compound or conjugated peptide of formula (I) or formula (II) comprising a cationic alkyl moiety, for use in the manufacture of a pharmaceutical (or medical composition) (or for use in a method of manufacture).

[0131] Another embodiment of the present invention is a compound of formula (I) comprising a cationic alkyl moiety for use as an excipient in the manufacture of medical compositions.

[0132] For example, a compound of formula II for use in the manufacture of a medical composition, by adding one or more pharmaceutically acceptable carriers or excipients, such as a bulking agent (e.g., sugar), a buffer, a stabilizer (e.g., cyclodextrin), and / or a preservative.

[0133] A compound of formula II for use in treating a disease or condition in a target that requires treatment, wherein the compound of formula II is used for treatment by administering a therapeutically effective bolus dose to a target in the range of 10.0 μmol / kg or less and / or from 10.0 μmol / kg to 0.0001 μmol / kg (including both ends of the range).

[0134] Another embodiment of the present invention is a medical compound comprising a cationic moiety or conjugated peptide of formula (I) and / or formula (II), further comprising one or more pharmaceutically acceptable excipients.

[0135] Another embodiment of the present invention is a compound comprising a conjugated peptide of formula (II) for use in a) the manufacture of a medical composition or b) the treatment of a disease in a subject.

[0136] Another embodiment of the present invention provides compounds or conjugated peptides of formula (I) and / or formula (II) comprising a cationic moiety, for use in the manufacture of pharmaceuticals (or medical compositions) for the treatment of diseases 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 (or for use in a method of manufacture).

[0137] The disclosure also provides compounds or conjugated peptides comprising a cationic moiety of formula (I) and / or formula (II) for use in methods of treating diseases (may be multiple) affecting the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, testes, ovaries, uterus, and / or blood vessels, the method comprising parenterally administering a pharmaceutical composition comprising a compound comprising a cationic moiety or conjugated peptide of formula (I) and / or formula (II) in a therapeutically effective bolus dose of 3.0 μmol / kg or less.

[0138] Another embodiment of the present invention provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of a 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, the method comprising adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II).

[0139] In some embodiments, pharmaceuticals or medical compositions for the treatment of diseases affecting the lungs treat diseases selected from ALI, ARDS, COVID (e.g., COVID-19), inflammation, sepsis, fibrosis, or cancer. In some embodiments, pharmaceuticals or medical compositions for the treatment of diseases affecting the liver treat diseases selected from non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), inflammation, fibrosis, or cancer. In some embodiments, pharmaceuticals or medical compositions for the treatment of diseases affecting the heart treat diseases selected from left ventricular fraction-preserving heart failure (HFpEF), left ventricular fraction-depleted heart failure (HFrEF), acute heart failure, or congestive heart failure. In some embodiments, pharmaceuticals or medical compositions for the treatment of diseases affecting the bones and / or joints treat diseases selected from osteoporosis, osteoarthritis, rheumatoid arthritis, inflammatory cancer, or dwarfism. In some embodiments, pharmaceuticals or medical compositions for the treatment of diseases affecting the kidneys treat 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, pharmaceuticals or medical compositions for the treatment of diseases affecting the prostate treat diseases selected from benign prostatic hyperplasia or prostate cancer.

[0140] Another embodiment of the present disclosure relates to a medical composition comprising a conjugated peptide of formula (II) for use in the treatment of (or in a method of treatment of) 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 treatment comprises administering a compound comprising the conjugated peptide of formula (II) to a target of interest in 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 or a derivative thereof, and the portion corresponding to the cationic alkyl portion of formula (I) is selected from SEQ ID NOs: 10-22 and 51-69.

[0141] In some embodiments, this disclosure provides the use of a conjugated peptide of formula II for the treatment of a disease, condition, or disorder. In some embodiments, the disease, condition, or disorder affects the lungs (e.g., a disease selected from ALI, ARDS, COVID (e.g., COVID-19), inflammation, sepsis, fibrosis, or lung cancer). In some embodiments, the disease, condition, or disorder affects the liver (e.g., a disease selected from non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), inflammation, fibrosis, or cancer). In some embodiments, the disease, condition, or disorder affects the heart (e.g., a disease selected from left ventricular fraction-preserving heart failure (HFpEF), left ventricular fraction-depleted heart failure (HFrEF), acute heart failure, or congestive heart failure). In some embodiments, the disease, condition, or disorder affects the bones and / or joints (e.g., a disease selected from osteoporosis, osteoarthritis, rheumatoid arthritis, inflammatory cancer, or dwarfism). In some embodiments, the disease, condition, or disorder affects the kidneys (e.g., a disease 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, condition, or disorder affects the prostate, such as benign prostatic hyperplasia or prostate cancer.

[0142] Another embodiment of the present disclosure relates to a medical composition comprising a conjugated peptide of formula (II) for use in the treatment of ALI, ARDS, COVID, sepsis, and / or fibrosis (or use in a method of treatment), wherein the treatment comprises administering a compound comprising the conjugated peptide of formula (II) to a subject of need in a therapeutically effective bolus dose of 3.0 μmol / kg or less, wherein the peptide portion of the conjugated peptide is a natriuretic peptide [e.g., SEQ ID NOs: 32, 44, 48, 75] or a derivative thereof, wherein one or more methionine residues in the natriuretic peptide are substituted with glutamine (Q), norleucine (Nle), methoxynine (Mox), or lucin (L), and the cationic alkyl portion of the formula (I) portion is selected from [SEQ ID NOs: 10-22 and 51-69].

[0143] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of ALI, ARDS, COVID, sepsis, and / or pulmonary fibrosis, 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 NOs: 29-31, 33-43, 45-47, and 49-50.

[0144] Another embodiment of the present disclosure relates to a medical composition comprising a conjugated peptide of formula (II) for use in the treatment of ALI, ARDS, COVID, sepsis, and / or fibrosis (or use in a method of treatment), wherein the treatment comprises administering a therapeutically effective bolus dose of the conjugated peptide of formula (II) of 3.0 μmol / kg or less to a subject in need, and the conjugated peptide of formula (II) is selected from SEQ ID NOs. 29-31, 33-43, 45-47, and 49-50.

[0145] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of ALI, ARDS, COVID, sepsis, and / or pulmonary fibrosis, wherein the manufacture or manufacture comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), and the conjugated peptide of formula (II) is selected from SEQ ID NOs: 29-31, 33-43.

[0146] Another embodiment of the present disclosure relates to a medical composition comprising a conjugated peptide of formula (II) for use in the treatment of ALI, ARDS, COVID, sepsis, and / or fibrosis (or use in a method of treatment), wherein the treatment comprises administering the conjugated peptide of formula (II) to a subject in need at a therapeutically effective bolus dose of 3.0 μmol / kg or less, and the conjugated peptide of formula (II) is selected from SEQ ID NOs. 29-31, 33-43.

[0147] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of ALI, ARDS, COVID, sepsis, and / or pulmonary fibrosis, wherein the manufacture or manufacture comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), and the conjugated peptide of formula (II) is selected from SEQ ID NOs: 29-31.

[0148] Another embodiment of the present disclosure relates to a medical composition comprising a conjugated peptide of formula (II) for use in the treatment of ALI, ARDS, COVID, sepsis, and / or fibrosis (or use in a method of treatment), wherein the treatment comprises administering the conjugated peptide of formula (II) to a subject in need at a therapeutically effective bolus dose of 3.0 μmol / kg or less, the conjugated peptide of formula (II) being selected from SEQ ID NOs. 29 to 31.

[0149] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of ALI, ARDS, COVID, sepsis, and / or pulmonary fibrosis, wherein the treatment comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being Sequence ID No. 29.

[0150] Another embodiment of the present disclosure relates to a medical composition comprising a conjugated peptide of formula (II) for use in the treatment of ALI, ARDS, COVID, sepsis, and / or fibrosis (or use in a method of treatment), wherein the treatment comprises administering the conjugated peptide of formula (II) to a subject in need at a therapeutically effective bolus dose of 3.0 μmol / kg or less, the conjugated peptide of formula (II) being Sequence ID No. 29.

[0151] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of ALI, ARDS, COVID, sepsis, and / or pulmonary fibrosis, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being Sequence ID No. 30.

[0152] Another embodiment of the present disclosure relates to a medical composition comprising a conjugated peptide of formula (II) for use in the treatment of ALI, ARDS, COVID, sepsis, and / or fibrosis (or use in a method of treatment), wherein the treatment comprises administering the conjugated peptide of formula (II) to a subject in need at a therapeutically effective bolus dose of 3.0 μmol / kg or less, the conjugated peptide of formula (II) being Sequence ID No. 30.

[0153] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of ALI, ARDS, COVID, sepsis, and / or pulmonary fibrosis, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being Sequence ID No. 31.

[0154] Another embodiment of the present disclosure relates to a medical composition comprising a conjugated peptide of formula (II) for use in the treatment of ALI, ARDS, COVID, sepsis, and / or fibrosis (or use in a method of treatment), wherein the treatment comprises administering the conjugated peptide of formula (II) to a subject in need at a therapeutically effective bolus dose of 3.0 μmol / kg or less, the conjugated peptide of formula (II) being SEQ ID NO: 31.

[0155] Another embodiment of the present disclosure relates to a medical composition for use in the treatment of (or in a method of treatment of) one or a combination thereof of medical conditions selected from hypoxemia, 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 in need at a therapeutically effective bolus dose of 3.0 μmol / kg or less, wherein the peptide portion of the conjugated peptide is a natriuretic peptide (e.g., SEQ ID NOs: 32, 44, 48, 75) or a derivative thereof, wherein one or more methionine residues in the natriuretic peptide are substituted with glutamine (Q), norleucine (Nle), methoxynine (Mox), or lucin (L), and the portion corresponding to the cationic alkyl portion of formula (I) is selected from SEQ ID NOs: 10-22 and 51-69.

[0156] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of one or a combination thereof of medical conditions selected from hypoxemia, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being selected from SEQ ID NOs: 29-31, 33-43, 45-47, 49-50.

[0157] Another embodiment of the present disclosure relates to a medical composition for use in the treatment of (or in a method of treatment of) one or a combination thereof of medical conditions selected from hypoxemia, 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 in a therapeutically effective bolus dose of 3.0 μmol / kg or less, wherein the conjugated peptide of formula (II) is selected from SEQ ID NOs. 29-31, 33-43, 45-47, 49-50.

[0158] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of one or a combination thereof of medical conditions selected from hypoxemia, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being selected from SEQ ID NOs: 29-31 and 33-43.

[0159] Another embodiment of the present disclosure relates to a medical composition for use in the treatment of (or in a method of treatment of) one or a combination thereof of medical conditions selected from hypoxemia, 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 in need at a therapeutically effective bolus dose of 3.0 μmol / kg or less, the conjugated peptide of formula (II) being selected from SEQ ID NOs. 29-31 and 33-43.

[0160] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of one or a combination thereof of medical conditions selected from hypoxemia, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), and the conjugated peptide of formula (II) is selected from SEQ ID NOs: 29-31.

[0161] Another embodiment of the present disclosure relates to a medical composition for use in the treatment of (or in a method of treatment of) one or a combination thereof of medical conditions selected from hypoxemia, 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 in 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 NOs. 29 to 31.

[0162] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of one or a combination thereof of medical conditions selected from hypoxemia, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being Sequence ID No. 29.

[0163] Another embodiment of the present disclosure relates to a medical composition for use in the treatment of (or in a method of treatment of) one or a combination thereof of medical conditions selected from hypoxemia, 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 in need at a therapeutically effective bolus dose of 3.0 μmol / kg or less, wherein the conjugated peptide of formula (II) is Sequence ID No. 29.

[0164] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of one or a combination thereof of medical conditions selected from hypoxemia, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being Sequence ID No. 30.

[0165] Another embodiment of the present disclosure relates to a medical composition for use in the treatment of (or in a method of treatment of) one or a combination thereof of medical conditions selected from hypoxemia, 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 in need at a therapeutically effective bolus dose of 3.0 μmol / kg or less, wherein the conjugated peptide of formula (II) is Sequence ID No. 30.

[0166] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of one or a combination thereof of medical conditions selected from hypoxemia, elevated levels of inflammatory cells in the lungs, pulmonary edema, sepsis, and / or bacteremia, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being Sequence ID No. 31.

[0167] Another embodiment of the present disclosure relates to a medical composition for use in the treatment of (or in a method of treatment of) one or a combination thereof of medical conditions selected from hypoxemia, 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 in need at a therapeutically effective bolus dose of 3.0 μmol / kg or less, wherein the conjugated peptide of formula (II) is Sequence ID No. 31.

[0168] Another embodiment of the present disclosure relates to a medical composition for use in the treatment of (or in a method of treatment of) metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate, testes, ovaries, and / or uterus, wherein the treatment comprises administering the conjugated peptide of formula (II) to a target of interest in a therapeutically effective bolus dose of 3.0 μmol / kg or less, wherein the peptide portion of the conjugated peptide is a natriuretic peptide (e.g., SEQ ID NOs: 32, 44, 48, 75) or a derivative thereof, wherein one or more methionine residues in the natriuretic peptide are substituted with glutamine (Q), norleucine (Nle), methoxynine (Mox), or lucin (L), and the portion of the conjugated peptide corresponding to the cationic alkyl portion of formula (I) is SEQ ID NOs: 10-22 and 51-69.

[0169] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for use in a method of manufacture of a pharmaceutical (or medical composition) for the treatment of metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate, testes, ovaries, and / or uterus, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being selected from SEQ ID NOs: 29-31, 33-43, 45-47, 49-50, and the medical composition.

[0170] Another embodiment of the present disclosure relates to a medical composition comprising a conjugated peptide of formula (II) for use in the treatment (or use in a method of treatment) of metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate, testes, ovaries, and / or uterus, wherein the treatment comprises administering the conjugated peptide of formula (II) to a target of interest in a bolus dose of 3.0 μmol / kg or less, and the conjugated peptide of formula (II) is selected from SEQ ID NOs. 29-31, 33-43, 45-47, 49-50.

[0171] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate, testes, ovaries, and / or uterus, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), and the conjugated peptide of formula (II) is selected from SEQ ID NOs: 29-31, 33-43.

[0172] Another embodiment of the present disclosure relates to a medical composition for use in the treatment (or in a method of treatment) of metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate, testes, ovaries, and / or uterus, wherein the medical composition comprises a prior treatment of administering the conjugated peptide of formula (II) to the subject of interest in a bolus dose of 3.0 μmol / kg or less, wherein the conjugated peptide of formula (II) is selected from SEQ ID NOs. 29-31, 33-43.

[0173] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate, testes, ovaries, and / or uterus, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being SEQ ID NOs. 29-31.

[0174] Another embodiment of the present disclosure relates to a medical composition for use in the treatment of (or in a method of treatment of) metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate, testes, ovaries, and / or uterus, wherein the treatment comprises administering the conjugated peptide of formula (II) to a target of interest in a therapeutically effective bolus dose of 3.0 μmol / kg or less, wherein the conjugated peptide of formula (II) is SEQ ID NOs. 29-31.

[0175] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate, testes, ovaries, and / or uterus, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being Sequence ID No. 29.

[0176] Another embodiment of the present disclosure relates to a medical composition for use in the treatment of (or in a method of treatment of) metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate, testes, ovaries, and / or uterus, wherein the treatment comprises administering the conjugated peptide of formula (II) to a target of interest in a therapeutically effective bolus dose of 3.0 μmol / kg or less, wherein the conjugated peptide of formula (II) is Sequence ID No. 29.

[0177] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate gland, testes, ovaries, and / or uterus, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being Sequence ID No. 30.

[0178] Another embodiment of the present disclosure relates to a medical composition for use in the treatment of (or in a method of treatment of) metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate, testes, ovaries, and / or uterus, wherein the treatment comprises administering the conjugated peptide of formula (II) to a target of interest in a therapeutically effective bolus dose of 3.0 μmol / kg or less, wherein the conjugated peptide of formula (II) is Sequence ID No. 30.

[0179] Another embodiment of the present disclosure provides a conjugated peptide of formula (II) for use in the manufacture of a pharmaceutical (or medical composition) for the treatment of metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate gland, testes, ovaries, and / or uterus, wherein the manufacture or method comprises adding one or more pharmaceutically acceptable excipients to the conjugated peptide of formula (II), the conjugated peptide of formula (II) being Sequence ID No. 31.

[0180] Another embodiment of the present disclosure relates to a medical composition for use in the treatment of (or in a method of treatment of) metastatic cancer located in one or more organs selected from the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, prostate, testes, ovaries, and / or uterus, wherein the treatment comprises administering the conjugated peptide of formula (II) to a target of interest in a therapeutically effective bolus dose of 3.0 μmol / kg or less, wherein the conjugated peptide of formula (II) is Sequence ID No. 31.

[0181] definition The following list of definitions clearly defines the inventions in this disclosure. Terms present in this disclosure but not listed herein have the same meaning as understood by those skilled in the art.

[0182] As used herein, the term "alkyl" refers to a linear (e.g., linear) or branched saturated hydrocarbon group. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. 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, alkyl groups are written using the formulas 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 linking the alkyl group to the rest of the molecule. In various parts of this specification, substituents of the compounds of this disclosure are disclosed as groups or ranges. This disclosure is specifically intended to include each and all individual partial combinations of members of such groups and ranges. sub-(CH2)x-"(x is 10-18), specifically each of the following: 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-C The intention is to disclose, but is not limited to, H2-CH2, CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2, and CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2.

[0183] As used herein, “cationic alkyl,” “cationic alkyl group,” or “cationic alkyl moiety” refers to an alkyl group derived from a fatty acid, covalently bonded to one or more positively charged groups or moieties by a spacer or linker. The spacer or linker is a chain of several amino acids, each of which may be unnatural (meaning not typically found in higher organisms, such as the D type) or natural (meaning typically found in organisms). The linker may be derived from amino acids such as 2-[2-(2-(aminoethoxy)ethoxy]acetic acid (also known as 8-amino-3,6-dioxaoctanoic acid), γ-aminobutyric acid (γAbu), or natural amino acids such as glutamic acid (E) or γ-linked glutamic acid (γE). Here, γ-linked glutamic acid (γE) refers to a glutamic acid residue in which the side-chain carboxyl group (γ position) is used to bind to the nitrogen of the molecule or peptide to be linked, and which is linked via this γ-position carboxyl group rather than the usual α-position carboxyl group. The positively charged group(s) are provided by amino acids having a positively charged side chain containing an amine group. In this specification, the term “fatty acid” refers to a molecule having a carboxylic acid with a long alkyl chain (or aliphatic chain), whether saturated or unsaturated. The carboxylic acid portion is a reactive portion that can form an “acylamide bond” with the linker.

[0184] As used herein, the terms “acylamide” or “fatty acid amide” refer 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, -CONH- is the amide moiety, and “-(CH2)x-CONH-” is an acylamide or fatty acid amide.

[0185] The term "modifying" means that, within the bounds of appropriate medical judgment, it is suitable for use in contact with human and lower animal tissues without causing excessive toxicity (ataxia), irritation, allergic reactions, or other adverse effects, and that the benefit-risk ratio is reasonable.

[0186] As used herein, “allometric scaling” refers to a tool used by pharmaceutical developers to predict human pharmacokinetics based on animal data. Predictive methods like allometric scaling allow for a “peek” at how a drug will behave in humans before clinical trials are conducted. This is important information for pharmaceutical developers and regulatory authorities (such as the FDA) because it provides a data-driven basis for establishing a safe bolus starting dose in humans. For the purposes of this disclosure, the exponent used in allometric scaling is 0.7 (i.e., dose in other species = rat dose ÷ ((rat body weight / average body weight of other species) raised to the power of 0.7)). A dose of 2.0 mg / kg in mouse corresponds to approximately 1.0 mg / kg in rat, approximately 0.33 mg / kg in dog, and 0.17 mg / kg in human. In molar-molar terms, 3.0 μmol / kg in rat is interpreted as 6.0 μmol / kg in mouse, 1.0 μmol / kg in dog, and 0.5 μmol / kg in human. A dose of 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. Conversely, 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 stated doses are derived from rats and should be understood to be applicable to other species after appropriate allometric scaling.

[0187] In this specification, “bolus,” “bolus dose,” or “bolus administration” refers to a single dose of a drug or other substance given or administered over a short period of time, for example, 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, but is not limited to, injection into any site of the body (including, but not limited to, intravascular, subcutaneous, intrathecal, or intradermal), enteral administration (e.g., oral administration as a dosage form), inhalation administration (e.g., intratracheal inhalation where the subject is exposed to a high concentration of aerosol and the active pharmaceutical ingredient is deposited directly into the lower respiratory tract), or nasal administration (e.g., as an aerosol, liquid, or powder). For the purposes of this disclosure, bolus administration is distinguished from intravenous infusion, which typically takes 30 minutes or more to complete.

[0188] As used herein, the terms “acute lung injury” or “ALI,” and the more severe “acute respiratory distress syndrome” or “ARDS,” refer to pulmonary manifestations of an acute systemic inflammatory process clinically characterized by pulmonary infiltration, hypoxemia, and edema, without evidence of left atrial hypertension. (See, for example, Bernard, GR, et al., J. Crit. Care, 1994.9(1):p.72-81; Rubenfeld, GD, et al., N Engl J Med, 2005.353(16):p.1685-93; Brun-Buisson, C., et al., Intensive Care Med, 2004.30(1):p.51-61; and Phua, J., et al., Am J Respir Crit Care Med, 2009.179(3):p.220-7). ALI is an acute onset of severe arterial hypoxemia (decreased blood oxygen concentration due to ventilatory abnormalities), characterized by a PaO2 / FiO2 ratio of 300 Torr or less, while ARDS is an acute onset of severe arterial hypoxemia (decreased blood oxygen concentration due to ventilatory abnormalities), characterized by a PaO2 / FiO2 ratio of 200 Torr or less. Signs and symptoms of ALI and ARDS often begin within two hours of the triggering event, but are also known to take 1 to 3 days. Diagnostic criteria require the onset of known impairment within 7 days of the syndrome. Signs and symptoms include shortness of breath, rapid breathing, muscle fatigue or general weakness, hypotension, dry cough, and fever. ARDS is an overwhelming inflammatory response to certain primary and secondary adverse stimuli, such as pneumonia (aseptic pneumonia, viral pneumonia, bacterial pneumonia, etc.), sepsis, aspiration, inhalation injury, pre-drowning conditions, and lung resection surgery (see, for example, Alam, N., et al, Ann Thorac Surg, 2007.84(4):p.1085-91). ARDS is characterized by rapidly developing respiratory failure requiring hospitalization in the intensive care unit (ICU) and mechanical ventilation support.When patients survive ALI / ARDS, lung scarring often negatively impacts their long-term quality of life (see, for example, Rubenfeld, GD, et al., N Engl J Med, 2005.353(16):p.1685-93, and Dowdy, DW, et al., Intensive Care Med, 2006.32(8):p.1115-24). To date, no effective drugs have been identified to treat acute lung injury (ALI) or ARDS, and there is a great need for such drugs.

[0189] As used herein, “pneumonia” is an infection that causes inflammation of the air sacs in one or both lungs. Fluid or pus (purulent material) may accumulate in the air sacs, causing a cough with sputum and pus, fever, chills, and difficulty breathing.

[0190] As used herein, “COVID” or “coronavirus-induced illness” is a general term for illnesses caused by coronaviruses. Coronaviruses are a family of various types of viruses, some of which cause illnesses and COVID. For example, coronavirus 19 (COVID-19) was identified in 2019 as causing COVID, which affects the lungs and other parts of the body, and is therefore called COVID-19.

[0191] As used herein, the term “cancer” refers to a malignant tissue mass. Malignant tumor cells can “metastasize” (=spread) through the blood and lymphatic systems to various organs in the body, including the lungs, liver, heart, bones / joints, kidneys, prostate, brain, eyes, skin, muscles, blood, digestive tract, bladder, prostate gland, testes, ovaries, and uterus. The affected organs will exhibit dysfunction and / or disease, and such organs can be treated by using the compounds or compositions of this disclosure.

[0192] 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. In peptides, the carboxyl group and the amino group form an amide bond, also called a peptide bond, and two or more amino acids linked by a peptide bond are called a peptide. The term “residue” refers to a part of a peptide derived from an amino acid. Amino acids that have an amino group on the alpha side of the carboxyl group are called α-amino acids. These α-amino acids, as well as other types of amino acids, typically have another substituent at the α position, which is known as a “side chain” or “R group.” The chemical properties of the R group greatly influence the broad chemical properties of the amino acid. In this invention, “cationic amino acid” means having a cation in its side chain, preferably due to a protonated or alkylated amine group. Amino acids may be natural or proteogenic, meaning they exist in nature and are used in the synthesis of polypeptides and proteins. Amino acids useful in this disclosure may be unnatural or not naturally occurring. For the purposes of this disclosure, non-natural amino acids that can be used to construct peptides are organic compounds less than 500 Da having a carboxyl group at one end and a primary amino group at the other. These bifunctional molecules can 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 the compounds of formula (II) in this disclosure, “cationic alkyl moiety” refers to the part of formula (II) corresponding to formula (I).

[0193] In this specification, we use single-letter codes 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 stereoisomers of amino acids, i.e., either L-amino acids or D-amino acids. For the purposes of this disclosure, γE is glutamic acid, and the side-chain carboxyl (gamma, γ) is a moiety used to ligate to the N-terminal portion of a peptide, instead of a typical alpha-carboxyl.

[0194] As used herein, the term “derivative” means a modified peptide resulting from any one or combination of the following: 1) the covalent addition of an amino acid(s), moiety(s), or peptide having a different biological activity from the modified peptide; 2) the cleavage or removal of one or more amino acids in the peptide backbone sequence; and / or 3) the substitution of one or more amino acids in the peptide backbone sequence, wherein the derivative retains the biological activity of the original peptide.

[0195] In this specification, the term "biological activity" refers to the activity of a peptide, which is a measurable and intrinsic characteristic of the peptide. The biological activity of a native peptide is the effect that occurs after it binds to a receptor, before any modifications or structural changes occur. In other words, it is the peptide's intrinsic biological activity that can be measured after exposure to a receptor in vitro or in vivo. If a modified peptide has measurable biological activity similar to that of the native or unmodified peptide, it cannot be considered that the modification has caused the loss of the peptide's biological activity. In other words, it can be considered that the modified peptide retains the characteristic, intrinsic, or endogenous biological activity of the native peptide.

[0196] As used herein, the term “bioavailability” refers to the proportion of a drug or other substance that, once introduced into the body, enters the bloodstream and exerts its active effect. Improvements in bioavailability can be measured using assays that measure the blood level of a drug at corresponding time points after administration. One method of measuring bioavailability is to plot the blood concentration of a drug over time and determine the area under the curve. In this specification, a derivative of a drug that has a much higher blood level than the parent drug (undenatured drug) at corresponding time points after administration is considered to have higher bioavailability.

[0197] As used herein, “apathy” means a clinical sign observed as an inability or unwillingness to resume normal activities characterized by exploration and attentiveness. In this disclosure, apathetic rats are readily observed by exhibiting a posture of near-inactivity and / or unwillingness to explore their surroundings, with eyes half-closed and a hunched back.

[0198] As used herein, “swelling” means a clinical sign observed as the enlargement or congestion of a body part due to blood or fluid, accompanied by visible redness. It is caused by the accumulation of fluid in the tissue. Swelling may occur throughout the body (systemic) or in only a part of the body (focal). In this disclosure, rat swelling induced by cationic alkyl-containing molecules or peptides is readily observed in the feet and nose and is accompanied by redness of the skin.

[0199] As used herein, “practical or reasonable large dose” refers to a therapeutic dose that is practical to administer, after allometric adjustment to a human dose, particularly in terms of the dose of an injectable preparation that must remain liquid for parenteral bolus administration. For non-intravenous parenteral bolus administration, a reasonable injection volume for humans is 2 mL or less. Volumes exceeding 2 mL are possible but are not considered practical for non-intravenous parenteral administration to humans. Due to the limited solubility of the peptide at therapeutically effective doses, injection volumes may exceed 2 mL. Therefore, considering the practical limitations of dose and solubility, the therapeutic dose must be reached within these dose parameters, and anything outside this range is considered unrealistic. Doses can be evaluated across species using allometric scaling.

[0200] As used herein, “subject” includes humans, 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.), as well as non-domesticated animals. In some embodiments, the subject is humans.

[0201] As used herein, the term “therapeutic dose” means a bolus dose of a therapeutic agent (i.e., a drug or therapeutic compound in units of μmol / kg or mg / kg) that elicits a biological or medical response desired by a researcher, veterinarian, physician, or other clinician in a tissue, system, animal, individual, or human, and includes one or more of the following:

[0202] (1) Changing the levels of analytes produced from tissues and / or blood as markers of biological responses that can mitigate disease progression,

[0203] (2) In individuals who have a predisposition to disease, condition, or disability but have not yet experienced or shown the pathology or overall symptoms of that disease, to prevent disease, condition, or disability.

[0204] (3) In an individual experiencing or exhibiting the pathology or overall symptoms of a disease, condition or disorder, inhibiting the disease, condition or disorder, and

[0205] (4) To improve the disease, condition, or disorder (i.e., to restore the pathology and / or overall symptoms) in an individual experiencing or exhibiting the pathology or overall symptoms of a disease, condition, or disorder, such as reducing the severity of the disease, to prolong survival, and / or prevent death.

[0206] As used herein, the term “composition” means a substance formulated, mixed, suspended, miscible, solvated, and / or cocrystallized with excipients, carriers, or solvents, in particular a therapeutic substance. This disclosure relates to compositions comprising formula (I) or formula (II) formulated with one or more pharmaceutically acceptable excipients. The compositions described herein can be used according to the uses and / or methods described herein, for example, to supply a compound of formula (I) or formula (II) for administration to a subject. Examples of compositions include pharmaceutical compositions and medical compositions.

[0207] As used herein, the terms “peptide” and “polypeptide” refer to polymers of amino acids. A “peptide” refers to a polypeptide in which three or more amino acids are covalently linked by an amide bond via an alpha-amino group and an alpha-carboxyl group. The number of amino acid residues in a peptide can range from 3 to about 100. Amino acid residues in a polypeptide or peptide can be canonical or non-canonical, and can be modified or unmodified. As used herein, the term “protein” refers to a polypeptide large enough to have a three-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, SEQ ID NOs. 23–50.

[0208] As used herein, the terms “subcutaneous administration,” “subcutaneously administered,” “sc,” “sc administration,” “SC,” and “SC administration” refer to the direct delivery of a drug, usually in liquid form, to the adipose tissue just beneath the skin. Delivery is typically done by direct injection. These injections are shallower than injections into muscle tissue. For drugs that are better suited to slow, steady absorption into the bloodstream, healthcare providers often use subcutaneous injection.

[0209] As used herein, the terms “intravenous administration,” “IV administration,” and “IV injection” refer to the direct delivery of a drug, typically in liquid form, to an animal or human vein. The method of delivery is usually direct injection. Intravenous administration routes can be either injections performed at high pressure using a syringe or infusions that utilize pressure supplied by gravity, for example.

[0210] As used herein, the terms “intramuscular administration,” “IM ​​administration,” and “IM injection” refer to the direct delivery of a drug, usually in liquid form, to the muscles of an animal or human. The method of delivery is usually direct injection, which allows the drug to be rapidly absorbed into the bloodstream. In some cases, IM injections may be self-administered. In some embodiments, IM injections may be used instead of intravenous injections, for example, when a particular therapeutic agent irritates a vein or when a suitable vein cannot be found.

[0211] As used herein, “nasal administration” means the delivery of a therapeutic agent (e.g., in the form of a gel, liquid, aerosol, gas, or powder) to the nasal cavity of an animal or human, for example, by topical application, liquid dripping, or inhalation, by spraying or misting. Depending on the formulation, this mode of administration may be used to deliver the therapeutic agent to the nasal cavity or lungs (depending on the device used). The therapeutic agent may not be absorbed systemically (purely topical administration), may be completely absorbed systemically (purely systemic administration), or may be partially absorbed both locally and systemically. Nasal sprays may contain drugs that act locally, and their systemic effects are typically minimal.

[0212] As used herein, the terms “inhalation administration” and “administration by inhalation” refer to the delivery of therapeutic drugs through the mouth or nose, often in the form of gas or aerosol (insufflation). In inhalation, therapeutic drugs can reach body tissues rapidly because they can come into contact with the blood supplied to the alveoli (air sacs) of the lungs almost instantaneously. In laboratory animals, inhalation is similar to intratracheal administration (IT) and is distinguished from intranasal administration because it avoids the nasal cavity. As used herein, intratracheal administration (IT) is similar to administration by inhalation.

[0213] In this specification, the terms “parenteral” and “non-gastrointestinal” refer to routes of administration that are not enteral or gastrointestinal. Examples of parenteral administration include subcutaneous, intravenous, intra-arterial, intramuscular, intraperitoneal (injection into the peritoneum), inhalation (e.g., endotracheal or inhalation into the lower respiratory tract), nasal, sublingual and buccal administration, intrathecal, intracerebral, intraventricular, intradermal, or other routes of administration that do not involve the gastrointestinal tract. In this specification, the term “enteral” means administration to any region of the gastrointestinal tract, including the mouth, pharynx, esophagus, stomach, small intestine, large intestine, rectum, anus, and any artificial opening in any of these regions.

[0214] As used herein, the term “excipient” refers to a substance formulated with an active pharmaceutical ingredient for the purpose of providing long-term stabilization, increasing the volume of a formulation containing a potent active ingredient in small amounts (hence often called “volume extender,” “filler,” or “diluent”), and / or conferring therapeutic enhancement to the active pharmaceutical ingredient in the final dosage form, such as improving drug absorption, potency, dose, viscosity, solubility, and / or duration of action or presence of the active pharmaceutical ingredient in the blood. The selection of an appropriate excipient depends on the route of administration, dosage form, active pharmaceutical ingredient, and other factors. Examples of excipients include sugars, amino acids, buffers, antioxidants, chelating agents, solvents, vehicles, and / or composite polymers that bind and stabilize the active pharmaceutical ingredient in vitro and / or in vivo. Excipients were once considered “inactive” components, but are now understood to sometimes be important factors determining the performance of a dosage form. In other words, the influence of excipients on pharmacological action and pharmacokinetics is significant and may require extensive investigation and research. In reality, the effect of excipients on the delivery of active pharmaceutical ingredients is often unpredictable.

[0215] For the purposes of this disclosure, “ataxia” means “toxicity” and is characterized as a clinical sign observed as inadequate muscle control resulting in clumsy voluntary movements and / or death. This is observed as difficulties with motor function, coordination, and eye movements. In toxicity determination (where observation of ataxia is used as a marker of toxicity), the bolus dose was increased until ataxia (i.e., toxicity) was observed, up to 10 μmol / kg (the practical maximum bolus dose of a peptide administered parenterally in a small amount of injection). As used in this disclosure, “ataxia” is also a clinical sign observed as inadequate muscle control and coordination, resulting in clumsy, unmanageable, or awkward voluntary movements. Examples show rats with ataxia exhibiting difficulties with motor function, coordination, and / or eye movements. The dose that causes ataxia and / or death is considered the adverse effect dose. As used herein, the determination of the “maximum tolerated dose” or “MTD” refers to the in vivo safety assessment of the compound under consideration. In this disclosure, the MTD of the test compound is the highest bolus dose at which no undesirable effects or ataxia are observed. Ataxia is reversible and not considered undesirable. In other words, the MTD is the highest bolus dose at which, after administration to an animal group, no visible or observable toxicity occurs compared to a control group. The control group is the vehicle group when testing the MTD of a cationic alkyl moiety or a peptide-cationic alkyl conjugate. In this disclosure, commonly observed effects after administration of a cationic alkyl moiety include, primarily, reversible swelling, changes in skin color, and reversible ataxia.

[0216] 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 is the ratio of the highest bolus dose that does not cause an undesirable effect, such as ataxia or apathy, to the highest bolus dose that does not cause observable peripheral discoloration. The highest bolus dose that does not cause an undesirable effect compared to a control is also called the “no observed adverse effect level (NOAEL).” The highest bolus dose that does not cause observable peripheral discoloration compared to an untreated control is also called the “no observed effect level (NOEL).” Therefore, TI can also be interpreted as NOAEL / NOEL. Peptides with a high therapeutic index exhibit a better safety profile than peptides with a low therapeutic index because they offer a wider safety margin when administered for the treatment of a disease. The NOAEL / NOEL ratio is a quantitative measure of relative safety, comparing the bolus dose that triggers the onset of a therapeutic effect (e.g., vasodilation in the context of the examples herein) with the highest bolus dose prior to the dose that causes toxicity.

[0217] For the purposes of this specification, peripheral discoloration includes redness of the extremities (e.g., the skin of the hands, feet, ears, and / or lips). In rats, peripheral vasodilation is associated with redness, which may or may not be accompanied by swelling, while peripheral vasoconstriction is associated with pallor of the extremities. Redness and pallor are determined by comparing treated subjects with untreated control subjects side by side. The Sprague-Dolly rats used in this example are white rats, making changes in color easily visible.

[0218] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Similar or equivalent methods and materials may be used in the practice or testing of this disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, this specification, including definitions, shall prevail. In addition, materials, methods, and examples are illustrative and not intended to limit the scope. It will be readily apparent that all aspects of this disclosure generally described herein and shown in the figures can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations expressly intended herein.

[0219] 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 each element shown in each figure and / or table. Furthermore, some of the illustrated elements may be combined or omitted. Additionally, exemplary embodiments may include elements not shown in the figures and / or tables. As used herein, “approximately” means ±5% with respect to measurements. As used herein, the stated range includes endpoints, so 0.5 mol percent to 99.5 mol percent includes both 0.5 mol percent and 99.5 mol percent.

[0220] Non-limiting embodiments of this disclosure Embodiment 1. Formula (I): J-(CH2)x(CO)-(A)y-(B)z- (I) A compound comprising a cationic alkyl moiety represented by, J is either HOOC or CH3. x is between 10 and 16. A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aeea), gamma-aminobutyric acid (γAbu), gamma-linked glutamic acid (γE), and alpha-linked glutamic acid (E). y is between 2 and 4, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is between 2 and 4, -(B)z- contains two or fewer Dab residues, and the Dap residues or Dab residues are linked via alpha-amino acids. compound.

[0221] Embodiment 2. 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 gamma-aminobutyric acid (γAbu), y is 3, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is either 2 or 3. The compound described in Embodiment 1.

[0222] Embodiment 3. 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 gamma-linked glutamic acid (γE). y is 3, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), and z is 2 or 3. The compound described in Embodiment 1.

[0223] Embodiment 4. 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 diaminobutanoic acid (Dab), z is 2 or 3. The compound described in Embodiment 1.

[0224] Embodiment 5. J is CH3, x is 10, 12, 14, or 16, A is gamma-aminobutyric acid (γAbu), y is 3, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is 2 or 3. The compound described in Embodiment 1.

[0225] Embodiment 6. 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 gamma-aminobutyric acid (γAbu), y is 3, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is either 2 or 3. The compound described in Embodiment 1.

[0226] Embodiment 7. 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 gamma-linked glutamic acid (γE). y is 3, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is either 2 or 3. The compound described in Embodiment 1.

[0227] Embodiment 8. J is 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 diaminobutanoic acid (Dab), z is either 2 or 3. The compound described in Embodiment 1.

[0228] Embodiment 9. J is HOOC, x is 10, 12, 14, or 16, A is gamma-bound glutamate (γE), y is 3, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is either 2 or 3. The compound described in Embodiment 1.

[0229] Embodiment 10. 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; (B)z is Dap-Dap, Dab-Dab, Dab-Dap, or Dap-Dab. The compound described in Embodiment 1.

[0230] Embodiment 11. 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, (B) z is Dap - Dap, Dab - Dab, Dab - Dap, or Dap - Dab, The compound according to Embodiment 1.

[0231] Embodiment 12. The compound according to Embodiment 1, wherein the cationic alkyl moiety is selected from SEQ ID NOs: 10 - 22 and 51 - 69.

[0232] Embodiment 13. The compound according to Embodiment 1, wherein the cationic alkyl moiety is selected from SEQ ID NOs: 10 - 22.

[0233] Embodiment 14. The compound according to any one of Embodiments 1 - 13, wherein when the compound is conjugated to a peptide, it does not have clinically observable ataxia after parenteral bolus administration at a dose of 10 μmol / kg or less in rats.

[0234] Embodiment 15. The compound according to any one of Embodiments 1 - 14, for use in covalent peptide modification.

[0235] Embodiment 16. Formula (II): CH3(CH2)x(CO)-(A)y-(B)z - peptide moiety (II) which is a conjugated peptide represented by where x is 10 - 16, A is independently selected from the group consisting of 2 - [2 - (2 - aminoethoxy)ethoxy]acetic acid (Aeea), gamma - aminobutyric acid (γAbu), gamma - linked glutamic acid (γE), alpha - linked glutamic acid (E), y is 2 - 4, B is independently diaminopropionic acid (Dap) or diaminobutyric acid (Dab), z is 2 - 4, -(B)z- contains two or fewer Dab residues, and the Dap or Dab residues are linked via alpha-amino acids. CH3(CH2)x(CO)-(A)y-(B)z- is covalently bonded to the N-terminus of the peptide portion, or is linked to one of the side-chain amino groups of the peptide portion. Selectively, the conjugated peptide has equivalent or greater biological activity than the unmodified peptide at an equivalent bolus dose, and / or The conjugated peptide has a blood level equivalent to or higher than that of the unconjugated peptide at the same time point after bolus administration at an equivalent dose. Conjugated peptide.

[0236] Embodiment 17. The conjugated peptide according to Embodiment 16, wherein the conjugated peptide binds to a natriuretic peptide receptor and does not cause undesirable effects or ataxia in rats at a bolus dose of 3.0 μmol / kg or less.

[0237] Embodiment 18. The conjugated peptide according to Embodiment 16 or 17, wherein the CH3(CH2)x(CO)-(A)y-(B)z- portion is covalently bonded to the N-terminus of the peptide portion.

[0238] Embodiment 19. The conjugated peptide according to any one of Embodiments 16 to 18, wherein the peptide portion is the natriuretic peptide of SEQ ID NO: 32, 44, 48, or 75, or a natriuretic peptide derivative.

[0239] Embodiment 20. The conjugated peptide according to any one of Embodiments 16 to 19, wherein the CH3(CH2)x(CO)-(A)y-(B)z- portion is selected from SEQ ID NOs. 10 to 22 and 51 to 69.

[0240] Embodiment 21. The conjugated peptide according to Embodiment 20, wherein the CH3(CH2)x(CO)-(A)y-(B)z- portion is selected from SEQ ID NOs. 10 to 22.

[0241] Embodiment 22. The conjugated peptide according to any one of Embodiments 16 to 20, wherein the peptide portion is a natriuretic peptide derivative in which one or more methionine residues are substituted with glutamine (Q), leucine (L), norleucine (Nle), or methoxynine (Mox).

[0242] Embodiment 23. The conjugated peptide according to any one of Embodiments 16 to 22, wherein the peptide portion is a natriuretic peptide according to SEQ ID NO: 32, or a derivative thereof in which one or more methionine residues are substituted with glutamine (Q), and the CH3(CH2)x(CO)-(A)y-(B)z- portion is selected from SEQ ID NOs: 10 to 22.

[0243] Embodiment 24. The conjugated peptide according to any one of Embodiments 16 to 23, wherein the conjugated peptide is selected from SEQ ID NOs. 29-31, 33-43, 45-47, and 49-51.

[0244] Embodiment 25. The conjugated peptide according to Embodiment 24, wherein the conjugated peptide is selected from SEQ ID NOs: 29-31 and 33-43.

[0245] Embodiment 26. The conjugated peptide according to Embodiment 25, wherein the conjugated peptide is selected from SEQ ID NOs: 29-31.

[0246] Embodiment 27. The conjugated peptide according to Embodiment 26, wherein the conjugated peptide is Sequence ID No. 29.

[0247] Embodiment 28. The conjugated peptide according to Embodiment 26, wherein the conjugated peptide is Sequence ID No. 30.

[0248] Embodiment 29. The conjugated peptide according to Embodiment 26, wherein the conjugated peptide of formula (II) is Sequence ID No. 31.

[0249] Embodiment 30. The conjugated peptide according to any one of Embodiments 16 to 29, wherein the conjugated peptide binds to natriuretic peptide receptor B (NPRB), natriuretic peptide receptor C (NPRC), or a combination thereof.

[0250] Embodiment 31. The conjugated peptide according to any one of Embodiments 16 to 30, wherein the conjugated peptide is an NPRB agonist.

[0251] Embodiment 32. The conjugated peptide according to any one of Embodiments 16 to 31, wherein the conjugated peptide is an NPRC agonist.

[0252] Embodiment 33. The conjugated peptide according to any one of Embodiments 16 to 32, wherein the conjugated peptide produces a physiological effect selected from an extension of an increase in blood cGMP, a change in cAMP, a change in blood pressure, an improvement in survival rate from sepsis, an improvement in survival rate from acute lung injury, an improvement in survival rate from acute respiratory distress syndrome, a decrease in MPO-positive cells, a decrease in the number of cells in alveolar fluid or bronchoalveolar lavage fluid, a decrease in the amount of protein in alveolar fluid or bronchoalveolar lavage fluid, a decrease in endothelial permeability, a decrease in lung weight per body weight, a decrease in monocyte chemoattractant protein-1 (MCP-1), a decrease in IL-6, a decrease in TNF-alpha, a decrease in A1008 / A9, a decrease in fibrosis, a decrease in tumor volume, a decrease in metastasis, a decrease in inflammation, an antiproliferative effect, a decrease in cancer burden, an inhibition of the expression of cyclooxygenase 2 (COX-2), an antagonism of the renin-angiotensin-aldosterone system, an inhibition of cardiac hypertrophy, or a combination thereof.

[0253] Embodiment 34. The compound according to any one of Embodiments 1 to 15, or the conjugated peptide according to any one of Embodiments 16 to 33, for use in the manufacture of a pharmaceutical composition.

[0254] Embodiment 35. The compound or conjugated peptide according to Embodiment 34, wherein the pharmaceutical composition contains one or more pharmaceutically acceptable carriers or excipients.

[0255] Embodiment 36. The compound or conjugated peptide for use according to Embodiment 35, wherein the one or more pharmaceutically acceptable carriers or excipients include a bulking agent, a buffer, a stabilizer, a preservative, or a combination thereof.

[0256] Embodiment 37. A compound according to any one of Embodiments 1 to 15, or a conjugated peptide according to any one of Embodiments 16 to 33, for use in treating a disease or condition in a subject that requires treatment.

[0257] Embodiment 38. The compound or conjugated peptide is a) One of the following sequence numbers: 29-31, 33-43, 45-47, or 49-51, or b) One of the following sequence numbers: 29-31, 33-43, or 45-47, or c) One of sequence numbers 29-31 or 33-43, or d) Any one of sequence numbers 29-31, or e) Sequence ID 29, or f) Sequence ID 30, or g) Sequence ID 31. Compounds or conjugated peptides for use in Embodiment 37.

[0258] Embodiment 39. A compound for use in either Embodiment 37 or 38, wherein the compound or conjugated peptide comprises SEQ ID NO: 31.

[0259] Embodiment 40. The disease or condition is one of the following: lung (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), bone / joint (e.g., osteoporosis, degenerative) Compounds or conjugated peptides for use according to any one of Embodiments 37 to 39, affecting the kidneys (e.g., chronic kidney disease (CKD), acute kidney injury (AKI), drug-induced kidney injury, inflammation / nephritis, renal fibrosis, glomerulosclerosis, or renal cancer), the prostate (e.g., benign prostatic hyperplasia, or prostate cancer), the brain, eyes, skin, muscles, blood, gastrointestinal tract, bladder, testes, ovaries, uterus, and / or blood vessels.

[0260] Embodiment 41. The compound or conjugated peptide for use according to any one of Embodiments 37 to 39, wherein the disease or condition is cancer before or after metastasis.

[0261] Embodiment 42. The compound or conjugated peptide for use according to Embodiment 41, wherein the cancer is a cancer of one or more organs selected from the lungs, lung pleura, liver, heart, bones / joints, kidneys, prostate, breast, brain, eye, skin, muscle, blood, blood vessels, gastrointestinal tract, bladder, testes, ovaries, and / or uterus.

[0262] Embodiment 43. A compound or conjugated peptide for use according to any one of Embodiments 37 to 39, wherein the disease or condition is pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), or COVID in the target population.

[0263] Embodiment 44. A compound or conjugated peptide for use according to any one of Embodiments 37 to 39, wherein the disease or pathological condition is fibrosis.

[0264] Embodiment 45. The compound or conjugated peptide for use according to any one of Embodiments 37 to 44, wherein the treatment is administered to a therapeutically effective bolus dose of 10.0 μmol / kg or less and / or in the range of 10.0 μmol / kg to 0.0001 μmol / kg (including both ends).

[0265] Embodiment 46. The compound or conjugated peptide for use according to any one of Embodiments 37 to 45, wherein the compound is administered as a monotherapy or in combination with one or more additional agents or treatments.

[0266] Embodiment 47. The compound or conjugated peptide for use according to Embodiment 46, wherein the one or more additional agents or treatments are selected from immune checkpoint inhibitors, surgery / amputation, radiation, chemotherapy, or a combination thereof.

[0267] Embodiment 48. The compound for use according to any one of Embodiments 37 to 46, wherein the compound is administered subcutaneously, by injection, inhalation, nasal spray, orally, by eye drops, and / or topically.

[0268] Embodiment 49. The compound or conjugated peptide for use according to any one of Embodiments 37 to 42, wherein the compound or conjugated peptide is administered to the subject by subcutaneous, injection, inhalation, nasal spray, oral, ophthalmic, and / or topical application.

[0269] Embodiment 50. A composition comprising a compound described in any one of Embodiments 1 to 15 or a conjugated peptide described in any one of Embodiments 16 to 33, and one or more pharmaceutically acceptable carriers or excipients.

[0270] Embodiment 51. The composition according to Embodiment 49, wherein the one or more pharmaceutically acceptable carriers or excipients include a bulking agent, a buffer, a stabilizer, a preservative, or a combination thereof. [Examples]

[0271] Example 1: Clinically observable adverse effects of various cationic alkyl moieties tested at 3.0, 5.0, or 10 μmol / kg indicate that cationic alkyl moieties containing Dap and / or Dab as cationic residues are far less toxic than those containing cationic amino acids with longer R groups.

[0272] The following experimental protocol was followed to generate the data in Table 1. Animal care and treatment: Male Sprague Dawley rats (8-10 weeks old, Charles River, Hollister, CA) were housed in pairs in disposable polypropylene cages lined with rodent cob bedding at the PharmaIN animal facility (n=3 per group). The animals were given free intake of feed (LabDiet Pico Rodent #5053 [Animal Specialties, Woodburn, OR]) and water. Temperature (68-74°F) and humidity (30-60%) were maintained within controlled ranges, and a 12-hour light-dark cycle was used. Drug administration and observation: Each rat received subcutaneous injection of a cationic alkyl modifier (SEQ ID NOs. 1-22, listed in Table 1 below) in sterile water buffer adjusted to pH 4.5-5, with an initial bolus dose of 5.0 μmol / kg and an injection volume of 1.0 mL / kg. Bolus doses were adjusted based on observation, and animals were carefully monitored for lethargy, ataxia, swelling, and discoloration during the first four hours after injection, and then every hour thereafter. As used herein, “ataxia” means a clinical sign observed as inadequate muscular control resulting in clumsy voluntary movements. Ataxia can result in difficulties with motor function, coordination, and eye movements. As used herein, “lethargy” means a clinical sign observed as an inability or unwillingness to resume normal activities characterized by exploration and attentiveness. Lethargic rats are easily observed by exhibiting little activity and / or unwillingness to explore their surroundings, half-closed eyes, and a hunched posture. As used herein, “swelling” means a clinical sign observed as enlargement or congestion of a body part due to blood or fluid, accompanied by visible redness (discoloration). If swelling was absent, the dose level was increased to 10 μmol / kg; if swelling was present, the bolus dose level was decreased to 3.0 μmol / kg. A one-week washout period was observed between bolus administrations, and the animals were humanely euthanized by carbon dioxide asphyxiation after final observation. [Table 1-1] [Table 1-2]

[0273] Example 2: Alkylated natriuretic peptides containing alkylated Dap / Dab showed fewer clinically observable adverse effects and ataxia in rats compared to those containing cationic amino acids with long R groups, such as lysine and arginine, or their unnatural D isomers. Furthermore, alkylated peptides exhibited improved pharmacokinetic and / or pharmacological properties compared to unmodified peptides (see Example 3, Table 3).

[0274] The following experimental protocol was followed to generate the data in Table 2. Animal care and treatment: Male Sprague Dawley rats (8-10 weeks old, Charles River, Hollister, CA) were housed in pairs in disposable polypropylene cages lined with rodent cob bedding at the PharmaIN animal facility (n=3 per group). The animals were given free intake of feed (LabDiet Pico Rodent #5053 [Animal Specialties, Woodburn, OR]) and water. Temperature (68-74°F) and humidity (30-60%) were maintained within controlled ranges, and a 12-hour light-dark cycle was used. Drug Administration and Observation: In these dose-ranging studies, rats were administered a subcutaneous bolus of 1.0 mL / kg of cationic alkyl-modified CNP (SEQ ID NOs. 23-31 [listed in Table 2 below, PharmaIN, Bothell, WA]) in lead buffer up to a maximum of 10 mg / kg. Animals were carefully monitored for 4 hours after injection, and thereafter every hour throughout the day for signs of swelling or discoloration (side effect due to diastolic effect), lethargy (side effect), and ataxia (ataxia is used as an indicator of toxicity). Rats were given a 1-week washout period between bolus administrations. After the final observation, animals were humanely euthanized by carbon dioxide asphyxiation. Cationic amino acids with longer R groups containing cationic alkyl groups (both natural and unnatural forms), 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]), resulted in conjugates with greater toxicity compared to CNPs modified with the cationic alkyl moiety of formula (I) [e.g., SEQ ID NOs. 29-31 (see Table 2)]. [Table 2]

[0275] Example 3: Pharmacokinetic and pharmacological data of several natriuretic peptides (NPs) modified with alkylated cationic Dap or Dab residues. These modifications exhibit improved pharmacological effects, as evidenced by the higher 2-hour and 6-hour plasma cGMP concentrations. When administered subcutaneously to mice at a bolus dose of 1.0 mg / kg (≤0.45 μmol / kg), pharmacokinetics were also improved compared to the corresponding natural NPs, as evidenced by the significant presence in the bloodstream (NP levels). The following experimental protocol was followed to generate the data in Table 3. Animal care and treatment: CD-1 male mice (6-9 weeks old, Charles River, Hollister, CA) were placed in groups of 5-6 in disposable polypropylene cages lined with rodent Cob bedding and given LabDiet Pico Rodent #5053 feed and water as needed. The animals were kept at a controlled temperature (68-74°C). oThe animals were housed in PharmaIN's animal facility with F) and humidity (30-60%) and a 12-hour light-dark cycle. Drug administration and blood collection: 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 (listed in Table 3, PharmaIN, Bothell, WA). A bolus dose of 1.0 mg / kg (≤0.45 μmol / kg) was administered subcutaneously between the scapulae. The test substance was mixed or dissolved in sterile water for injection on the day of administration. No adverse effects were observed in any animal at this bolus dose. Blood samples were collected by post-orbital puncture 2 and 6 hours post-injection. Two blood samples were collected from each animal at two different time points. Samples were collected in K2EDTA tubes and centrifuged (2000 × g; 15 minutes, 4°C) within 30 minutes of collection. The resulting plasma samples were stored at -80°C. Biochemical analysis: Plasma concentrations of each natriuretic peptide were analyzed using commercially available ELISA kits from Phoenix Pharmaceuticals (Burlingame, CA) (ANP ELISA (cat# EKE-005-06), BNP ELISA (cat# EKE-011-03), CNP ELISA (cat# EKE-012-03)). The ANP and BNP kits detect only their respective derivatives, while the CNP kit detects the cyclic structure of CNP and all CNP derivatives with the same level of reactivity. Plasma cGMP was analyzed using a commercially available kit from Abcam (ab133052, Waltham, MA). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0276] Example 4: Subcutaneous administration of cationic alkyl-modified CNP resulted in prolonged plasma residence time and enhanced physiological activity compared to natural CNP.

[0277] The following experimental protocol was followed to generate the data shown in Figure 1. Animal care and treatment: CD-1 male mice (6-9 weeks old, Charles River, Hollister, CA) were placed in groups of 5-6 in disposable polypropylene cages lined with rodent Cob bedding and given LabDiet Pico Rodent #5053 feed and water as needed. The animals were kept at a controlled temperature (68-74°C). o The mice were housed in a PharmaIN animal facility with F) and humidity (30-60%) and a 12-hour light-dark cycle. Drug-administered CD-1 male mice were given either natural human CNP (SEQ ID NO: 32, sequence: GLSKGCFGLKLDRIGSMSGLGC, Chempep Inc., Wellington, FL) or a cationic alkyl-modified CNP derivative synthesized with PharmaIN (SEQ ID NO: 29, sequence: CH3(CH2) 14 (C=O)-Aeea-Aeea-Aeea-Dab-Dab-GLSKGCFGLKLDRIGSQSGLGC, or sequence number 31, sequence CH3(CH2). 14(C=O)-Aeea-Aeea-Aeea-Dap-Dap-GLSKGCFGLKLDRIGSQSGLGC) was administered as a single subcutaneous injection at a dose of 1.0 mg / kg. Lead buffer was used to combine or dissolve all test substances on the day of administration. Blood samples were collected at several time points (0, 0.5, 1, 2, 4, 6, 8, and 24 hours) by posterior orbital puncture and processed in K2 EDTA tubes (2000 × g; 15 minutes, 4°C, within 30 minutes of 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. The CNP ELISA kit detects the cyclic structure of CNP and all CNP derivatives that show comparable reactivity to the ELISA kit. CNP derivatives are modified human CNP molecules in which the methionine in the cyclic portion of natural CNP (i.e., GLSKGCFGLKLDRIGSMSGLGC) is replaced with glutamine (Q) (i.e., GLSKGCFGLKLDRIGSQSGLGC), and the N-terminus is CH3(CH2) relative to SEQ ID NO: 29. 14 (C=O)-Aeea-Aeea-Aeea-Dab-Dab[Sequence No. 10] is CH3(CH2) for Sequence No. 31. 14 (C=O)-Aeea-Aeea-Aeea-Dap-Dap[SEQ ID NO: 14] is an extended form of cGMP. For testing the pharmacological or biological activity of cGMP, plasma aliquots were thawed at 4°C and analyzed using a commercially available cGMP kit from Abcam (ab133052, Waltham, MA).

[0278] Example 5: Single and repeated administration of cationic alkyl-modified C-type natriuretic peptide enhances survival in LPS-induced sepsis and ALI animal models.

[0279] Both SEQ ID NOs: 31 and 29 are effective in improving survival rates from ALI in mice, whether administered subcutaneously (SC) or intratracheally (IT). The following experimental protocol was followed to generate the data in Figure 2. Animal care and treatment: C57BL / 6J male mice (6 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Drug administration in Figure 2A): Mice were intraperitoneally (IP) injected with LPS (15 mg / kg, Sigma-Aldrich) and further treated with various test substances, including cationic alkyl-modified CNP (SEQ ID NOs: 31 and 29, 0.3 mg / kg (0.1 μmol / kg) SC). The control group received LPS treatment without test substances. Test substances were administered immediately after LPS administration. Survival rates were monitored every two hours from 8 to 56 hours, after which the mice were euthanized under isoflurane anesthesia. Statistical analysis was based on the Gehan-Breslow-Wilcoxon test performed using GraphPad Prism (n=10, 10, and 10; control, SEQ ID NO: 31, and SEQ ID NO: 29). **P<0.01, *P<0.05 (compared to the control group). Drug administration in Figure 2B): Mice were administered intratracheally (IT) with LPS (20 mg / kg; Sigma-Aldrich) and further treated with various test substances including cationic alkyl-modified CNP (SEQ ID NO: 31 and 29, 0.3 mg / kg (0.1 μmol / kg) IT). The control group received LPS treatment without the test substances. The test substances were administered repeatedly every 24 hours starting immediately after LPS administration, for a total of three bolus doses. Survival rates were monitored every 8 hours until 72 hours, after which the mice were euthanized under isoflurane anesthesia. Statistical analysis was performed using the Gehan-Breslow-Wilcoxon test with 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).

[0280] Example 6: Bolus administration of cationic alkyl-modified C-type natriuretic peptide suppresses lung injury and resolves ALI / ARDS.

[0281] The following experimental protocol was followed to generate the data shown in Figure 3. Animal care and treatment: C57BL / 6J male mice (6 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). After administering LPS (0.05 mg / kg IT, Sigma-Aldrich) to drug-treated mice, they were treated with various test substances, including intraperitoneal (IP) injection of the human neutrophil elastase inhibitor sivelestat (150 mg / kg; Nipro, Osaka, Japan) and cationic alkyl-modified CNP (SEQ ID NOs. 31, 29, and 30; 0.3 mg / kg (0.1 μmol / kg) SC), as positive controls. The test substances were administered immediately after LPS injection. In addition, a normal control (NC) group that did not receive LPS and a control group that received only LPS without any test substances were also included. After 24 hours, the mice were euthanized under isoflurane anesthesia, and their lungs were collected for analysis. Biochemical analysis as shown in Figure 3B): The lungs were finely chopped with Tri-Reagent (Cosmo Bio, Tokyo, Japan), and 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 removed. Then, 75% EtOH was added, and the sample was centrifuged (12,000 × g, 4°C, 5 minutes). The supernatant was removed, and the pellet was dissolved in nuclease-free water (Ambion, MA, USA). The gene expression levels of S100A8 and S100A9 were measured by qRT-PCR analysis using a cDNA synthesis kit (Qiagen, Venlo, the Netherlands). In several types of inflammatory lung diseases, the expression of bone marrow-derived proteins (S100A8 / A9) is elevated. Statistical analysis was performed using Dunnett's test with 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 (for each corresponding control group).Biochemical analysis related to Figures 3C,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 detection using peroxidase-labeled anti-rabbit IgG goat polyclonal antibody (Nichirei Biosciences Co., Ltd., Tokyo, Japan), and then 3,3'-diaminobenzidine-4HCl (DAB) (Agilent Technologies, Santa Clara, CA). The number of myeloperoxidase-positive (MPO+) cells was quantified in each field of view. An increase in neutrophil count is often seen in ALI and ARDS. MPO. + Cells serve as an indicator that directly measures the presence of neutrophils. 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 (for each corresponding control group).

[0282] Example 7: Cationic alkyl-modified CNP derivatives reduced neutrophil infiltration in the lungs and showed resolution of ALI / ARDS.

[0283] The following experimental protocol was followed to generate the data in Figure 4. Animal care and treatment: C57BL / 6J male mice (6 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Drug administration: Mice were administered LPS (0.05 mg / kg IT, Sigma-Aldrich) and then treated with various test substances, including IP-injected human neutrophil elastase inhibitor sivelestat (150 mg / kg; Nipro, Osaka, Japan) and cationic alkyl-modified CNP (SEQ ID NOs. 31, 29, and 30; 0.3 mg / kg (0.1 μmol / kg) SC), as positive controls. Test substances were administered immediately after LPS injection. In addition, a normal control (NC) group that did not receive LPS and a control group that received only LPS without any test substances were also included. After 24 hours, mice were euthanized under isoflurane anesthesia, and bronchoalveolar lavage fluid (BALF) was collected. Biochemical analysis: The total number of cells in BALF was counted in a counting chamber. The total protein concentration in BALF was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). 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 (for each corresponding control group). ALI and ARDS are associated with an increase in cells, particularly neutrophils, in bronchoalveolar lavage fluid (BALF). To assess the resolution of ALI / ARDS in animal models, it is common to measure the number of cells (Figure 4B) and total protein (Figure 4C), which serve as neutrophil markers. A decrease in these markers indicates the resolution of ALI / ARDS.

[0284] Example 8: Table showing that treatment with cationic alkyl-modified CNP derivatives suppresses LPS-induced upregulation of inflammatory cytokines in BALF.

[0285] To generate the data in Table 4, the following experimental protocol was followed. Animal care and treatment: C57BL / 6J male mice (6 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Drug administration: Mice were administered LPS (0.05 mg / kg IT, Sigma-Aldrich) and then treated with various test substances, including IP-injected human neutrophil elastase inhibitor sivelestat (150 mg / kg; Nipro, Osaka, Japan) and cationic alkyl-modified CNP (SEQ ID NOs. 31, 29, and 30; 0.3 mg / kg (0.1 μmol / kg) SC), as positive controls. Test substances were administered immediately after LPS injection. In addition, a normal control (NC) group that did not receive LPS and a control group that received only LPS without any test substances were also included. After 24 hours, the mice were euthanized under isoflurane anesthesia, and BALF (baby fat cells) were collected. Biochemical analysis: ALI and ARDS are often characterized by elevated levels of inflammatory cytokines in BALF, including macrophage chemoattractant protein-1 (MCP1), interleukin-6 (IL-6), and tissue necrosis factor α (TNFα). A decrease in these concentrations indicates resolution 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, e.g., PLoS One, 2014 Jul22;9(7):e102967), and have shown upregulation of TNFα and IL-6 in non-surviving groups (see, e.g., Chest, 1997:111:1306-21). On the other hand, MCP-1 levels were elevated in the group that developed ALI / ARDS (see, for example, International Journal of Molecular Sciences, 2019:20(9):2218). Statistical analysis was performed using Dunnett's test with 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 (for each corresponding control group). [Table 4]

[0286] Example 9: Effect of cationic alkyl-modified CNP derivatives on the inflammatory state during acute exacerbation (IPF-AE) of idiopathic pulmonary fibrosis in the lungs. Shows resolution of ALI / ARDS.

[0287] The following experimental protocol was followed to generate the data in Figure 5. Animal care and treatment: C57BL / 6J male mice (6 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Drug administration: Mice were administered bleomycin (Bleo, 1.0 mg / kg, Nippon Kayaku, Tokyo, Japan) via intra-artificial administration, and LPS (0.025 mg / kg, Sigma Aldrich, St. Louis, MO, USA) via intra-artificial administration three weeks later. As shown in Figure 5A, either SEQ ID NO: 29 or SEQ ID NO: 31 was administered subcutaneously at 0.3 mg / kg (0.1 μmol / kg). In addition, a normal control (NC) group without LPS / Bleo treatment, a Bleo group without LPS treatment, and a control group without test substance treatment were also included. On the final day, mice were euthanized under isoflurane anesthesia, and lung tissue was collected and weighed (Figure 5B). An increase in lung weight / body weight ratio is a commonly measured parameter indicating lung damage. Biochemical analysis in Figure 5C): Lung tissue was collected and fixed with 4% paraformaldehyde. Paraffin sections of the fixed lung tissue were immunohistochemically stained with anti-MPO rabbit polyclonal antibody (Agilent Technologies Santa Clara, CA), horseradish peroxidase (HRP)-labeled anti-rabbit IgG goat polyclonal antibody (Nichirei Biosciences Co., Ltd., Tokyo, Japan), and 3,3'-diaminobenzidine-4HCl (DAB) (Agilent Technologies, Santa Clara, CA). MPO expression was evaluated using Image J (NIH, Bethesda, MD, USA). Biochemical analysis in Figure 5D): Lung tissue was finely chopped in lysis buffer and diluted with PBS (Fujifilm, Tokyo, Japan). Macrophage chemoattractant protein-1 (MCP1) was measured using an ELISA kit (R&D SYSTEMS, Minneapolis MN). Biochemical analysis (Figure 5E): Lung tissue was finely chopped with Tri-Reagent (Cosmo Bio, Tokyo, Japan), and 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 incubating at room temperature for 10 minutes, the sample was centrifuged (12,000×g, 4°C, 15 minutes), and the supernatant was removed. Then, 75% EtOH was added and the sample was centrifuged (12,000×g, 4°C, 5 minutes). The supernatant was removed, and the pellet was dissolved in nuclease-free water (Ambion, MA, USA). The gene expression level of IL-6 was measured by qRT-PCR analysis using a cDNA synthesis kit (Qiagen, Venlo, the Netherlands). In previous reports, an increase in the number of neutrophils (see, for example, Kona M., et al., Respir. Med., 2021, vol. 186), upregulation of MCP1 (see, for example, Arai T., et al., BMJ Open Respir. Res., 2021, vol. 8, 1), and upregulation of IL-6 in the non-surviving group (see, for example, Lee J., et al., PloS one, 2021, vol. 16, 7) have been shown. Statistical analysis was based on Student's t-test 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.

[0288] Example 10: Repeated subcutaneous administration of a cationic alkyl-modified CNP derivative exhibited significant antitumor activity in a breast cancer orthotopic transplantation mouse model using E0771 cells.

[0289] The group treated with cationic alkyl-modified CNP (SEQ ID NOs: 29, 30, and 31) showed a significant reduction in tumor volume compared to the control group at the end of the study. Cationic alkyl sequences without CNP (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 CNP attachment is essential for antitumor activity. The following experimental protocol was followed to generate the data in Figure 6. Animal care and treatment: C57BL / 6J female mice (6 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan, or PicoLab Rodent Diet 20, LabDiet Corp., [St. Louis, Missouri]). Transplantation and Drug Administration: E0771 mammary cancer cells (250,000 cells / mouse, Cosmo Bio Tokyo, Japan) were transplanted into the left mammary gland of mice, and they were randomly assigned to one of n=10 groups. From day 4 post-inoculation, cationic alkyl-modified CNP derivatives (SEQ ID NOs. 29, 30, 31) were administered subcutaneously once daily for 5 days (5 days administration, 2 days rest) in a bolus dose of 0.3 mg / kg (0.1 μmol / kg, administration volume 10 mL / kg) in sterile water for injection (Otsuka Pharmaceutical, Tokushima, Japan) for 3 cycles. The control group (received buffer only) was administered in the same manner as the other groups to establish baseline tumor growth dynamics. Tumor size was measured using calipers. Statistical analysis was based on Dunnett's test performed using GraphPad (n=10, 10, 10, and 10; control, SEQ ID NOs. 29, SEQ ID NOs. 30, and SEQ ID NOs. 31). ****P<0.0001 (compared to the control group).

[0290] Example 11: Fluorescence polarization (FP) assay of natriuretic peptide receptor B (NPRB) and natriuretic peptide receptor C (NPRC)

[0291] C-type natriuretic peptide (CNP) alone (5nM CNP-F*) conjugated to a 5(6)-carboxyfluorescein (F*)-based probe exhibits rapid rotation and a low fluorescence polarization (FP) signal. As shown in Figure 7, the addition of human NPRB or NPRC (50nM) causes the CNP probe to bind to these receptors, resulting in slower rotation and a higher FP signal. No change in FP signal was detected in the presence of NPRA, suggesting that the CNP probe does not bind to NPRA. The low FP signal in the presence of [SEQ ID NO: 31] indicates that the CNP probe binds to both NPRB and NPRC.

[0292] Fluorescence polarization (FP) assay The CNP-F* probe was incubated at a final concentration of 5 nM in assay buffer containing PBS, pH 7.4, and 0.01% Triton® X-100, in the presence of 50 nM human NPRB or NPRC. Prior to FP measurement, 100 μL volumes of CNP-F* probe and NPR were dispensed into a black 96-well Costar flat-bottom polystyrene plate. Next, 1 μL volume of SEQ ID NO: 31 (final concentration 150 nM) was added to the pre-mixed probe and NPR. The plate was incubated at room temperature for 10 minutes. Subsequently, fluorescence polarization was measured using Flexstation 3 (excitation wavelength: 480 nm, emission wavelength: 525 nm, cutoff: 515 nm).

[0293] Synthesis of CNP-F* probes. CNP-F* was prepared using solid-phase peptide synthesis. Briefly, a linear peptide (sequence: F*-GLSKGCFGLKLDRIGSMSGLGC, where F* is 5(6)-carboxyfluorescein) was synthesized using 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 precipitated with ether. Disulfide bond formation of the peptide was performed at high dilution in 10% DMSO. Finally, the product was purified and characterized by reverse-phase HPLC (1260 Infinity II Preparative LC Systems, Agilent Technologies, Santa Clara, CA), and its mass was confirmed by an LC / MS system (6100 Series Single Quadrupole LC / MS, Agilent Technologies, Santa Clara, CA).

[0294] Recombinant NPR-Fc fusion protein The extracellular domains (ECDs) of recombinant human NPRA, NPRB, and NPRC were expressed as soluble human IgG1 Fc fusion proteins in mammalian cells. The method basically followed the previously reported method (Bennet et al, JBC 266(34)23060-23067, 1991). In short, the human mature NPR ECD peptide sequences NPRA N1-L439 (GenBank Accn.#XP_005245275.1), NPRB R1-T433 (GenBank Accn.#NP_003986.2), and NPRC Q1-S434 (GenBank Accn.#NP_001191304.1) were synthetically ligated to amino acids E216-G446 (IMGT.org, EU numbering) of the human IgG1 heavy chain (Accn.P01857.2) (Azenta, Burlington, MA), and then cloned into pCMV6-a-puro (Invitrogen [Carlsbad, CA]) for mammalian expression. To eliminate disulfide bond aggregation, a single C>S substitution was introduced at position C232 (an unpaired cysteine ​​residue) of NPRA (Olympic Protein Technologies, PA1 Report, unpublished). All NPR-Fc plasmid sequences were validated, and plasmids were constructed using endotoxin-free reagents (Azenta, Burlington, MA).

[0295] Each NPR-Fc was transfected into Expi293 cells (A-14635, Thermo-Fisher Scientific) in 200 ml scale. After 4 days of growth, the transfection supernatant was collected, filtered, and protein A was captured (MabSelect SuRe, Cytiva). NPRA-Fc and NPRC-Fc were eluted with 0.1 M citrate buffer (pH 6.0 / 3.5 mM MgCl2 / 2% glycerol). NPRB-Fc was eluted at pH 6.6 (Pierce Gentle Elution buffer, Thermo Fisher catalog #21027). SDS-PAGE analysis revealed an 80-85 kDa band under reducing conditions, which was larger than the expected 75 kDa band, likely due to glycosylation in the ECD of all three NPRs (Potter LR, et al. 2009; (191):341-366). SEC analysis confirmed that the main peak was larger than the 153 kDa molecular weight marker. Preparative SEC removed high molecular weight (HMW) proteins from both NPRA and NPRC-Fc, purifying both to 93% monomer and exchanging buffer to PBS. NPRB-Fc was 98% monomer, so no further purification was required (Olympic Protein Technologies, PA1 Report, unpublished).

[0296] Example 12: The cationic alkyl-modified CNP derivative alone or in combination with pirfenidone showed a reduction in pulmonary fibrosis (inversely proportional to alveolar area) in a mouse model of idiopathic pulmonary fibrosis (IPF).

[0297] The following experimental protocol was followed to generate the data in Figures 8A and 8B. Animal care and treatment: C57BL / 6J male mice (6 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). To induce pulmonary fibrosis, mice were administered bleomycin (Bleo, 1.0 mg / kg, Nippon Kayaku, Tokyo, Japan) via I / O. Drug administration was initiated after 7 days. SEQ ID NO: 31 (0.3 mg / kg (0.1 μmol / kg) at SC), pirfenidone (100 mg / kg at PO), or a combination of pirfenidone (Pir, 100 mg / kg at PO) and SEQ ID NO: 31 (0.3 mg / kg (0.1 μmol / kg) at SC) was administered as shown in Figure 8A. Furthermore, a normal control group (NC) that did not undergo Bleo treatment and a Bleo control group that did not undergo treatment with the test substance were also included in this study. Mice were euthanized under isoflurane anesthesia on day 21, and their lung tissue was collected. Biochemical analysis in Figure 8B): Lung tissue was collected, fixed with 4% paraformaldehyde, and stained with Azan at Kyushu University (Fukuoka, Japan). Alveolar area was inversely proportional to fibrotic area and was measured by Image J in a blinded manner (NIH, Bethesda, MD, USA). Statistical analysis was performed using GraphPad based on Student's t-test (n=3, 7, 7, 7, and 7), for NC, Bleo control, SEQ ID NO: 31, Pir, and combined (SEQ ID NO: 31 and Pir). ***P<0.001, ns=not significant (no significant difference) (compared to the control group). The groups treated with Sequence ID No. 31 alone, or in combination with pirfenidone, showed significantly higher alveolar area, indicating the presence of healthy tissue and reduced fibrosis.

[0298] Example 13: Repeated subcutaneous administration of cationic alkyl-modified CNP derivatives, either as monotherapy or in combination with immune checkpoint inhibitors, demonstrated significant antitumor activity in a mouse model of breast cancer using E0771 cells.

[0299] The following experimental protocol was followed to generate the data shown in Figure 9. Animal care and treatment: C57BL / 6J female mice (6 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (CRF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Transplantation and drug administration: E0771 mammary cancer cells (250,000 cells / mouse, Cosmo Bio Tokyo, Japan) were orthotopically transplanted into the left mammary gland of the mice, and they were randomly assigned to groups of n=7-8. Drug administration was started on day 4 after inoculation. Anti-PD1 Ab (aPD1, BioX cell, West Lebanon, NH, #RMP1-14)) was administered intraperitoneally at a dose of 5 mg / kg twice a week for 2 cycles. Sequence ID 31 was administered subcutaneously once daily for 5 days (5 days of administration, 2 days of rest) in a bolus dose of 0.3 mg / kg (0.1 μmol / kg) in sterile water for injection (Otsuka Pharmaceutical, Tokushima, Japan) for 3 cycles. Alternatively, a combination of aPD1 (5 mg / kg IP administration twice weekly for 2 cycles) and Sequence ID 31 (0.3 mg / kg SC administration once daily for 5 days, 2 days of rest, 3 cycles) was used. The control group, which received sterile water for injection only, was administered in the same manner as Sequence ID 31 to establish baseline tumor growth dynamics. Tumor size was measured using calipers. Statistical analysis was based on Dunnett's test performed using GraphPad (n=8, 7, 8, and 7; control, aPD1, SEQ ID NO: 31, combination (aPD1 and SEQ ID NO: 31)). ****P<0.0001, *P<0.05 (compared to the control group). At the end of this study, the group treated with cationic alkyl-modified CNP (SEQ ID NO: 31) showed a significant reduction in tumor volume compared to the control group and the group treated with the immune checkpoint inhibitor aPD1 alone.

[0300] Example 14: In a mouse model of orthotopic bone metastasis using breast cancer E0771 cells, repeated subcutaneous administration of radiation, immune checkpoint inhibitors, and cationic alkyl-modified CNP derivatives reduced bone metastasis and significantly improved overall survival.

[0301] The following experimental protocol was followed to generate the data shown in Figure 10. Animal care and treatment: C57BL / 6J female mice (6 weeks old) were purchased from Oriental Bio Service Co., Ltd. (Kobe, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (CRF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Transplantation and drug administration: E0771 mammary cancer cells (250,000 cells / mouse in RPMI1640 medium (Fujifilm, Tokyo, Japan)) were orthotopically transplanted into the left mammary gland of mice, and E0771 mouse mammary cancer cells (500,000 cells / mouse in 50% Matrigel (Corning, NY, USA, #354234)) were orthotopically transplanted into the femur. The mice were then randomly assigned to 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 10A shows the drug administration schedule, which began 5 days after inoculation. Sequence ID 31 was administered subcutaneously once daily for 5 days (5 days of administration, 2 days of rest) over 4 cycles at a bolus dose of 0.3 mg / kg (0.1 μmol / kg) in buffer solution (15 mM succinate (TCI, Tokyo, Japan, #S0100), 4% (w / v) D-mannitol (TCI, Tokyo, Japan, #M0044), 10 mM hydroxypropyl-beta-cyclodextrin (TCI, Tokyo, Japan, #H0979), pH 4.4). aPD1 (BioX cell, West Lebanon, NH, #RMP1-14) was administered intraperitoneally at a dose of 5 mg / kg twice a week for two cycles. On days 5, 8, and 12, mice were anesthetized with a mixture of three anesthetics (0.3 mg / kg medetomidine, 4.0 mg / kg midazolam, and 5.0 mg / kg butorphanol (M / M / B: 0.3 / 4 / 5SC; Oriental Bioservices, Kobe, Japan)) and their bones were irradiated with 5 Gy of radiation in three sets using an X-ray irradiation system (mediXtec Japan Corporation, Chiba, Japan, MX-160Labo).The control group (received only buffer solution) was administered in the same manner as SEQ ID NO: 31 to establish baseline tumor growth dynamics. Survival rates were monitored until day 33 after cell inoculation (Figure 10B). After the remaining mice were euthanized as planned, tumors were collected and measured in size using calipers. Statistical analysis was performed using the Log-rank (Mantel-Cox) test with GraphPad (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))**P<0.01 (compared to group 5). At the end of this study, the combination of SEQ ID NO: 31, radiation, and aPD1 (immune checkpoint inhibitor) showed a significant improvement in survival rates. Furthermore, the addition of Sequence ID No. 31 reduced the incidence of bone metastases in all groups.

[0302] Example 15: In an orthotopic lung metastasis model mouse using osteosarcoma LM8 cells, repeated subcutaneous administration of a cationic alkyl-modified CNP derivative in combination with cleavage showed a significant reduction in lung metastasis compared to cleavage alone.

[0303] The following experimental protocol was followed to generate the data shown in Figures 11A-11C. Animal care and treatment: CH3 / He male mice (7 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd. [Tokyo, Japan]). Transplantation and drug administration: LM8 osteosarcoma cells (RCB, Tsukuba, Japan, #RCB1450) (1,000,000 cells / mouse) were orthotopically transplanted into the femurs of the mice. The mice were then randomly assigned to groups of n=7. Figure 11A shows the drug administration plan, which began on day 4 after inoculation. Sequence ID No. 31 was administered subcutaneously at a bolus dose of 0.3 mg / kg (0.1 μmol / kg) in buffer (15 mM succinate (TCI, Tokyo, Japan, #S0100), 4% (w / v) D-mannitol (TCI, Tokyo, Japan, #M0044), 10 mM hydroxypropyl-beta-cyclodextrin (TCI, Tokyo, Japan, #H0979), pH 4.4) once daily for 5 days (5 days on, 2 days rest), for more than three cycles. On day 7 after inoculation, all mice underwent amputation surgery to remove the primary tumor under isoflurane anesthesia and sutured with sutures (Alfresa Pharma Co., Ltd., Osaka, Japan, #HR0806NW45-KF2). The amputated control group also received the buffer once daily for 5 days (5 days on, 2 days rest) for more than three cycles instead of treatment. Mice were euthanized on day 34, and lung tissue was collected. Biochemical analysis (Figure 11B): The collected lung tissue was immersed in 4% paraformaldehyde (Fujifilm, Tokyo, Japan, #163-20145). The tissue was then embedded in paraffin, and the resulting 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 evaluated by directly counting the present metastatic nodules (Figure 11C). Outliers were identified using the ROUT test (Q=1%). All statistical analyses were based on Dunnett's test performed using GraphPad (n=7 and 7; control, combined (amsection and SEQ ID NO: 31))**P<0.01 (compared to the control group). In addition to chemotherapy, surgical interventions such as amputation may be used to prevent lung metastases.In conclusion, repeated SC administration of SEQ ID NO: 31 with cleavage reduced the incidence of lung metastases compared to cleavage alone.

[0304] Example 16: Repeated subcutaneous administration of cationic alkyl-modified CNP derivatives, either as monotherapy or in combination with immune checkpoint inhibitors, showed a significant reduction in tumor volume in a subcutaneous mouse model of colon cancer using MC38 cells.

[0305] To generate the data shown in Figure 12, the following experimental protocol was followed. Animal care and treatment: C57BL / 6J male mice (6 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and housed under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Transplantation and drug administration: MC38 colon cancer cells (1,000,000 cells / mouse) were subcutaneously transplanted into the right flank of the mice, and they were randomly assigned to groups of n=8-9. Drug administration was started on day 4 after inoculation. Anti-TIGIT antibody (Absolute antibody, Shirley, MA, USA, #Ab01258-1.1-VXX, clone 1B4) was administered intraperitoneally at a dose of 5 mg / kg twice a week for two cycles. Sequence ID 31 was administered subcutaneously at a bolus dose of 0.3 mg / kg (0.1 μmol / kg) in buffer (15 mM succinate (TCI, Tokyo, Japan, #S0100), 4% (w / v) D-mannitol (TCI, Tokyo, Japan, #M0044), 10 mM hydroxypropyl-beta-cyclodextrin (TCI, Tokyo, Japan, #H0979), pH 4.5) once daily for 3 cycles over 5 days (5 days of administration, 2 days of rest). Alternatively, a combination of anti-TIGIT antibody (5 mg / kg IP administration twice weekly for 2 cycles) and Sequence ID 31 (0.3 mg / kg SC administration once daily for 5 days, 2 days of rest, 3 cycles) was used. The control group (received buffer only) was administered in the same manner as Sequence ID 31 to establish baseline tumor growth dynamics. Tumor size was measured using calipers on days 4, 7, 14, and 22 after inoculation. After the final tumor measurement on day 22, the mice were humanely euthanized. Statistical analysis was based on Dunnett's test performed using GraphPad (n=9, 8, 8, and 8; control, anti-TIGIT antibody, SEQ ID NO: 31, combination (Combo) (anti-TIGIT antibody and SEQ ID NO: 31). *P<0.05 (compared to the anti-TIGIT antibody monotherapy group). At the end of this study, the group treated with cationic alkyl-modified CNP (SEQ ID NO: 31) showed a significant reduction in tumor volume compared to the control group and the group treated with the immune checkpoint anti-TIGIT antibody alone.

[0306] Example 17: HeLa cells treated with cationic alkyl-modified CNP derivatives showed significant inhibition of baseline cyclic adenosine monophosphate levels.

[0307] The following experimental protocol was followed to generate the data shown in Figure 13. In vitro protocol: HeLa cells were purchased from ATCC (Manassas, VA, USA). The cells were cultured at 37°C under conditions of 100% humidity and 5% CO2 in Dulbecco's modified Eagle medium (DMEM) (Fujifilm, Tokyo, Japan, #044-29765) supplemented with 10% FBS (Sigma Aldrich, St. Louis, MO, USA, #F2442). The cells were harvested and placed in ENGS (Lifeline, San Diego, CA, USA, #LEC-LL0002) for 10 minutes. 7 Cells were suspended at a concentration of cells / mL and 5 μL of the cell suspension was added to each well of a 96-well low-adhesion plate (PerkinElmer, Waltham, MA, USA, #66PL96005). Next, cells (n=4 wells) were treated for 10 minutes with 5 μL of SEQ ID NO: 31 (final concentration 10 μg / mL) in ENGS containing 1 mM IBMX (Fujifilm, Tokyo, Japan, #099-03411). cAMP levels were assessed using the PerkinElmer (Waltham, MA, USA, #62AM4PEB) cAMP assay kit according to the manufacturer's protocol and using a plate reader (PerkinElmer, Waltham, MA, USA, #Nivo). Statistical analysis was performed using GraphPad Prism on untreated control wells (n=4 wells). *P<0.05. The NPR-C receptor has been suggested to inhibit adenylyl cyclase activity and reduce cyclic adenosine monophosphate (cAMP) production. From this study, it can be concluded that baseline cAMP levels in HeLa cells treated with SEQ ID NO: 31 (known to express NPR-C) were altered (inhibited), indicating binding to NPR-C.

[0308] All patents, published applications, and references cited herein are incorporated in their entirety by reference.

[0309] While exemplary embodiments have been specifically shown and described, it will be understood by those skilled in the art that various modifications can be made to the form and details within the scope of embodiments covered by the appended claims without departing from that scope.

Claims

1. Equation (I): J-(CH2)x(CO)-(A)y-(B)z- (I) A compound comprising a cationic alkyl moiety represented by, J is either HOOC or CH3, x is between 10 and 16, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aea), gamma-aminobutyric acid (γAbu), gamma-linked glutamic acid (γE), and alpha-linked glutamic acid (E). y is between 2 and 4, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is between 2 and 4, -(B)z- contains two or fewer Dab residues, and the Dap residues or Dab residues are linked via alpha-amino acids. compound.

2. 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 (Aea) and gamma-aminobutyric acid (γAbu), y is 3, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is 2 or 3, The compound according to claim 1.

3. 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 (Aea) and gamma-linked glutamic acid (γE), y is 3, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), and z is 2 or 3. The compound according to claim 1.

4. 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 diaminobutanoic acid (Dab), z is 2 or 3, The compound according to claim 1.

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

6. 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 (Aea) and gamma-aminobutyric acid (γAbu), y is 3, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is 2 or 3, The compound according to claim 1.

7. 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 gamma-linked glutamic acid (γE), y is 3, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is 2 or 3, The compound according to claim 1.

8. J is 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 diaminobutanoic acid (Dab), z is 2 or 3, The compound according to claim 1.

9. J is HOOC, x is 10, 12, 14, or 16, A is gamma-bound glutamate (γE), y is 3, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is 2 or 3, The compound according to claim 1.

10. J is CH3, x is 14, (A) y is Aeea-Aeea-Aeea, γAbu-γAbu-γAbu, γAbu-Aeea-Aeea, γE-Aeea-Aeea, E-Aeea-Aeea; E-Aeea, (B)z is Dap-Dap, Dab-Dab, Dab-Dap, or Dap-Dab. The compound according to claim 1.

11. 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, (B)z is Dap-Dap, Dab-Dab, Dab-Dap, or Dap-Dab. The compound according to claim 1.

12. The compound according to claim 1, wherein the cationic alkyl moiety is selected from SEQ ID NOs: 10-22 and 51-69.

13. The compound according to claim 1, wherein the cationic alkyl portion is selected from SEQ ID NOs. 10 to 22.

14. The compound according to any one of claims 1 to 13, wherein, when the compound is bound to a peptide, it does not exhibit clinically observable ataxia after parenteral bolus administration at a dose of 10 μmol / kg or less in rats.

15. A compound according to any one of claims 1 to 14, for use in peptide modification via covalent bonding.

16. Formula (II): CH3(CH2)x(CO)-(A)y-(B)z-peptide moiety (II) A conjugated peptide represented by, x is between 10 and 16, A is independently selected from the group consisting of 2-[2-(2-aminoethoxy)ethoxy]acetic acid (Aea), gamma-aminobutyric acid (γAbu), gamma-linked glutamic acid (γE), and alpha-linked glutamic acid (E). y is between 2 and 4, B is independently diaminopropionic acid (Dap) or diaminobutanoic acid (Dab), z is between 2 and 4, -(B)z- contains two or fewer Dab residues, and the Dap or Dab residues are linked via alpha-amino acids. CH3(CH2)x(CO)-(A)y-(B)z- is covalently bonded to the N-terminus of the peptide portion, or is linked to one of the side-chain amino groups of the peptide portion. The conjugated peptide has biological activity equal to or greater than that of the unmodified peptide at an equivalent bolus dose, and / or The conjugated peptide has a blood level equivalent to or higher than that of the unconjugated peptide at the same time point after bolus administration at an equivalent dose. Conjugated peptide.

17. The conjugated peptide according to claim 16, wherein the conjugated peptide binds to a natriuretic peptide receptor and does not cause undesirable effects or ataxia in rats at a bolus dose of 3.0 μmol / kg or less.

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

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

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 NOs. 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 NOs. 10 to 22.

22. The conjugated peptide according to any one of claims 16 to 20, wherein the peptide portion is a natriuretic peptide derivative in which one or more methionine residues are substituted with glutamine (Q), leucine (L), norleucine (Nle), or methoxynine (Mox).

23. The conjugated peptide according to any one of claims 16 to 22, wherein the peptide portion is a natriuretic peptide according to SEQ ID NO: 32, or a derivative thereof in which one or more methionine residues are substituted with glutamine (Q), and the CH3(CH2)x(CO)-(A)y-(B)z- portion is selected from SEQ ID NOs: 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 NOs: 29-31, 33-43, 45-47, and 49-51.

25. The conjugated peptide according to claim 24, wherein the conjugated peptide is selected from SEQ ID NOs: 29-31 and 33-43.

26. The conjugated peptide according to claim 25, wherein the conjugated peptide is selected from SEQ ID NOs: 29-31.

27. The conjugated peptide according to claim 26, wherein the conjugated peptide is Sequence ID No.

29.

28. The conjugated peptide according to claim 26, wherein the conjugated peptide is Sequence ID No.

30.

29. The conjugated peptide according to claim 26, wherein the conjugated peptide of formula (II) is Sequence 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 aforementioned conjugated peptide has been shown to have an extended increase in blood cGMP, changes in cAMP, changes in blood pressure, improved survival rates from sepsis, improved survival rates from acute lung injury, improved survival rates from acute respiratory distress syndrome, a decrease in MPO-positive cells, a decrease in the number of cells in alveolar fluid or bronchoalveolar lavage fluid, a decrease in the amount of protein in alveolar fluid or bronchoalveolar lavage fluid, decreased endothelial permeability, a decrease in lung weight per unit body weight, and monocyte chemoattractant-1 The conjugated peptide according to any one of claims 16 to 32 produces a physiological effect selected from among: a decrease in Protein-1, MCP-1), a decrease in IL-6, a decrease in TNF-alpha, a decrease in A1008 / A9, a decrease in fibrosis, a decrease in tumor volume, a decrease in metastasis, a decrease in inflammation, an antiproliferative effect, a decrease in tumor load, inhibition of cyclooxygenase-2 (COX-2) expression, antagonism of the renin-angiotensin-aldosterone system, suppression of cardiac hypertrophy, or a combination thereof.

34. A compound according to any one of claims 1 to 15, or a conjugated peptide according to any one of claims 16 to 33, for use in the manufacture of a medical composition.

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

36. The compound or conjugated peptide for use according to claim 35, wherein the one or more pharmaceutically acceptable carriers or excipients include bulking agents, buffers, stabilizers, preservatives, or combinations thereof.

37. A compound according to any one of claims 1 to 15, or a conjugated peptide according to any one of claims 16 to 33, for use in treating a disease or pathological condition in a subject that requires such treatment.

38. The aforementioned compound or conjugated peptide a) Any one of sequence numbers 29-31, 33-43, 45-47, or 49-51, b) Any one of sequence numbers 29-31, 33-43, or 45-47, c) Any one of sequence numbers 29-31 or 33-43, d) Any one of sequence numbers 29-31, e) Sequence ID 29, or, f) Sequence ID 30, or g) Sequence ID 31, Compounds or conjugated peptides for use in Embodiment 37.

39. A compound for use in either claim 37 or 38, wherein the compound or conjugated peptide comprises SEQ ID NO:

31.

40. The aforementioned diseases or conditions include: lung (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 preserving ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), acute heart failure, or congestive heart failure), and bone / joint (e.g., osteoporosis, osteoarthritis). Compounds or conjugated peptides for use according to any one of claims 37 to 39, affecting the kidneys (e.g., chronic kidney disease (CKD), acute kidney injury (AKI), drug-induced kidney injury, inflammation / nephritis, renal fibrosis, glomerulosclerosis, or renal cancer), the prostate (e.g., benign prostatic hyperplasia, or prostate cancer), the 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 condition is pre-metastatic or post-metastatic cancer.

42. The compound or conjugated peptide for use according to claim 41, wherein the cancer is a cancer of one or more organs selected from the lungs, lung pleura, liver, heart, bones / joints, kidneys, prostate, breast, brain, eye, skin, muscle, blood, blood vessels, gastrointestinal tract, bladder, testes, ovaries, and / or uterus.

43. The compound or conjugated peptide for use according to any one of claims 37 to 39, wherein the disease or condition is, in the target population, pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), or COVID-19.

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

45. The compound or conjugated peptide for use according to any one of claims 37 to 44, wherein the treatment is administered to a therapeutically effective bolus dose of 10.0 μmol / kg or less and / or in the range of 10.0 μmol / kg to 0.0001 μmol / kg (including both ends).

46. The compound or conjugated peptide for use according to any one of claims 37 to 45, wherein the compound is administered as a monotherapy or applied in combination with one or more additional 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 a combination thereof.

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

49. The compound or conjugated peptide for use according to any one of claims 37 to 42, wherein the compound or conjugated peptide is administered to the subject by subcutaneous, injection, inhalation, nasal spray, oral, ophthalmic, and / or 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 according to claim 49, wherein the one or more pharmaceutically acceptable carriers or excipients include a bulking agent, a buffer, a stabilizer, a preservative, or a combination thereof.

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