Alpha-1-microglobulin-derived peptide fragments and uses thereof

Abstract

The present disclosure relates to drugs comprising polypeptides and their use in medicine. Specifically, the disclosure relates to fragments of alpha-1-microglobulin. Also disclosed herein are fusion proteins comprising the polypeptides, polynucleotides encoding the polypeptides, vectors comprising the polynucleotides, and cells comprising the polynucleotides or vectors.

Description

[Technical Field]

[0001] The present disclosure relates to drugs comprising polypeptides and their use in medicine. In particular, the present disclosure relates to polypeptide fragments of alpha-1-microglobulin or variants thereof. [Background technology]

[0002] Alpha-1-microglobulin (α1-microglobulin, A1M, protein HC) is an extracellular protein with tissue-clearing functions (Ekstrerom et al., 1977; Akerstrom and Gram, 2014). It is present in many animals, including fish, birds, rodents, mammals, and other vertebrates. A1M is synthesized primarily in the liver, but at a lower rate in most other cells in the body. It is encoded by the α1-microglobulin-bikunin precursor gene (AMBP) and is co-translated with another protein, bikunin, as a continuous peptide precursor in all cells and species (Kaumeyer et al., 1986).

[0003] A1M is a physiological antioxidant active in both intracellular and extracellular compartments. Four distinct molecular mechanisms have been described that form the basis for A1M's protective effects: 1) catalytic reductase activity, 2) radical scavenging, 3) heme binding and degradation, and 4) mitochondrial binding and protection. A1M utilizes its redox properties to function as a "circulatory trash can" capable of binding and removing reactive oxygen species (ROS) and other pathological oxidation products by transporting them to the kidney for degradation. In particular, the heme-scavenging properties of A1M enable it to protect cells from heme-induced damage.

[0004] As described in WO2010 / 006809 and WO2017 / 158181, the following residues were identified as important for the antioxidant effect of A1M: Y22, C34, K69, K92, K118, K130, Y132, L180, I181, P182, and R183.

[0005] A recombinant human form of A1M (rA1M, rhA1M) has been developed for pharmaceutical evaluation (WO2017 / 158181). Several variants of rA1M with altered amino acid sequences but retaining the same biological effects have been developed and evaluated. Recombinant human A1M (rA1M) has been shown to be fully functional compared to endogenous A1M derived from human plasma or urine.

[0006] Although these proteins can be expressed in E. coli, they lack glycosylation and are less soluble and stable than human A1M purified from plasma. It is also expected that A1M or rA1M, or variants thereof, must be administered parenterally to have the desired therapeutic effect, and that recombinant proteins lacking glycosylation have a high risk of undesirable immunogenic responses. However, it would be advantageous to provide A1M-mimetic peptides that are easy to prepare, have a size that allows for the formulation of pharmaceutical compositions, and have suitable physicochemical, pharmacokinetic, and / or biodistribution properties.

[0007] A1M and its variants have shown great potential for the treatment of a range of diseases and disorders (WO2016 / 135214, WO2017 / 158181, and WO2019 / 086569).

[0008] Therefore, there is a need to develop peptides that have the desired properties of A1M but are significantly smaller in size. Such peptides would offer the possibility of long-term administration via subcutaneous or parenteral routes, would pose a lower risk of immunogenicity compared to the full-length complex protein, and would be chemically synthesized, resulting in lower production costs compared to recombinant proteins produced in E. coli. Summary of the Invention

[0009] As discussed above, A1M and its variants have shown great potential as therapeutic biologics. However, certain drawbacks are associated with the use of proteins as active pharmaceutical ingredients (APIs), including the complexity of protein production and their stability and shelf life. Therefore, there is an unmet need for novel active pharmaceutical ingredients (APIs) that exhibit similar therapeutic activity to A1M but have different properties, such as pharmacokinetic profiles and tissue distribution characteristics, or the ability to administer different routes with reduced potential for immunogenicity.

[0010] The inventors have found that certain fragments of A1M maintain the therapeutic activity of native A1M when they satisfy the structural requirements outlined herein. While several residues have been reported to be important for the function of A1M as outlined herein above, the inventors have discovered that only a limited number of structural features appear to be critical for A1M function. Accordingly, the inventors provide herein polypeptides comprising one or more of the above structural features, which have A1M activity. The polypeptides disclosed herein are substantially shorter than previously reported A1M and its variants. Therefore, polypeptide production and handling are relatively feasible. The polypeptides can also be lyophilized, which is not possible with many proteins. Furthermore, these peptides are contemplated to have improved properties, such as improved shelf life / stability, over A1M and some previously reported A1M variants. The disclosed polypeptides can also be administered via routes not feasible for A1M. The A1M fragments disclosed herein are also long enough to allow for administration routes not feasible for full-length A1M and its variants. The route of administration can be, for example, oral, subcutaneous, or topical.

[0011] One aspect of the present disclosure provides a medicament comprising a polypeptide comprising: a. A polypeptide comprising 12 to 80 amino acid residues, wherein the polypeptide has the amino acid sequence GSTCPWLKKIX 1(SEQ ID NO: 20), where X 1 is M, K, R, or Nle, or b. A polypeptide comprising 30 to 80 amino acid residues, wherein the polypeptide has the amino acid sequence GATEAEISMTSTX 2 WRKGVCEETSGAYEKTD (SEQ ID NO: 21), where X 2 is H, R, or K.

[0012] One aspect of the present disclosure provides a pharmaceutical agent comprising a polypeptide consisting of 10 to 80 amino acid residues, the polypeptide comprising SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29; X 3 is selected from D, N, or G, X 4 is selected from M, K, R, or Nle, X 5 is selected from H, R, or K; X 6 is selected from D, N, or E, X 7 is selected from M or Nle, the fragment is at least 10 amino acid residues in length, The fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO:30.

[0013] Polypeptides falling within this definition are shown herein to have antioxidant activity, and are also shown to protect cells from heme-induced cell death.

[0014] One aspect of the present disclosure provides a medicament comprising a polypeptide of the present disclosure and an additional moiety.

[0015] One aspect of the present disclosure provides a medicament for use in treating or preventing a disease or disorder, comprising a polypeptide comprising: a. A polypeptide comprising 12 to 80 amino acid residues, wherein the polypeptide has the amino acid sequence GSTCPWLKKIX 1(SEQ ID NO: 20), where X 1 is M, K, R, or Nle, or b. A polypeptide comprising 30 to 80 amino acid residues, wherein the polypeptide has the amino acid sequence GATEAEISMTSTX 2 WRKGVCEETSGAYEKTD (SEQ ID NO: 21), where X 2 is H, R, or K, or the polypeptide has 10 to 80 amino acid residues, and the polypeptide comprises SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29; X 3 is selected from D, N, or G, X 4 is selected from M, K, R, or Nle, X 5 is selected from H, R, or K; X 6 is selected from D, N, or E, X 7 is selected from M or Nle, the fragment is at least 10 amino acid residues in length, The fragment comprises amino acid residues corresponding to C34 or C72 of SEQ ID NO:30.

[0016] One aspect of the present disclosure provides a method of treating a disease or disorder comprising administering a therapeutically effective amount of an agent disclosed herein.

[0017] One embodiment of the present disclosure provides the use of an agent disclosed herein in the manufacture of a medicament for the treatment of a disease or disorder. [Brief explanation of the drawings]

[0018] [Figure 1]Cytochrome c reduction profiles of RMC-035 and GTI-1 through GTI-15. Concentrations are shown in μM. The corresponding 4PL regression curves are overlaid. GTI-1 through GTI-4 and GTI-6 through GTI-14 all demonstrate potency relative to RMC-035. Notably, the polypeptides exhibit greater than 30% of the potency of RMC-035. Furthermore, the EC50 values ​​were found to be potent, with GTI-6 and GTI-11 through GTI-14 being particularly potent. The negative controls GTI-5 and GTI-15 exhibited low activity, specifically less than 30% of the activity of RMC-035. [Figure 2] The cytochrome c reducing activity of RMC-035 and polypeptides GTI-1 to GTI-15 is shown as a percentage (%) of the cytochrome c reducing activity of RMC-035. [Figure 3] EC50 values ​​obtained in the cytochrome c reduction assay for RMC-035 and GRI-1 to GTI-15. Values ​​not obtained from fitting are indicated as na. [Figure 4] RBC lysis profiles of RMC-035 and GTI-1 through GTI-15 showing LDH release. Concentrations are shown in μM. Corresponding 4PL regression curves are overlaid. RMC-035, GTI-1 through GTI-4, and GTI-6 through GTI-12 all show a decrease in absorbance with increasing polypeptide concentration. GTI-5 and GTI-15 showed no change in absorbance even at high polypeptide concentrations. [Figure 5] RBC lysis profiles of RMC-035 and GTI-1 through GTI-15 showing hemoglobin release. Concentrations are shown in μM. Corresponding 4PL regression curves are overlaid. RMC-035, GTI-1 through GTI-4, and GTI-6 through GTI-14 all showed decreased hemoglobin absorption with increasing polypeptide concentration, indicating that these polypeptides can prevent RBC lysis. GTI-5 and GTI-15 showed no change in absorption even at high polypeptide concentrations. [Figure 6]Synthetic peptides of human A1M. (A) Amino acid sequences of two synthetic peptides covering portions of human A1M. Peptides are numbered and named based on the first three amino acids followed by the number of amino acids. (B) Alignment of the two A1M-peptides with the full-length amino acid sequence of human A1M (Kaumeyer et al., 1986). [Figure 7] Localization of A1M peptides in the three-dimensional structure of human A1M. The crystal structure of human A1M (Meining and Skerra, 2012) is visualized using UCSF Chimera free software (version 1.10.2). The main chain of peptide ENF-46 (dark gray), omega loop 1 (medium gray helix), and the side chain of Cys34 (space-filling atomic model) are shown. [Figure 8] SDS-PAGE of full-length A1M and peptide ENF-46. Two micrograms of A1M-wt, A1M-035, and 10 micrograms of peptide ENF-46 were incubated with sample buffer with or without β-mercaptoethanol, boiled for 1 min, separated on a 4–20% gradient polyacrylamide gel, and finally stained with Coomassie brilliant blue. [Figure 9] Native PAGE of full-length A1M and peptide ENF-46. Two micrograms of A1M-wt or A1M-035, or 10 micrograms of peptide ENF-46, were incubated alone or with a four-fold molar excess of heme and then separated by non-denaturing (native) PAGE. Gels were stained with Coomassie (left) or imaged by tryptophan fluorescence on a BioRad ChemiDoc instrument (right). [Figure 10] Reduction of cytochrome c by A1M-wt and peptide ENF-46. Absorbance spectra of 100 μM cytochrome c with or without either 10 μM A1M-wt or 20 μM peptide ENF-46 in 20 mM Tris-HCl, pH 8, 0.15 M NaCl + 100 μM NADPH, incubated at room temperature for 20 min. Buffer alone was used as a blank. [Figure 11A]Protection of cells against heme-induced cell death by A1M and A1M-peptides. (A) K562 cells seeded at 1 × 10 cells / well in 96-well microtiter plates were incubated with 100 μM heme for 1 h at 37°C in the presence of a dilution series of either ovalbumin (control), A1M-wt, A1M-035, or SRI-36 or ovalbumin. Incubations were performed in quadruplicate. Cell death was measured as LDH release into the medium by subtracting LDH values ​​from viable cells. Means (n ​​= 4) ± SD are shown, and statistical significance was calculated using ANOVA with Dunnett's correction for multiple comparisons. [Figure 11B] HK-2 cells were seeded at 5x104 cells / well and incubated with heme, A1M-035, ENF-46, and / or SWT-21 (control peptide) at the concentrations indicated in the figure for 2 hours at 37°C. Incubations were performed in quadruplicate. After incubation, cells were analyzed for cell viability using the WST-1 assay. Mean values ​​(n=4) ± SD are shown, and statistical significance was calculated by ANOVA with Turkey's correction for multiple comparisons. [Figure 11C] HK-2 cells were seeded at 5x104 cells / well and incubated with heme, A1M-035, ENF-46, and / or SWT-21 (control peptide) at the concentrations indicated in the figure for 2 hours at 37°C. Incubations were performed in quadruplicate. After incubation, cells were analyzed for cell viability using the WST-1 assay. Mean values ​​(n=4) ± SD are shown, and statistical significance was calculated by ANOVA with Turkey's correction for multiple comparisons. [Figure 11D] HK-2 cells were seeded at 5x104 cells / well and incubated with heme, A1M-035, or ENF-46 at the concentrations indicated in the figure for 1 hour at 37°C. Incubations were performed in quadruplicate. Cell death was measured as LDH release into the medium by subtracting LDH values ​​from viable cells. Mean values ​​(n=4) ± SD are shown, and statistical significance was calculated by ANOVA with Turkey's correction for multiple comparisons. [Figure 11E]Red blood cells (RBCs) were suspended at 1% (v / v) and incubated with heme, A1M-035, and / or ENF-46 at the concentrations indicated in the figure for 3 hours (excluding the control time of 0 hours) at room temperature with rotation. Incubations were performed in triplicate. Cell death was measured as LDH release into the medium. Mean values ​​(n = 3) ± SD are shown, and statistical significance was calculated by ANOVA with Turkey's correction for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 12] Viability profiles of the human kidney-2 (HK-2) proximal tubule cell line for RMC-035 and GTI-1 through GTI-15, GTI-24, GTI-25, GTI-29 through GTI-31, GTI-46, GTI-50, GTI-61 through GTI-64, and GTI-73, showing lactate dehydrogenase (LDH) release. Concentrations are shown in μM. Corresponding 4PL regression curves are overlaid. All tested peptides, except for RMC-035 and GTI-5, GTI-7, GTI-13, GTI-14, and GTI-15, showed a decrease in LDH absorption with increasing polypeptide concentration, indicating their ability to inhibit heme-induced cell viability loss. GTI-5 and GTI-15 showed no change in absorption even at high polypeptide concentrations. [Figure 13] HO-1 mRNA expression determined by RT-qPCR. [Figure 14] Serum creatinine and blood urea nitrogen (BUN) after unilateral nephrectomy and renal ischemia / reperfusion injury, and after treatment with the peptide GTI-11. Serum creatinine and BUN were measured preoperatively at baseline, 1, 2, 3, and 5 days after unilateral nephrectomy, and 40 minutes of unilateral renal pedicle clamping in rats. n = 7 / 8, values ​​are presented as mean ± SEM. Serum creatinine and BUN were measured preoperatively at baseline, 1, 2, 3, and 5 days after unilateral nephrectomy and 40 minutes of unilateral renal pedicle clamping in rats. n = 7 / 8, values ​​are presented as mean ± SEM. Differences between GTI-11-treated animals and vehicle were analyzed using the Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001. [Figure 15] Plasma creatinine (mg / dL) and blood urea nitrogen (BUN, mg / dL) in rats on days 1 and 3 after unilateral nephrectomy and ischemia-reperfusion injury, followed by treatment with saline vehicle or peptide GTI-2, administered IV or SC. Values ​​are shown as mean ± SEM. Differences between GTI-2-treated animals and vehicle were analyzed using the Mann-Whitney U test. *P<0.05, **P<0.01. [Figure 16] Glomerular filtration rate (GFR) in rats 3 days after unilateral nephrectomy and ischemia-reperfusion injury and treatment with saline vehicle or peptide GTI-2 administered IV or SC. Values ​​are shown as mean ± SEM. Differences between GTI-2-treated animals and vehicle were analyzed using the Mann-Whitney U test. *P<0.05, **P<0.01. [Figure 17] Plasma creatinine (pCreatinine, mg / dL), blood urea nitrogen (BUN, mg / dL), and aspartate aminotransferase (AST, nmol / mL / min) in mice at baseline before LPS administration, 8 hours and 24 hours after LPS administration, and after treatment with saline vehicle or peptide GTI-2 administered intravenously. Values ​​are shown as mean ± SEM. Differences between GTI-2-treated animals and vehicle were analyzed using the Mann-Whitney U test. *P<0.05, ***P<0.001, ****P<0.0001. [Figure 18] Plasma creatinine (pCreatinine, mg / dL) and blood urea nitrogen (BUN, mg / dL) in mice at baseline and 4 days after cisplatin-induced renal injury and after treatment with PBS vehicle, peptide GTI-2, or GTI-86 administered IV or SC. Values ​​are shown as mean ± SEM. Differences between GTI-2-treated animals and vehicle were analyzed using the Mann-Whitney U test. *P<0.01, ***P<0.001. [Figure 19]Plasma creatinine and urinary albumin / creatinine ratio (UACR, mg / mmol) in mice on day 0 (day 25 after STZ treatment) and days 14 and 28 of treatment with SC administration of the A1M tool peptide (RMC-035). Values ​​are shown as mean ± SEM. Differences between STZ- and A1M tool peptide-treated animals and vehicle were analyzed using the Mann-Whitney U test. *P<0.05, **P<0.01, ***P<0.001. [Figure 20] Adriamycin treatment significantly increased plasma creatinine, BUN, and UACR levels in adriamycin-treated and vehicle-treated mice on both days 9 and 14 after adriamycin challenge (Figure 20). The adriamycin-induced increases in plasma creatinine (Figure 20A), BUN (Figure 20B), and UACR (Figure 20C) were all significantly inhibited by once-daily treatment with GTI-86 in a dose-dependent manner. *P<0.05, and **P<0.01. [Figure 21] A1M-035 (A) and peptides GTI-86, GTI-111, and GTI-115 (B–D) bind to free heme, as shown by fluorescence microscopy, where an absorbance shift is observed between free heme and heme complexed with A1M-035 or peptides. E: GTI-86 binds to heme, as evidenced by an increase in the absorbance peak at 383 nm. GTI-86 also increased reductase activity in a dose-dependent manner, as evidenced by the absorbance at 400–470 nm. DETAILED DESCRIPTION OF THE INVENTION

[0019] definition As used herein, the phrase "consisting of X to Y amino acid residues" is accepted to mean a polypeptide consisting of X to Y amino acid residues, inclusive. That is, the polypeptide may consist of X amino acid residues, Y amino acid residues, or a number of amino acid residues falling between X and Y, but may not consist of more than Y amino acid residues. By way of example, a polypeptide containing 3 to 6 amino acid residues consists of exactly 3, 4, 5, or 6 amino acid residues.

[0020] As used herein, a "fragment," when used in reference to a reference polypeptide, is a polypeptide having some, but not all, of the amino acid sequence of the reference polypeptide.

[0021] By "variant" is meant that the peptide fragment does not share 100% amino acid sequence identity with the reference sequence. That is, one or more amino acid residues of the peptide fragment are mutated compared to the reference sequence and / or there are one or more gaps (i.e., deletions) of one or more amino acid residues relative to the reference sequence. For example, the peptide fragment may have at least 80% sequence identity to the reference sequence.

[0022] A "variant comprising X consecutive amino acid residues of a reference sequence" means that the variant comprises a sequence of X amino acid residues that has 100% sequence identity to the reference sequence.

[0023] As used herein, the terms "polypeptide fragment," "A1M fragment," and "RMC-035 fragment" are used interchangeably.

[0024] Amino acid residue numbering throughout this specification refers to SEQ ID NO: 30, and when other sequences are described, one of skill in the art will understand how to identify amino acid residues that correspond to amino acid residues in SEQ ID NO: 30, based on context and sequence overlap.

[0025] As used herein, compound GTI-1 corresponds to the polypeptide having SEQ ID NO:1. As used herein, compound GTI-2 corresponds to the polypeptide having SEQ ID NO:2. As used herein, compound GTI-3 corresponds to the polypeptide having SEQ ID NO:3. Compound GTI-4 corresponds to the polypeptide having SEQ ID NO:4. As used herein, compound GTI-5 corresponds to the polypeptide having SEQ ID NO:5. As used herein, compound GTI-6 corresponds to the polypeptide having SEQ ID NO:6. As used herein, compound GTI-7 corresponds to the polypeptide having SEQ ID NO:7. As used herein, compound GTI-8 corresponds to the polypeptide having SEQ ID NO:8. As used herein, compound GTI-9 corresponds to the polypeptide having SEQ ID NO:9. As used herein, compound GTI-10 corresponds to the polypeptide having SEQ ID NO:10. As used herein, compound GTI-11 corresponds to the polypeptide having SEQ ID NO:11. As used herein, compound GTI-12 corresponds to the polypeptide having SEQ ID NO:12. As used herein, compound GTI-13 corresponds to the polypeptide having SEQ ID NO:13. As used herein, the compound GTI-14 corresponds to the polypeptide having SEQ ID NO: 14. As used herein, the compound GTI-15 corresponds to the polypeptide having SEQ ID NO: 15. As used herein, the compound RMC-035 (A1M-035) corresponds to the polypeptide having SEQ ID NO: 16.

[0026] As used herein, the term "treatment" or "treating" refers to an approach to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, the alleviation or amelioration of one or more symptoms or pathological conditions, whether detectable or undetectable, attenuation of the extent of a disease or disorder, stabilization (i.e., not worsening) of the disease or disorder state, prevention of the disease or disorder state, delay or slowing of the progression of a disease or disorder, improvement or palliation of the disease state, and remission (partial or complete).

[0027] As used herein, the term "A1M activity" refers to the property of a compound to act similarly to A1M. Specifically, the compound may be an antioxidant, may bind heme, may reduce cytochrome c, or may exhibit any other physiological or therapeutic effect exhibited by A1M.

[0028] "CLIPS" refers to a modification with 1,3-bis(bromomethyl)benzene, which allows a nucleophilic moiety of an amino acid to react at the bromomethyl carbon, substituting Br for the nucleophilic moiety of the amino acid.

[0029] Unless otherwise specified, the peptides disclosed herein have N-terminal acetylation and C-terminal amidation.

[0030] The names "A1M-035" and "RMC-035" are used interchangeably to refer to the same protein.

[0031] As used herein, "oxidative stress" refers to the imbalance between the production and expression of excessive levels of molecular oxygen or reactive oxygen species (ROS) and the ability of biological systems to readily detoxify these reactive intermediates or repair the resulting damage through endogenous antioxidant systems. Disturbances in the normal redox state of tissues can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA, through mechanisms including lipid peroxidation, protein oxidation, and aggregation and DNA damage. Some reactive oxygen species may even act as messengers through a phenomenon called redox signaling.

[0032] As used herein, the term "hematuria" refers to the presence of red blood cells (RBCs) in urine and is defined as three or more RBCs per high-power field (HPF) in a urine sample by microscopic analysis or a positive dipstick test in a urinalysis. Hematuria can be macroscopic (visible blood) or microscopic (blood detected by urinalysis or urine microscopy). Hematuria can be intermittent or persistent. Other methods for assessing hematuria include renal parameters (e.g., glomerular filtration rate (GFR), serum creatinine, urea nitrogen (BUN)), imaging (ultrasound, CT, MRI), cystoscopy, urine cytology, and / or renal biopsy.

[0033] As used herein, the term "glomerular hematuria" is defined to mean hematuria of glomerular origin.

[0034] As used herein, the term "glomerulonephritis" is defined to mean inflammation of the glomerulus or clusters of glomeruli and / or small blood vessels of the kidney.

[0035] As used herein, the term "acute kidney injury" (AKI), formerly known as "acute renal failure" (ARF), refers to an acute clinical syndrome characterized by a rapid decline in renal function caused by several factors, including reduced renal blood flow, glomerulonephritis, and the use of nephrotoxic antibiotics or anticancer drugs. Oxidative stress and inflammation are the primary pathogenic mediators of AKI. The duration of AKI can last up to 7 days.

[0036] As used herein, the term "acute kidney disease" (AKD) refers to AKI lasting between 7 and 90 days.

[0037] As used herein, the term "chronic kidney disease" (CKD) refers to the presence of kidney damage or an estimated glomerular filtration rate (eGFR) of less than 60 ml / min per 1.73 square meters that persists for 90 days or more. This is a state of progressive loss of kidney function, ultimately necessitating renal replacement therapy (dialysis or transplantation). CKD has its general meaning in the art and is used to classify a number of conditions that affect the kidney, resulting in destruction of the renal parenchyma and loss of functional nephrons or glomeruli. Examples of causes of CKD include, but are not limited to, cardiovascular disease, hypertension, diabetes, glomerulonephritis, polycystic kidney disease, and patients who have undergone kidney transplants.

[0038] Polypeptide fragments and variants thereof The present invention relates to novel polypeptides that are fragments of A1M or variants thereof. The present disclosure aims to provide A1M fragments that have A1M activity. A1M and its variants, such as the disclosed RMC-035, are effective treatments for diseases and disorders associated with heme and oxidative stress. A1M therapeutic mechanisms include antioxidant activity, heme binding, reductase activity, and mitochondrial protection. The following residues have been reported to be important for A1M function: Y22, C34, K69, K92, K118, K130, Y132, L180, I181, P182, and R183. However, the present inventors have discovered that some A1M fragments that do not contain most of these residues retain A1M activity.

[0039] The present inventors have surprisingly discovered that certain peptide fragments of A1M maintain A1M activity. Specifically, the present inventors have found A1M activity in A1M fragments designed to include an 11-aa fragment corresponding to residues G31 to M41 and / or a 30-aa fragment corresponding to residues G54 to D83 of SEQ ID NO: 30. Specifically, the fragments may include the variable sites disclosed herein. The present inventors have also discovered that the properties of the A1M fragments can be further improved, e.g., by introducing additional non-natural modifications into the fragments, e.g., to enhance potency and / or stability.

[0040] One embodiment of the present disclosure provides a medicament comprising a polypeptide disclosed herein, hi one embodiment, the medicament consists of a polypeptide disclosed herein.

[0041] One embodiment of the present disclosure provides a medicament comprising a polypeptide consisting of: a. A polypeptide comprising 12 to 80 amino acid residues, wherein the polypeptide has the amino acid sequence GSTCPWLKKIX 1 (SEQ ID NO: 20), where X 1 is M, K, R, or Nle, or b. A polypeptide comprising 30 to 80 amino acid residues, wherein the polypeptide has the amino acid sequence GATEAEISMTSTX 2 WRKGVCEETSGAYEKTD (SEQ ID NO: 21), where X 2 is H, R, or K.

[0042] One embodiment of the present disclosure provides a pharmaceutical agent comprising a polypeptide consisting of 10 to 80 amino acid residues, the polypeptide comprising a fragment of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29; X 3 is selected from D, N, or G, X 4 is selected from M, K, R, or Nle, X 5 is selected from H, R, or K; X 6 is selected from D, N, or E, X 7 is selected from M or Nle, the fragment is at least 10 amino acid residues in length, The fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO:30.

[0043] One embodiment of the present disclosure provides an agent comprising a polypeptide consisting of at least 10 amino acid residues, the polypeptide comprising SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29; X 3 is selected from D, N, or G, X 4 is selected from M, K, R, or Nle, X 5 is selected from H, R, or K; X 6 is selected from D, N, or E, X 7 is selected from M or Nle, the fragment is at least 10 amino acid residues in length, The fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO:30.

[0044] In one embodiment, the polypeptide has the amino acid sequence GSTCPWLKKIX 1 (SEQ ID NO: 20), where X 1 is M, K, R, or Nle.

[0045] In one embodiment, the polypeptide has the amino acid sequence GATEAEISMTSTX 2 WRKGVCEETSGAYEKTD (SEQ ID NO: 21), where X 2 is H, R, or K.

[0046] In one embodiment, the polypeptide comprises the amino acid sequence GSTCPWLKKIM (SEQ ID NO: 22). In one embodiment, the polypeptide comprises the amino acid sequence GSTCPWLKKIK (SEQ ID NO: 23). In one embodiment, the polypeptide comprises the amino acid sequence GSTCPWLKKIR (SEQ ID NO: 24). In a preferred embodiment of the present disclosure, the polypeptide comprises the amino acid sequence GSTCPWLKKIM (SEQ ID NO: 22). In a preferred embodiment of the present disclosure, the polypeptide comprises the amino acid sequence GSTCPWLKKI[Nle] (SEQ ID NO: 145).

[0047] In one embodiment, the polypeptide comprises the amino acid sequence GATEAEISMTSTHWRKGVCEETSGAYEKTD (SEQ ID NO: 25). In one embodiment, the polypeptide comprises the amino acid sequence GATEAEISMTSTRWRKGVCEETSGAYEKTD (SEQ ID NO: 26). In one embodiment, the polypeptide comprises the amino acid sequence GATEAEISMTSTKWRKGVCEETSGAYEKTD (SEQ ID NO: 27).

[0048] In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 28. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 29. The polypeptide sequences corresponding to SEQ ID NO: 28 and SEQ ID NO: 29 comprise variable residues. When a polypeptide fragment disclosed herein comprises two or more of the variable residues of SEQ ID NO: 28 or SEQ ID NO: 29, any combination of variable residues defined herein is considered to be part of the disclosure.

[0049] In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO:28 or SEQ ID NO:29, 3 is selected from the group consisting of D, N, and G. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 28 or SEQ ID NO: 29, 4 is selected from the group consisting of M, K, R, and Nle. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 28 or SEQ ID NO: 29, 5 is selected from the group consisting of H, R, and K. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 28 or SEQ ID NO: 29, 6 is selected from the group consisting of D, N, R, and E. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 28 or SEQ ID NO: 29, 7 In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 28 or SEQ ID NO: 29, and X is selected from the group consisting of M and Nle. 3 is selected from the group consisting of D, N, and G; 4 is selected from the group consisting of M, K, and R; X5 is selected from the group consisting of H, R, and K; X 6 is selected from the group consisting of D, N, and E; X 7 is selected from the group consisting of M and Nle.

[0050] In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 16. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 17. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 18. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 19.

[0051] It is contemplated that certain residues in the polypeptides of the present disclosure may be replaced with chemically similar residues, i.e., such residues may be replaced with other residues according to the principle of "conservative substitution." In one embodiment of the present disclosure, a polypeptide of the present disclosure may differ from a polypeptide defined herein by the presence of one conservative amino acid substitution. In one embodiment of the present disclosure, a polypeptide of the present disclosure may differ from a polypeptide defined herein by the presence of two conservative amino acid substitutions. In one embodiment of the present disclosure, a polypeptide of the present disclosure may differ from a polypeptide defined herein by the presence of three conservative amino acid substitutions. In one embodiment of the present disclosure, a polypeptide of the present disclosure may differ from a polypeptide defined herein by the presence of four conservative amino acid substitutions. In one embodiment of the present disclosure, a polypeptide of the present disclosure may differ from a polypeptide defined herein by the presence of five conservative amino acid substitutions. Those skilled in the art will recognize that individual additions, deletions, insertions, and / or substitutions in an amino acid sequence that alter a single amino acid or a small percentage of amino acids preserve the properties of the amino acid side chain. This is therefore referred to as a "conservative substitution" or "conservative modification," and the modification of the protein results in a protein with similar function. Conservative substitution tables provide functionally similar amino acids and are well known in the art. Examples of amino acid side chain characteristics include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains sharing the following functional groups or characteristics: aliphatic side chains (G, A, V, L, I, P), side chains containing hydroxyl groups (S, T, Y), side chains containing sulfur atoms (C, M), side chains containing carboxylic acids and amides (D, N, E, Q), side chains containing bases (R, K, H), and side chains containing aromatic groups (H, F, Y, W).Furthermore, the following eight groups include amino acids that can be conservatively substituted for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); 8) cysteine ​​(C), methionine (M). However, in preferred embodiments of the present disclosure, the cysteine ​​moieties defined herein, i.e., C34 and C72, are not substituted.

[0052] The relative hydrophobicity characteristics of amino acids are believed to determine the secondary structure of the resulting polypeptide, which in turn defines the polypeptide's interactions with other molecules, such as enzymes, substrates, receptors, antibodies, and antigens. It is known that an amino acid can be substituted for another amino acid with a similar hydrophobicity index and still obtain a functionally equivalent polypeptide. In such changes, substitutions of amino acids with hydrophobicity indices within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0053] In one embodiment of the present disclosure, the polypeptide consists of 12 to 80 amino acid residues. In a further embodiment of the present disclosure, the polypeptide consists of 12 to 80 amino acid residues and has the amino acid sequence GSTCPWLKKIX. 1 (SEQ ID NO: 20), wherein X 1 is as defined herein.

[0054] In one embodiment of the disclosure, the polypeptide consists of 30 to 80 amino acid residues. In a further embodiment of the disclosure, the polypeptide consists of 30 to 80 amino acid residues and has the amino acid sequence GATEAEISMTSTX 2 WRKGVCEETSGAYEKTD (SEQ ID NO: 21), 2 is defined herein.

[0055] In one embodiment of the present disclosure, the polypeptide consists of at least 12 amino acid residues. In one embodiment, the polypeptide consists of at least 13 amino acid residues. In one embodiment, the polypeptide consists of at least 14 amino acid residues. In one embodiment, the polypeptide consists of at least 15 amino acid residues, such as at least 16, such as at least 17, such as at least 18, such as at least 19, such as at least 20, such as at least 21, such as at least 22, such as at least 23, such as at least 24 amino acid residues.

[0056] In one embodiment of the disclosure, the polypeptide consists of 170 or less, such as 160 or less, for example, 150 or less, such as 140 or less, for example, 130 or less, such as 120 or less, for example, 110 or less, such as 100 or less, for example, 90 or less amino acid residues.

[0057] In one embodiment of the disclosure, the polypeptide consists of 80 or fewer amino acid residues, e.g., 79 or fewer, 78 or fewer, 77 or fewer, 76 or fewer, 75 or fewer, 74 or fewer, 73 or fewer, 72 or fewer, 71 or fewer, 70 or fewer, 69 or fewer, 68 or fewer, 67 or fewer, 66 or fewer, 65 or fewer, 64 or fewer, 63 or fewer, 62 or fewer, 61 or fewer, 60 or fewer, 59 or fewer, 58 or fewer, 57 or fewer, 56 or fewer, 55 or fewer, 54 or fewer, 53 or fewer, 52 or fewer, 51 or fewer, 50 or fewer, 49 or fewer, 48 or fewer, 47 or fewer, 46 or fewer, 45 or fewer, 44 or fewer, 43 or fewer, 42 or fewer, 41 or fewer, 40 or fewer, 39 or fewer, 38 or fewer, 37 or fewer, 36 or fewer, 35 or fewer amino acid residues.

[0058] In one embodiment of the present disclosure, the polypeptide consists of 12 to 15 amino acid residues, for example, 12 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, or 75 to 80 amino acid residues.

[0059] In one embodiment of the disclosure, the polypeptide comprises at least 4 consecutive amino acid residues of the reference sequence, such as at least 5, for example at least 6, for example at least 7, for example at least 8, for example at least 9, for example at least 10, for example at least 11, for example at least 12 consecutive amino acid residues.

[0060] Human A1M (SEQ ID NO: 30) is a 183-residue protein. One embodiment of the present disclosure provides a pharmaceutical agent comprising a fragment of human A1M. In one embodiment of the present disclosure, the fragment consists of a sequence corresponding to residues located toward the N-terminus of A1M or a variant thereof. In one embodiment, the fragment consists of a sequence corresponding to positions 1-180 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1-170 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1-160 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1-150 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1-140 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1-130 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1-120 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1-110 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1-100 of SEQ ID NO: 30. In one embodiment, the fragment consists of the sequence corresponding to positions 1 to 90 of SEQ ID NO:30.

[0061] In a preferred embodiment of the present disclosure, the polypeptide comprises or consists of an amino acid sequence selected from the group consisting of: i. DDDDKQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 1), ii. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 2); iii. DDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIM (SEQ ID NO: 3), iv. KGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 4), v. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEETSGAYEK (SEQ ID NO: 6), vi. DDDDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRM (SEQ ID NO: 7), vii. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEET (SEQ ID NO: 8), viii. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRK (SEQ ID NO: 9), ix. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMT (SEQ ID NO: 10), x. GSTCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 11), xi. NLAIGSTCPWLKKIMDR (SEQ ID NO: 12), xii. GEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDG (SEQ ID NO: 13), and xiii. GATEAEISMTSTHWRKGVCEETSGAYEKTD (SEQ ID NO: 14).

[0062] In a preferred embodiment of the present disclosure, the polypeptide comprises or consists of the amino acid sequence GSTCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 11).

[0063] Based on the results outlined in the Examples herein below, it is contemplated that the cysteine ​​residues corresponding to C34 and C72 are particularly important for the function of the polypeptides disclosed herein. This consideration is based on the observations that i) polypeptides that exhibit A1M activity (i.e., cytochrome c reduction assay activity and / or RBC assay activity) contain residues corresponding to C34 and / or C72, and ii) polypeptides that do not contain residues corresponding to C34 and / or C72 do not exhibit A1M activity. Thus, in a preferred embodiment of the present disclosure, the polypeptide contains residues corresponding to C34. In another preferred embodiment of the present disclosure, the polypeptide contains residues corresponding to C72. In another preferred embodiment of the present disclosure, the polypeptide contains residues corresponding to C34 and residues corresponding to C72. One embodiment of the present disclosure provides a polypeptide that i) is a fragment of A1M, ii) contains a free cysteine ​​moiety, and ii) has A1M activity.

[0064] Polypeptides of the present disclosure can be produced using known techniques, for example, by solid phase synthesis or by production in genetically engineered organisms, ie, by recombinant production.

[0065] The present disclosure provides a peptide having a length of 10 to 92 amino acid residues, the peptide being human wild-type alpha-1-microglobulin, A1M, SEQ ID NO: 83: GPVPTPPDNI QVQENFNISR IYGKWYNLAI GSTCPWLKKI MDRMTVSTLV 1-50 a fragment selected from positions 1 to 92 of LGEGATEAEI SMTSTRWRKG VCEETSGAYE KTDTDGKFLY HK; The fragments have a length of 10 to 92 amino acid residues, and the fragments include one or more of Y22, C34, K69, or K92, where these positions refer to positions in human wild-type A1M (SEQ ID NO: 30). The fragments can be selected from positions 1 to 92 of human wild-type A1M, and the fragments have at least 50% identity to the corresponding fragment of human wild-type A1M. The fragments can have a length of 15 to 80 amino acid residues, e.g., 20 to 75 amino acid residues. The fragments can be selected from positions 22 to 92 of human wild-type A1M, SEQ ID NO: 84: YGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK, in which case the fragments have a length of 10 to 71 amino acid residues.

[0066] In one embodiment of the present disclosure, the fragment is selected from one of the following positions of human wild-type A1M: i) positions 22 to 62 (SEQ ID NO: 85); ii) positions 26 to 62 (SEQ ID NO: 86); iii) positions 30 to 62 (SEQ ID NO: 87); iv) positions 31 to 56 (SEQ ID NO: 88); v) positions 26 to 56 (SEQ ID NO: 89); vi) positions 26 to 92 (SEQ ID NO: 90); vii) positions 30 to 92 (SEQ ID NO: 91), or viii) positions 31 to 92 (SEQ ID NO: 92).

[0067] The peptides of the present invention are up to 92 aa (amino acids) in length, particularly up to 80 aa, such as up to 75 aa, up to 71 aa, up to 60 aa, up to 55 aa, up to 50 aa, such as up to 49 aa, up to 48 aa, up to 47 aa, up to 46 aa, up to 45 aa, up to 44 aa, up to 43 aa, up to 42 aa, up to 41 aa, up to 40 aa, up to 39 aa, up to 38 aa, up to 37 aa, up to 36 aa, up to 35 aa, up to 34 aa, up to 33 aa, up to 32 aa, up to 31 aa, up to 30 aa, up to 29 aa, up to 28 aa, or up to 27 aa. Generally, the minimum length of peptides of the present disclosure is 10 aa. Generally, the length of peptides of the present disclosure is 10-80 aa, such as 10-70 aa, 10-60 aa, or 10-50 aa. Peptides referred to in the Examples herein have a length of 30-50 aa, for example 35-50 aa.

[0068] The peptides of the present disclosure have the following human wild-type A1M sequence: i) 22nd to 62nd place, ii) 26th to 62nd place, iii) 30th to 62nd place, iv) 31st~56th place, v) 26th to 56th place, vi) 26th to 92nd place, vii) Ranks 30th to 92nd, or viii) Contains an amino acid sequence having 50% or more sequence identity to one of positions 31 to 92.

[0069] The peptides of the present disclosure have the following human wild-type A1M sequence: i) 22nd to 62nd place, ii) 26th to 62nd place, iii) 30th to 62nd place, iv) 31st~56th place, v) 26th to 56th place, vi) 26th to 92nd place, vii) Ranks 30th to 92nd, or viii) It may contain an amino acid sequence corresponding to one of positions 31 to 92.

[0070] Additionally, peptides according to the present disclosure may comprise an amino acid sequence Z having a length of 1 to 20 amino acid residues at the N-terminus and / or C-terminus. In one embodiment, such an amino acid sequence is a tag, i.e., a polypeptide segment that can be attached to a peptide disclosed herein to provide additional benefits, such as peptide purification, solubility, or detection. As used herein, the term "peptide tag" generally refers to a small peptide fragment that may or may not be directly designed or derived from an isopeptide protein.

[0071] Z is i) one or more lysine, K, residue(s), one or more glutamic acid, E, residue(s), one or more arginine, R, residue(s) and / or one or more aspartic acid, D, residue(s); ii) one or more hydrophobic amino acid residue(s); iii) one or more of histidine, H, residue(s), one or more glutamine, G, residue(s), and / or one or more isoleucine I residue(s), and / or At the N-terminus, Z has the following sequence: i) HHHHHHHHGGGGGIEGR (8H5GIEGR) (SEQ ID NO: 93); ii) HHHHHHHHDDDDK (8H4DK) (SEQ ID NO: 94); iii) HHHHHHDDDDK (6H4DK) (SEQ ID NO: 95), or iv) HHHHHHHH (8H) (SEQ ID NO: 96), and / or At the C-terminus, Z has the following sequence: i) RGIEGGGGGHHHHHHHH (RGIE5G8H) (SEQ ID NO: 97) ii) KDDDDHHHHHHHH(K4D8H) (SEQ ID NO: 98) iii) KDDDDHHHHHH(K4D6H) (SEQ ID NO: 99), or iv) HHHHHHHH(8H) (SEQ ID NO: 96).

[0072] The peptides according to the present disclosure comprise: Y-Π1-K, Y-Π2-C, or C-Π3-K, Π1 is, GKWYNLAI GSTCPWLKKI MDRMTVSTLV has at least 50% sequence identity with SEQ ID NO: 100 (corresponding to positions 23 to 91 of wtA1M), which is LGEGATEAEI SMTSTRWRKG VCEETSGAYE KTDTDGKFLY H; Π2 is, has 50% sequence identity with SEQ ID NO: 101, GKWYNLAI GST (corresponding to positions 23-33 of wtA1M), and Π3 is, It has at least 50% sequence identity with SEQ ID NO: 102, which is LGEGATEAEI SMTSTRWRKG VCEETSGAYE KTDTDGKFLY H (corresponding to positions 35 to 91 of wtA1M).

[0073] In an embodiment of the disclosure, the peptide has at least 50% sequence identity to SEQ ID NO:83.

[0074] As is apparent from the examples herein, the peptides of the present disclosure: i) an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 103, which is SR IYGKWYNLAI GSTCPWLKKI MDRMTVSTLV LGEG (corresponding to positions 19 to 54 of SRI-36, wtA1M); ii) ENFNISR IYGKWYNLAI GSTCPWLKKI MDRMTVSTLV It comprises an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 104, which is LEGATEAE (ENF-46, corresponding to positions 14 to 59 of wtA1M).

[0075] The present disclosure provides peptides having a length of 10 to 92 amino acid residues, including fragments selected from positions 1 to 92 of human wild-type alpha-1-microglobulin (A1M) as defined by SEQ ID NO: 83. In one embodiment of the disclosure, the fragments have a length of 10 to 92 amino acid residues. In one embodiment of the disclosure, the fragments include one or more of Y22, C34, K69, or K92, which positions refer to positions in human wild-type A1M (SEQ ID NO: 30). In one embodiment of the disclosure, the fragments may be selected from positions 1 to 92 of human wild-type A1M, and the fragments have at least 50% identity to the corresponding fragment of human wild-type A1M. In one embodiment of the disclosure, the fragments have a length of 15 to 80 amino acid residues, e.g., 20 to 75 amino acid residues.

[0076] In one embodiment of the present disclosure, the fragment is the sequence defined in SEQ ID NO: 84, which corresponds to positions 22 to 92 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is selected from the sequence defined in SEQ ID NO: 84, and the fragment has a length of 10 to 71 amino acid residues.

[0077] In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 85, which corresponds to positions 22-62 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 86, which corresponds to positions 26-62 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 87, which corresponds to positions 30-62 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 88, which corresponds to positions 31-56 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 89, which corresponds to positions 26-56 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 90, which corresponds to positions 26-92 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 91, which corresponds to positions 30-92 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 92, which corresponds to positions 31-92 of human wild-type A1M.

[0078] In one embodiment of the present disclosure, the peptide is at most 92 aa (amino acids) in length, in particular at most 80 aa, for example at most 75 aa, at most 71 aa, at most 60 aa, at most 55 aa, at most 50 aa, for example at most 49 aa, at most 48 aa, at most 47 aa, at most 46 aa, at most 45 aa, at most 44 aa, at most 43 aa, at most 42 aa, at most 41 aa, at most 40 aa, at most 39 aa, at most 38 aa, at most 37 aa, at most 36 aa, at most 35 aa, at most 34 aa, at most 33 aa, at most 32 aa, at most 31 aa, at most 30 aa, at most 29 aa, at most 28 aa, or at most 27 aa. In one embodiment of the present disclosure, the minimum length of the peptide is 10 aa. In one embodiment of the present disclosure, the peptide length is 10-80 aa, for example, 10-70 aa, 10-60 aa, or 10-50 aa.

[0079] The peptides referred to in the examples herein have a length of 30 to 50 aa, for example 35 to 50 aa.

[0080] In one embodiment of the present disclosure, the fragment has at least 50% sequence identity to the sequence defined in SEQ ID NO: 85. In one embodiment of the present disclosure, the fragment has at least 50% sequence identity to the sequence defined in SEQ ID NO: 86. In one embodiment of the present disclosure, the fragment has at least 50% sequence identity to the sequence defined in SEQ ID NO: 87. In one embodiment of the present disclosure, the fragment has at least 50% sequence identity to the sequence defined in SEQ ID NO: 88. In one embodiment of the present disclosure, the fragment has at least 50% sequence identity to the sequence defined in SEQ ID NO: 89. In one embodiment of the present disclosure, the fragment has at least 50% sequence identity to the sequence defined in SEQ ID NO: 90. In one embodiment of the present disclosure, the fragment has at least 50% sequence identity to the sequence defined in SEQ ID NO: 91. In one embodiment of the present disclosure, the fragment has at least 50% sequence identity to the sequence defined in SEQ ID NO: 92. In one embodiment, the sequence identity threshold is 60%, 70%, 80%, 85%, 90%, or 95%. In one embodiment, the polypeptide does not differ from the reference sequence within the heme-binding site, but there may be mutations relative to the reference sequence outside the heme-binding site (Meining and Skerra 2012). In one embodiment, the polypeptide has the sequence GSTCPWLKKIX 1 (SEQ ID NO: 20). In one embodiment, the polypeptide does not differ from the reference sequence spanning the sequence GATEAEISMTSTX 2 (SEQ ID NO: 21). In one embodiment, the polypeptide contains a cysteine ​​residue corresponding to position 34 of SEQ ID NO: 30. In one embodiment, the polypeptide contains a cysteine ​​residue corresponding to position 72 of SEQ ID NO: 30. In one embodiment, the polypeptide differs from the reference sequence only by conservative mutations.

[0081] In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 100, which corresponds to positions 23-91 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 101, which corresponds to positions 23-33 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 102, which corresponds to positions 35-91 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is SRI-36, which corresponds to positions 19-54 of human wild-type A1M, which is defined as SEQ ID NO: 103. In one embodiment of the present disclosure, the fragment is ENF-46, which corresponds to positions 14-59 of human wild-type A1M, which is defined as SEQ ID NO: 104.

[0082] In one embodiment of the present disclosure, the peptide has at least 50% sequence identity with the sequence defined in SEQ ID NO: 83. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity with the sequence defined in SEQ ID NO: 103. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity with the sequence defined in SEQ ID NO: 104. In one embodiment, the sequence identity threshold is 60%, 70%, 80%, 85%, 90%, or 95%. In one embodiment, the polypeptide does not differ from the reference sequence within the heme-binding site, but there may be mutations relative to the reference sequence outside the heme-binding site (Meining and Skerra 2012). In one embodiment, the polypeptide has the sequence GSTCPWLKKIX. 1 (SEQ ID NO: 20). In one embodiment, the polypeptide does not differ from the reference sequence spanning the sequence GATEAEISMTSTX 2 (SEQ ID NO: 21). In one embodiment, the polypeptide contains a cysteine ​​residue corresponding to position 34 of SEQ ID NO: 30. In one embodiment, the polypeptide contains a cysteine ​​residue corresponding to position 72 of SEQ ID NO: 30. In one embodiment, the polypeptide differs from the reference sequence only by conservative mutations.

[0083] In one embodiment of the disclosure, the peptide has the sequence Y-Π1-K, where Π1 has at least 50% sequence identity to the sequence defined in SEQ ID NO: 100. In one embodiment of the disclosure, the peptide has the sequence Y-Π2-C, where Π2 has at least 50% sequence identity to the sequence defined in SEQ ID NO: 101. In one embodiment of the disclosure, the peptide has the sequence C-Π3-K, where Π3 has at least 50% sequence identity to the sequence defined in SEQ ID NO: 102.

[0084] The present disclosure provides peptides having a length of 10 to 100 amino acid residues, the peptides comprising a fragment selected from positions 1 to 183 of human wild-type alpha-1-microglobulin, A1M, SEQ ID NO: 30, the fragment having a length of 10 to 100 amino acid residues, and the fragment comprising one or more of Y22, C34, K69, K92, K118, H122, Y132, L180, I181, P182 and R183.

[0085] The fragment may be selected from positions 1 to 183 of human wild-type A1M, wherein the fragment has at least 50% identity to the corresponding fragment of human wild-type A1M.

[0086] Fragments typically have a length of 10 to 80 amino acid residues, such as 10 to 70, 15 to 66, or 20 to 75 amino acid residues.

[0087] The fragments selected are typically: i) one or more of Y22, C34, K69, K92, K118, H122, and Y132; ii) one or more of K92, K118, H122, Y132, L180, I181, P182, and R183; or iii) one or more of L180, I181, P182 and R183.

[0088] Typically, the fragments are i) positions 22 to 183 of human wild-type A1M, SEQ ID NO: 105; ii) positions 22 to 160 of human wild-type A1M, SEQ ID NO: 106; iii) positions 51 to 155 of human wild-type A1M, SEQ ID NO: 107; iv) positions 59 to 135 of human wild-type A1M, SEQ ID NO: 108; v) positions 92 to 183 of human wild-type A1M, SEQ ID NO: 109, or vi) positions 92 to 135 of human wild-type A1M, SEQ ID NO: 110.

[0089] The peptides of the present invention are at most 100 aa (amino acids) in length, particularly at most 80 aa, such as at most 75 aa, at most 71 aa, at most 60 aa, at most 55 aa, at most 50 aa, such as at most 49 aa, at most 48 aa, at most 47 aa, at most 46 aa, at most 45 aa, at most 44 aa, at most 43 aa, at most 42 aa, at most 41 aa, at most 40 aa, at most 39 aa, at most 38 aa, at most 37 aa, at most 36 aa, at most 35 aa, at most 34 aa, at most 33 aa, at most 32 aa, at most 31 aa, at most 30 aa, at most 29 aa, at most 28 aa, or at most 27 aa. Generally, the minimum length of peptides of the present disclosure is 10 aa. Generally, the length of peptides of the present disclosure is 10-80 aa, such as 10-70 aa, 10-60 aa, or 10-50 aa. Peptides referred to in the Examples herein have a length of 30-50 aa, for example 35-50 aa.

[0090] The peptides of the present disclosure may have an amino acid sequence that is identical to a portion of the amino acid sequence of human wild-type A1M (SEQ ID NO: 30), i.e., no changes other than length. Alternatively, the peptides of the present disclosure may have 50% or more, e.g., 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with a portion of the amino acid sequence of human wild-type A1M, which includes both changes in the length of the amino acid sequence and changes in specific amino acids. Alternatively, the peptides of the present disclosure may have 50% or more, e.g., 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity with a portion of the amino acid sequence of human wild-type A1M, which includes both changes in the length of the amino acid sequence and changes in specific amino acids.

[0091] When a particular amino acid sequence is referred to herein, it is understood that a peptide based on the disclosure (or where the sequence relates only to a portion of a peptide of the disclosure) may have 50% or more, e.g., 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the particular amino acid sequence. This applies, inter alia, to SEQ ID NOS: 30 and 103-116.

[0092] In certain embodiments, when a particular amino acid sequence is referred to, it is understood that a peptide based on the disclosure (or where the sequence is related only to a portion of a peptide of the disclosure) may have 50% or more, e.g., 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the particular amino acid sequence. This applies, among others, to SEQ ID NOS: 30 and 103-116.

[0093] Peptides according to the present disclosure may be used to identify human wild-type A1M in the following ranges: i) positions 22 to 135 (SEQ ID NO: 110); ii) positions 26 to 135 (SEQ ID NO: 111); iii) positions 30 to 135 (SEQ ID NO: 112); iv) positions 31 to 135 (SEQ ID NO: 113); v) positions 61 to 135 (SEQ ID NO: 114); vi) positions 66 to 135 (SEQ ID NO: 115); vii) positions 87 to 135 (SEQ ID NO: 116); viii) a fragment selected from one of positions 92 to 135 (SEQ ID NO: 117).

[0094] Peptides according to the present disclosure may be derived from the following human wild-type A1M sequence: i) 22nd to 135th place, ii) 26th to 135th place, iii) 30th to 135th place, iv) 31st~135th place, v) 59th to 135th place, vi) 66th to 135th place, vii) 87th to 135th place, viii) It may contain an amino acid sequence having 50% or more sequence identity with one of positions 92 to 135.

[0095] Peptides according to the present disclosure may be derived from the following human wild-type A1M sequence: i) 59th to 135th place, ii) 66th to 135th place, iii) 87th to 135th place, iv) an amino acid sequence selected from one of positions 92 to 135.

[0096] Furthermore, the peptide according to the present disclosure may comprise, at the N-terminus and / or C-terminus, an amino acid sequence Z having a length of 1 to 20 amino acid residues.

[0097] Z is iv) one or more lysine, K, residue(s), one or more glutamic acid, E, residue(s), one or more arginine, R, residue(s) and / or one or more aspartic acid, D, residue(s); v) one or more hydrophobic amino acid residue(s); vi) one or more of histidine, H, residue(s), one or more glutamine, G, residue(s), and / or one or more isoleucine I residue(s), and / or At the N-terminus, Z has the following sequence: v) HHHHHHHHGGGGGIEGR (8H5GIEGR) (SEQ ID NO: 93); vi) HHHHHHHHDDDDK(8H4DK) (SEQ ID NO: 94); vii) HHHHHHDDDDK(6H4DK) (SEQ ID NO: 95), or viii) HHHHHHHH(8H) (SEQ ID NO: 96), and / or At the C-terminus, Z has the following sequence: v) RGIEGGGGGHHHHHHHH (RGIE5G8H) (SEQ ID NO: 97) vi) KDDDDHHHHHHHH(K4D8H) (SEQ ID NO: 98) vii) KDDDDHHHHHH(K4D6H) (SEQ ID NO: 99), or viii) HHHHHHHH(8H) (SEQ ID NO: 96).

[0098] As is apparent from the examples herein, the peptides of the present disclosure: [ka]

[0099] In one embodiment of the present disclosure, the peptide has a length of 10 to 100 amino acid residues and comprises a fragment selected from positions 1 to 183 of human wild-type A1M as defined in SEQ ID NO: 30. In one embodiment of the present disclosure, the fragment has a length of 10 to 100 amino acid residues. In one embodiment of the present disclosure, the fragment comprises one or more of Y22, C34, K69, K92, K118, H122, Y132, L180, I181, P182, and R183.

[0100] In one embodiment of the present disclosure, the fragment has at least 50% sequence identity to a fragment selected from positions 1 to 183 of human wild-type A1M.

[0101] In one embodiment of the present disclosure, fragments typically have a length of 10 to 80 amino acid residues, such as, for example, 10 to 70, 15 to 66, or 20 to 75 amino acid residues.

[0102] In one embodiment of the present disclosure, the fragment typically includes one or more of Y22, C34, K69, K92, K118, H122, and Y132.

[0103] In one embodiment of the present disclosure, the fragment typically comprises one or more of K92, K118, H122, Y132, L180, I181, P182 and R183.

[0104] In one embodiment of the present disclosure, the fragment comprises one or more of L180, I181, P182 and R183.

[0105] In one embodiment of the present disclosure, the fragment is the sequence defined by SEQ ID NO: 105, which corresponds to positions 22-183 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is the sequence defined by SEQ ID NO: 106, which corresponds to positions 22-160 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is the sequence defined by SEQ ID NO: 107, which corresponds to positions 51-155 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is the sequence defined by SEQ ID NO: 108, which corresponds to positions 59-135 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is the sequence defined by SEQ ID NO: 109, which corresponds to positions 92-183 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is the sequence defined by SEQ ID NO: 110, which corresponds to positions 22-135 of human wild-type A1M.

[0106] In one embodiment of the disclosure, the peptide is at most 100 amino acid residues in length, in particular at most 80 residues, e.g., at most 75 residues, at most 71 residues, at most 60 residues, at most 55 residues, at most 50 residues, e.g., at most 49 residues, at most 48 residues, at most 47 residues, at most 46 residues, at most 45 residues, at most 43 residues, at most 41 residues, at most 40 residues, at most 39 residues, at most 38 residues, at most 37 residues, at most 36 residues, at most 35 residues, at most 34 residues, at most 33 residues, at most 32 residues, at most 31 residues, at most 30 residues, at most 29 residues, at most 28 residues, or at most 27 residues. In one embodiment of the disclosure, the minimum length of the peptide is 10 residues. In one embodiment of the present disclosure, the peptide length is 10 to 80 residues, for example, 10 to 70 residues, 10 to 60 residues, or 10 to 50 residues. The peptides referred to in the Examples herein have a length of 30 to 50 residues, such as 35 to 50 residues.

[0107] In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 111, which corresponds to positions 26-135 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 112, which corresponds to positions 30-135 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 113, which corresponds to positions 31-135 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 114, which corresponds to positions 61-135 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 115, which corresponds to positions 66-135 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 116, which corresponds to positions 87-135 of human wild-type A1M. In one embodiment of the present disclosure, the fragment is defined as SEQ ID NO: 117, which corresponds to positions 92-135 of human wild-type A1M. In one embodiment of the disclosure, the fragment is SRI-36 as defined in SEQ ID NO: 103, which corresponds to positions 19 to 54 of human wild-type A1M. In one embodiment of the disclosure, the fragment is ENF-46 as defined in SEQ ID NO: 104, which corresponds to positions 14 to 59 of human wild-type A1M.

[0108] In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 30. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 105. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 106. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 107. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 108. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 109. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 110. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 111. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 112. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 113. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 114. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 115. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 116. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 117. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 103. In one embodiment of the present disclosure, the peptide has at least 50% sequence identity to the sequence defined in SEQ ID NO: 104.

[0109] In one embodiment, a polypeptide of the present disclosure is an isolated polypeptide. The term "isolated" in this context is understood to mean that the polypeptide has been removed from or is not associated with some or all of the other components with which it is found in its natural state. For example, an "isolated" polypeptide may be removed from other amino acid sequences within a larger polypeptide sequence, or may be removed from natural components such as unrelated proteins. For clarity, an "isolated" polypeptide also includes polypeptides that are not derived from nature but are prepared de novo, for example, by chemical synthesis and / or recombinant methods. As described herein, the isolated polypeptides described herein may be included as components of a longer polypeptide or a fusion polypeptide.

[0110] An agent of the present disclosure can include a second polypeptide, e.g., a second polypeptide that is not A1M but is derived from A1M or an A1M variant. Thus, one embodiment provides an agent comprising a polypeptide disclosed herein and a second polypeptide, e.g., a second polypeptide that is not A1M but is derived from A1M or an A1M variant. "A1M, A1M-derived, or A1M variant" refers to a polypeptide having the sequence of A1M, an A1M variant having at least 60%, 70%, 80%, or 90% sequence identity to A1M, or a fragment of A1M, e.g., a fragment having a length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues. In one embodiment, the polypeptide does not have the sequence of SEQ ID NO:137.

[0111] qualification The polypeptides disclosed herein may optionally contain one or more modifications. As used herein, "modification" includes any functionalization of the polypeptide, for example, by forming a covalent bond between any atom in the polypeptide and an atom in another compound, by forming a covalent bond between two atoms in the polypeptide itself, or by forming a covalent bond with another polypeptide, whether similar or different.

[0112] In one embodiment, the polypeptides disclosed herein comprise one or more further modifications. In one embodiment, one or more amino acid residues of the polypeptide are optionally modified.

[0113] The modification may be due to other compounds derived from other peptides (e.g., comprising or consisting of one or more amino acids), or the modification may be due to other moieties that are not based on amino acids. In one embodiment of the present disclosure, the additional modification is a non-peptide modification. In one embodiment of the present disclosure, the additional modification is a peptide-based (i.e., amino acid-based) modification.

[0114] In one embodiment, the modifications disclosed herein are achieved by attachment to the disclosed polypeptides via a linking moiety.

[0115] In one embodiment, the polypeptide is provided as a TFA salt or a chloride salt.

[0116] replacement In one embodiment, the further modification is a substitution of one or more atoms or one or more moieties.

[0117] In one embodiment, the polypeptide is further modified at the N-terminus. In one embodiment, the polypeptide is further modified at the C-terminus. In one embodiment, the polypeptide is further modified at the side chain. In one embodiment, the polypeptide is modified at one position, for example, at two positions, three positions, four, five, six, seven, eight, nine, or ten positions.

[0118] Synthetically produced polypeptides, such as those synthesized on a solid support, may contain one or more modifications compared to the original amino acid sequence when released from the support. Such modifications may be due to specific protecting groups used to facilitate the synthesis of the polypeptide or due to specific linkers used to fix the polypeptide to the solid support. For example, some linkers release the synthesized polypeptide as a C-terminal amide. In one embodiment, the polypeptide is acetylated at the N-terminus. In one embodiment, the polypeptide is amidated at the C-terminus.

[0119] Unnatural amino acids can be used to modify the properties of a polypeptide. Thus, in one embodiment, a polypeptide comprises a substitution with an unnatural amino acid. In one embodiment, a polypeptide comprises an insertion of one or more unnatural amino acids into an original amino acid sequence. In one embodiment of the present disclosure, a polypeptide comprises a substitution with a D-amino acid. In one embodiment, a polypeptide comprises an insertion of a D-amino acid. In certain embodiments of the present disclosure, a residue in a polypeptide (e.g., an L-amino acid) is substituted with the same amino acid in the D-configuration. "The same amino acid in the D-configuration" means that the original amino acid is considered to be the same, but the two amino acids differ in the stereochemical configuration at the alpha carbon. As an example, an L-arginine residue can be replaced with a D-arginine residue. In one embodiment, a polypeptide comprises one, two, three, four, five, six, seven, eight, nine, ten, or more substitutions with D-amino acids.

[0120] In certain embodiments, the polypeptide is i. NLAIGSTCP[D-Trp]LKKIMDR (SEQ ID NO: 66), ii. NLAIGSTCPW[D-Leu]KKIMDR (SEQ ID NO: 67), iii. NLAIGSTCPWLK[D-Lys]IMDR (SEQ ID NO: 68); iv. GSTCPWLK[D-Lys]IMDRMTVSTLVLGEG (SEQ ID NO: 69); v. GSTCPWLKKIMD[D-Arg]MTVSTLVLGEG (SEQ ID NO: 70), vi. GSTCPWLKKIMDRMTVSTLV[D-Leu]GEG (SEQ ID NO: 71), vii. GSTCPWLKKIMDRMTVSTL[D-Asp]LGEG (SEQ ID NO: 72), viii. G[D-Ser]STCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 81), and ix. A sequence selected from the group consisting of: [D-Asn]LAIGSTCPWLKKIMDR (SEQ ID NO: 82); or a sequence having at least 70% sequence identity thereto, such as at least 75%, at least 80%, at least 85%, at least 90%, such as at least 95% sequence identity thereto.

[0121] In one embodiment, the polypeptide is i. NLAIGSTCP[D-Trp]LKKIMDR (SEQ ID NO: 66), ii. NLAIGSTCPW[D-Leu]KKIMDR (SEQ ID NO: 67); iii. NLAIGSTCPWLK[D-Lys]IMDR (SEQ ID NO: 68); iv. GSTCPWLK[D-Lys]IMDRMTVSTLVLGEG (SEQ ID NO: 69); v. GSTCPWLKKIMD[D-Arg]MTVSTLVLGEG (SEQ ID NO: 70), vi. GSTCPWLKKIMDRMTVSTLV[D-Leu]GEG (SEQ ID NO: 71), vii. GSTCPWLKKIMDRMTVSTL[D-Asp]LGEG (SEQ ID NO: 72), viii. G[D-Ser]STCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 81), and ix. [D-Asn]LAIGSTCPWLKKIMDR (SEQ ID NO: 82).

[0122] In one embodiment, one, two, or more methionine residues are substituted with norleucine (NLe or Nle). In a particular embodiment, the methionine residue corresponding to M41 of A1M is substituted with norleucine. In one embodiment, the methionine residue corresponding to M44 of A1M is substituted with norleucine. In one embodiment, both methionine residues corresponding to M41 and M44 of A1M are substituted with norleucine.

[0123] In one embodiment, the polypeptide is i. GSTCPWLKKI[Nle]DR[Nle]TVSTLVLGEG (SEQ ID NO: 139), ii. GSTCPWLKKI[Nle]DR[Nle]TVSTL[D-Asp]LGEG (SEQ ID NO: 140); iii. GSTCPWLKKI[Nle]DR[Nle]TVSTLVLGEG[D-Ala][D-Thr][D-Glu] (SEQ ID NO: 141), iv. GSTCPWLKKI[Nle]DR[Nle]TKSTLVLGEG (SEQ ID NO: 142), v. EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TVSTLVLGE (SEQ ID NO: 143), and vi. GSTCPWLKKI[Nle]DR[Nle]TVSTL[D-Asp]LGEG (SEQ ID NO: 144).

[0124] In one embodiment, the polypeptide is i. GSTCPWLKKIMDRMTKSTL[dD]LGEG (SEQ ID NO: 146) ii. EEKYNLAIGSTCPWLKKIMDRMTESTLDLGE (SEQ ID NO: 147) iii. EEKYNLAKGSTCPWLKKIMDRMTESTLDLGE (SEQ ID NO: 148) iv. GSTCPWLKKI[NLe]DR[NLe]TKSTL[dD]LGEG (SEQ ID NO: 149) v. EEKYNLAIGSTCPWLKKI[NLe]DR[NLe]TESTLDLGE (SEQ ID NO: 150) vi. EEKYNLAKGSTCPWLKKI[NLe]DR[NLe]TESTLDLGE (SEQ ID NO: 151) vii. GSTCPWLKKI[NLe]DR[NLe]TK[N-Meth S]TL[D]LGEG (SEQ ID NO: 152) viii. EEKYNLAIGSTCPWLKKI[NLe]DR[NLe]TE[N-Meth S]TLDLGE (SEQ ID NO: 153) ix. EEKYNLAKGSTCPWLKKI[NLe]DR[NLe]TE[N-Meth S]TLDLGE (SEQ ID NO: 154) x. Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-NH2 (SEQ ID NO: 155) xi. Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TESTLVLGE-NH2 (SEQ ID NO: 161).

[0125] PEGylation In one embodiment of the present disclosure, the polypeptide is PEGylated. PEGylation refers to -(CH2CH2O)n-, -(CH2OCH2)n-, or -(OCH2CH2) n means functionalization with a chemical moiety having the structure -.

[0126] In one embodiment, PEGylation is at the N-terminus, C-terminus, and / or side chains, hi one embodiment, PEGylation is at the C-terminus.

[0127] In one embodiment, the PEGylation is with PEG having a molecular weight of 60 kDa or less, such as with PEG having a molecular weight of 50 kDa or less, such as 40 kDa or less, for example, 30 kDa or less, such as 20 kDa or less, for example, 15 kDa or less, for example, 10 kDa or less, for example, 5 kDa or less. In one embodiment, the PEGylation is with any one of PEG1 to PEG50. In one embodiment, the PEGylation is with any one of PEG1 to PEG30. In one embodiment, the PEGylation is with any one of PEG2 to PEG20. In one embodiment, the PEGylation is with PEG2, PEG5, PEG10, or PEG20.

[0128] In one embodiment, the PEGylated polypeptide is i. GSTCPWLKKIMDRMTVSTLVLGEG-PEG2-CONH2 (SEQ ID NO: 34), ii. GSTCPWLKKIMDRMTVSTLDLGEG-PEG2-CONH2 (SEQ ID NO: 35), iii. Ac-NLAIGSTCPWLKKIMDR-CONH2-PEG2 (SEQ ID NO: 39); iv. Ac-DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT-PEG2 (SEQ ID NO: 40), v. NLAIGSTCPWLKKIMDR-PEG5 (SEQ ID NO: 60), vi. NLAIGSTCPWLKKIMDR-PEG10 (SEQ ID NO: 61), vii. GSTCPWLKKIMDRMTVSTLVLGEG-PEG5 (SEQ ID NO: 62); viii. GSTCPWLKKIMDRMTVSTLVLGEG-PEG10 (SEQ ID NO: 63), ix. NLAIGSTCPWLKKIMDR-PEG20 (SEQ ID NO: 64), and x. a structure selected from the group consisting of: GSTCPWLKKIMDRMTVSTLVLGEG-PEG20 (SEQ ID NO: 65); or a sequence having at least 70% sequence identity thereto, such as at least 75%, at least 80%, at least 85%, at least 90%, such as at least 95% sequence identity thereto.

[0129] In one embodiment of the disclosure, the PEGylated polypeptide of the disclosure is i. GSTCPWLKKIMDRMTVSTLVLGEG-PEG2-CONH2 (SEQ ID NO: 34) ii. GSTCPWLKKIMDRMTVSTLDLGEG-PEG2-CONH2 (SEQ ID NO: 35) iii. Ac-NLAIGSTCPWLKKIMDR-CONH2-PEG2 (SEQ ID NO: 39) iv. Ac-DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT-PEG2 (SEQ ID NO: 40) v. NLAIGSTCPWLKKIMDR-PEG5 (SEQ ID NO: 60) vi. NLAIGSTCPWLKKIMDR-PEG10 (SEQ ID NO: 61) vii. GSTCPWLKKIMDRMTVSTLVLGEG-PEG5 (SEQ ID NO: 62) viii. GSTCPWLKKIMDRMTVSTLVLGEG-PEG10 (SEQ ID NO: 63) ix. NLAIGSTCPWLKKIMDR-PEG20 (SEQ ID NO: 64), and x. GSTCPWLKKIMDRMTVSTLVLGEG-PEG20 (SEQ ID NO: 65).

[0130] Functionalization of the albumin binding domain The polypeptides of the present disclosure may be further modified with other polypeptides, proteins, or fragments thereof, hi one embodiment, the polypeptide further comprises an albumin binding domain or a fragment of an albumin binding domain.

[0131] In one embodiment, the albumin binding domain consists of or comprises the amino acid sequence of SEQ ID NO:118.

[0132] In one embodiment, the albumin-binding domain fragment consists of 10 to 50 amino acid residues. In one embodiment, the albumin-binding domain fragment consists of or comprises the amino acid sequence of SEQ ID NO:119.

[0133] In one embodiment of the present disclosure, the albumin binding domain or fragment thereof is attached to the N-terminus of the polypeptide, the C-terminus of the polypeptide, or a side chain of the peptide.

[0134] In one embodiment of the present disclosure, the albumin binding domain or fragment thereof is attached to the polypeptide via a linking moiety. In one embodiment, the linker is a PEG linker. In one embodiment, the linking moiety is a peptide linker. In one embodiment, the linking moiety is i. GGGGGSAS (SEQ ID NO: 121), and ii. GGGGSGGGGSGGGGSAS (SEQ ID NO: 122).

[0135] In one embodiment, the albumin binding domain fragment consists of or comprises the amino acid sequence SDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 119).

[0136] In one embodiment, the polypeptide is i. GSTCPWLKKIMDRMTVSTLVLGEGSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 36), ii. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 37), iii. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSGGGGSGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 38), iv. NLAIGSTCPWLKKIMDRGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 41), v. NLAIGSTCPWLKKIMDRGGGGSGGGGSGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 42), vi. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 43), and vii. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSGGGGSGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 44); or a sequence having at least 70% sequence identity thereto, such as at least 75%, at least 80%, at least 85%, at least 90%, for example at least 95% sequence identity thereto.

[0137] In one embodiment, the polypeptide is i. GSTCPWLKKIMDRMTVSTLVLGEGSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 36), ii. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 37), iii. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSGGGGSGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 38), iv. NLAIGSTCPWLKKIMDRGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 41), v. NLAIGSTCPWLKKIMDRGGGGSGGGGSGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 42), vi. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 43), and vii. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSGGGGSGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 44).

[0138] fatty acid bond The polypeptides of the present disclosure may be further modified by conjugation to a fatty acid, for example, to improve their albumin binding ability. In one embodiment, the polypeptide further comprises a fatty acid. In one embodiment, the polypeptide is conjugated to a fatty acid.

[0139] In one embodiment, the fatty acid is attached to the N-terminus of the polypeptide, the C-terminus of the polypeptide, or a side chain of the peptide.

[0140] In one embodiment, the fatty acid is linked to the polypeptide via a linking moiety. In one embodiment, the linking moiety is γGlu-2×OEG (OEG: 8-amino-3,6-dioxaoctanoic acid, γGlu: gamma-glutamic acid). In one embodiment, the fatty acid is a C20 fatty diacid moiety [C20DA], such as octadecanedioic acid. In one embodiment, the polypeptide is linked to γGlu-2×OEG-C20 diacid. Other fatty acids / linkers can be conjugated to the peptide to improve stability. See Zhao et al., 2022; Xu et al., 2023.

[0141] In one embodiment, the polypeptide is i. Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]T[K([C20DA]-[yGlu]-[OEG]-[OEG]-)]STLVLGE-NH2 (SEQ ID NO: 156 and SEQ ID NO: 164) ii. [C20DA]-[yGlu]-[OEG]-[OEG]-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-NH2 (SEQ ID NO: 157) iii. Ac-GSTCPWLK[K([C20DA]-[yGlu]-[OEG]-[OEG]-)]I[NLe]DR[NLe]TK[NMeSer]TL[D]LGEG-NH2 (SEQ ID NO: 158 and SEQ ID NO: 165) iv. [C20DA]-[yGlu]-[OEG]-[OEG]-GSTCPWLKKI[NLe]DR[NLe]TK[NMeSer]TL[D]LGEG-NH2 (SEQ ID NO: 159).

[0142] Amino acid modifications The polypeptides disclosed herein may contain one or more modifications of one or more amino acid residues within the polypeptide. In one embodiment, the polypeptide contains a modification of one or more amino acid residues.

[0143] In one embodiment, the modification is selected from methylation, such as N-methylation, dimethylation, such as N-dimethylation or C-dimethylation, side chain methylation, oxidation, such as oxidation of cysteine ​​or methionine, and dimerization, such as dimerization between two cysteine ​​moieties.

[0144] In one embodiment, the modification is carbamidomethylation of cysteine ​​(cysteine ​​CAM). In one embodiment, the polypeptide comprises or consists of the amino acid sequence GSTC(Cam)PWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 48), where Cam represents carbamidomethylation.

[0145] In one embodiment, the polypeptide comprises homocysteine.

[0146] In one embodiment, the cysteine ​​moieties of the polypeptide are substituted (replaced) with homocysteine. In one embodiment, the polypeptide has the amino acid sequence GST{h C}PWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 49), wherein hC is homocysteine.

[0147] In one embodiment, the polypeptide comprises a penicillamine residue. In one embodiment, a cysteine ​​moiety of the polypeptide is substituted (replaced) with penicillamine. In one embodiment, the polypeptide comprises or consists of the amino acid sequence GST{Penc}PWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 50), where Penc is a penicillamine residue.

[0148] In one embodiment, the polypeptide comprises one or more methionine sulfoxides. In one embodiment, the polypeptide comprises or consists of the amino acid sequence GSTCPWLKKIM(ox)DRM(ox)TVSTLVLGEG (SEQ ID NO: 51), where M(ox) represents methionine sulfoxide.

[0149] The polypeptides of the present disclosure can be alkylated, e.g., methylated. In one embodiment of the present disclosure, the polypeptide is methylated or dimethylated. In one embodiment, the polypeptide is i. arginine side chain methylation, ii. lysine methylation, iii. lysine dimethylation, iv. N-methylation of threonine, and v. N-methylation of isoleucine.

[0150] In one embodiment of the disclosure, the polypeptide comprises: i. GSTCPWLKKIMD[Arg methylated side chain]MTVSTLVLGEG (SEQ ID NO: 73), ii. GSTCPWLK[Lys dimethylated]IMDRMTVSTLVLGEG (SEQ ID NO: 74); iii. GSTCPWLKKIMDRM[Thr N-methylated]VSTLVLGEG (SEQ ID NO: 75); iv. GSTCPWLKK[Ile N-methylated]MDRMTVSTLVLGEG (SEQ ID NO: 76), v. NLAIGSTCPWLK[Lys dimethylated]IMDR (SEQ ID NO: 77), vi. NLAIGS[Thr N-methylated]CPWLKKIMDR (SEQ ID NO: 78), vii. NLA[Ile N-methylated]GSTCPWLKKIMDR (SEQ ID NO: 79), and viii. NLAIGSTCPWLKKIMD [Arg methylated side chain] (SEQ ID NO: 80); or comprising or consisting of a sequence having at least 70% sequence identity thereto, such as at least 75%, at least 80%, at least 85%, at least 90%, for example at least 95% sequence identity thereto.

[0151] Cyclic Polypeptides The polypeptides of the present disclosure may be cyclized. Cyclization can be achieved by any known technique, i.e., chemical modification of the polypeptide. Cyclization can be achieved, for example, by reaction with a compound that links two portions of the polypeptide. Cyclization can also be achieved by reaction with a compound that causes a reaction between two portions on the polypeptide. In one embodiment, the polypeptide is cyclized. In one embodiment, the polypeptide includes a linker moiety that links two amino acid residues of the polypeptide.

[0152] In one embodiment, the linking moiety has formula (I): [ka] , and wherein R can be 1, 2, 3, or 4 substitutions independently selected from the group consisting of H, F, Cl, Br, I, NO, NO, SH, SOH, SOH, SOH, alkyl, alkenyl, alkynyl, COOH, COOR, CHO, OH, R, and OR, where R is alkyl, alkenyl, or alkynyl; where the dashed lines indicate the points of attachment to amino acid residues of the polypeptide.

[0153] In one embodiment, the linking moiety has formula (Ia): [ka] and where the dashed lines indicate the points of attachment to amino acid residues of the polypeptide.

[0154] In one embodiment, the polypeptide comprises a linker moiety connecting two amino acid residues, and the linker moiety was prepared using 1,3-bis(bromomethyl)benzene.

[0155] In one embodiment, the two linked amino acid residues are linked via groups independently selected from an N-terminal amine, a side chain heteroatom selected from S, N, and O, a C-terminal acid, and a C-terminal amide.

[0156] In one embodiment, the polypeptide is i. C1-GSTCPWLKKIMDR-C1-TVSTLVLGEG (SEQ ID NO: 54), ii. C1-NLAIGSTCPWLKKIMDRMTVS-C1 (SEQ ID NO: 55); iii. C1-AIGSTCPWLKKIMDRMT-C1 (SEQ ID NO: 56), and iv. A structure selected from the group consisting of: C1-NLAIGSTCPWLKKIMDR-C1 (SEQ ID NO: 59); or comprising or consisting of a sequence having at least 70% sequence identity thereto, e.g., at least 75%, at least 80%, at least 85%, at least 90%, e.g., at least 95% sequence identity thereto; In the formula, C1 indicates the attachment point of the linker moiety.

[0157] In one embodiment, the polypeptide is i. C1-GSTCPWLKKIMDR-C1-TVSTLVLGEG (SEQ ID NO: 54), ii. C1-NLAIGSTCPWLKKIMDRMTVS-C1 (SEQ ID NO: 55); iii. C1-AIGSTCPWLKKIMDRMT-C1 (SEQ ID NO: 56), and iv. C1-NLAIGSTCPWLKKIMDR-C1 (SEQ ID NO: 59); In the formula, C1 indicates the attachment point of the linker moiety.

[0158] In one embodiment, the polypeptide is cyclized from the N-terminus to the C-terminus, such as via a peptide bond, hi one embodiment, the polypeptide comprises or consists of the amino acid sequence EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE (GTI-125, SEQ ID NO: 160) cyclized from the N-terminus to the C-terminus.

[0159] Properties of the Drugs of the Disclosure The agents of the present disclosure exhibit A1M activity. A1M activity can be assessed by several different assays. In particular, it is recognized that the activity of A1M can be assessed using a cytochrome c reduction assay. Thus, in one embodiment of the present disclosure, the agent is effective at reducing cytochrome c. The cytochrome c reduction activity can be compared to the activity of A1M (SEQ ID NO: 30) or a functional variant thereof, such as the A1M variant RMC-035 (SEQ ID NO: 16). In one embodiment of the present disclosure, the agent has at least 35% of the cytochrome c reduction activity of SEQ ID NO: 16, e.g., at least 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the cytochrome c reduction activity of SEQ ID NO: 16.

[0160] The cytochrome c reduction assay disclosed in the Examples below can be used to assess the EC of compounds exhibiting A1M activity, e.g., agents of the disclosure. 50 In one embodiment of the present disclosure, the agent can be used to assess the EC value of cytochrome c reduction. 50 It has a value of 500 μM or less, for example, 400 μM, 300 μM, 200 μM, 100 μM, or 50 μM or less.

[0161] The therapeutic effect of A1M is due in part to its ability to bind heme. Free heme is cytotoxic. Therefore, it is advantageous for A1M to bind heme, thereby reducing cytotoxic side effects. In one embodiment of the present disclosure, the agent of the present disclosure is capable of binding heme.

[0162] A1M is an antioxidant and therefore has the ability to reduce oxidative stress. In one embodiment of the present disclosure, the agent of the present disclosure is an antioxidant.

[0163] Because A1M and the agents disclosed herein have similar activities, it is believed that the agents disclosed herein will be effective in treating or preventing the same diseases and disorders that A1M and its variants are effective in treating or preventing, as described in the Examples below.

[0164] In one embodiment of the present disclosure, the agent is capable of preventing cell death. In one embodiment of the present disclosure, the agent is capable of preventing lysis of red blood cells (RBCs).

[0165] Drugs In one embodiment of the present disclosure, the agent further comprises a moiety, which in a further embodiment is capable of altering a biophysical property of the polypeptide.

[0166] One aspect of the present disclosure is a composition comprising a composition comprising: a. A polypeptide consisting of 12 to 80 amino acid residues, the amino acid sequence of which is GSTCPWLKKIX 1 (SEQ ID NO: 20), 1 is M, K, R, or Nle, or b. A polypeptide consisting of 30 to 80 amino acid residues and having the amino acid sequence GATEAEISMTSTX 2 WRKGVCEETSGAYEKTD (SEQ ID NO: 21), 2 is H, R, or K.

[0167] One embodiment of the present disclosure provides a pharmaceutical agent comprising a component and a biologically active polypeptide consisting of 10 to 80 amino acid residues, the polypeptide comprising SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or a fragment of SEQ ID NO: 29; X 3 is selected from D, N, or G, X 4 is selected from M, K, R, or Nle, X 5 is selected from H, R, or K; X 6 is selected from D, N, or E, X 7 is selected from M and Nle, the fragment is at least 10 amino acid residues in length, The fragment comprises amino acid residues corresponding to C34 or C72 of SEQ ID NO:30.

[0168] In one embodiment of the present disclosure, the moiety is attached to the N-terminus of the polypeptide. In one embodiment of the present disclosure, the moiety is attached to the C-terminus of the polypeptide. In one embodiment of the present disclosure, the moiety is attached to a side chain of the polypeptide. The additional moiety can be a second polypeptide, a protein, or another compound. When the moiety is a second polypeptide attached to the N-terminus or C-terminus of the polypeptide of the present disclosure, the moiety and the polypeptide together are not A1M or a variant thereof. Specifically, when the moiety is a polypeptide, the agent is an amino acid sequence having at most 95%, for example, at most 90%, 85%, or 80% sequence identity to human A1M (SEQ ID NO: 30). It has 75%, 70%, 65%, 60%, 55%, for example, at most 50% sequence identity to SEQ ID NO: 30. This sequence identity is calculated based on the length of SEQ ID NO: 30, i.e., not based on the length of the shorter of the two sequences.

[0169] In one embodiment, a description of an agent comprising a polypeptide consisting of a plurality of amino acid residues comprising a particular amino acid sequence should also be construed as an agent comprising a polypeptide consisting of a plurality of amino acid residues comprising the particular sequence, wherein the N-terminal residue is not bound to an amino acid residue at the N-terminus, and the C-terminal residue is not bound to an amino acid residue at the C-terminus. In certain embodiments, the N-terminal residue may be bound to an amino acid residue at the N-terminus that does not form part of the continuous sequence of A1M (e.g., SEQ ID NO: 30). In certain embodiments, the C-terminal residue may be bound to an amino acid residue at the N-terminus that does not form part of the continuous sequence of A1M (e.g., SEQ ID NO: 30).

[0170] In one embodiment, a description of a drug comprising a polypeptide consisting of multiple amino acid residues comprising a specific amino acid sequence should also be interpreted as a drug consisting of a polypeptide comprising the specific sequence and one or more excipients, in which one or more amino acid residues are optionally modified.

[0171] fusion proteins It is contemplated that the polypeptides disclosed herein can be fused to other molecules, such as other polypeptides, to form the disclosed drugs without losing the therapeutic activity of the drug. Thus, one embodiment of the present disclosure provides a drug comprising a polypeptide of the present disclosure and a second polypeptide. In one embodiment, the polypeptide of the present disclosure is fused to an albumin binding domain (SEQ ID NO: 118) or a fragment of an albumin binding domain (e.g., SEQ ID NO: 119).

[0172] In one embodiment, the second polypeptide is not a fragment of A1M (SEQ ID NO: 120). In one embodiment, the agent comprises up to 80 contiguous amino acid residues of human A1M (SEQ ID NO: 120), or up to 80 contiguous amino acid residues of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to human A1M (SEQ ID NO: 120). This means that the agent can have more than 80 amino acid residues, but the portion of the agent that corresponds to an A1M fragment or has such sequence identity is 80 amino acid residues or less.

[0173] In one embodiment, the second polypeptide is an albumin binding domain (ABD1, SEQ ID NO: 118). In one embodiment, the second polypeptide is a fragment of A1M (SEQ ID NO: 119). In one embodiment, the second polypeptide is a human Fc fragment, such as a human IgG Fc fragment, more particularly a human IgG1 Fc fragment, such as a human IgG1 Fc fragment having SEQ ID NO: 138.

[0174] Polynucleotides, vectors, and cells One embodiment of the present disclosure provides a polynucleotide encoding an agent or polypeptide disclosed herein.

[0175] One embodiment of the present disclosure provides a vector comprising the polynucleotide disclosed herein.

[0176] One embodiment of the present disclosure provides a cell comprising a polynucleotide disclosed herein or a vector disclosed herein.

[0177] Pharmaceutical Composition One embodiment of the present disclosure provides a pharmaceutical composition comprising an agent of the present disclosure and a pharmaceutically acceptable carrier, excipient, or diluent. In one embodiment of the present disclosure, the pharmaceutical composition is formulated for oral administration. In one embodiment, the pharmaceutical composition is formulated for subcutaneous administration, for example, subcutaneous injection. In one embodiment, the pharmaceutical composition is formulated for topical administration.

[0178] Therapy One embodiment provides an agent of the present disclosure for use in medicine.One embodiment provides an agent of the present disclosure for use in the treatment of a disease or disorder.

[0179] One embodiment of the present disclosure provides a method of treating a disease or disorder comprising administering a therapeutically effective amount of an agent of the present disclosure.

[0180] One embodiment of the present disclosure provides the use of an agent of the present disclosure for the manufacture of a medicament for the treatment of a disease or disorder.

[0181] Oxidative stress has been reported in renal diseases and is believed to be an important factor in the renal pathophysiology of AKI, AKD, and CKD. Therefore, one embodiment provides a medicament of the present disclosure for use in treating renal disease, injury, or damage. In one embodiment of the present disclosure, the renal disease, injury, or damage is induced and / or propagated by, or associated with, oxidative stress.

[0182] One embodiment provides a method of treating a kidney disease, disorder, or injury comprising administering an agent of the present disclosure to a subject in need thereof.

[0183] One embodiment provides a pharmaceutical composition for use in treating a renal disease, disorder, or injury, the composition comprising an agent of the present disclosure.

[0184] One embodiment provides the use of the agents herein in the treatment of a renal condition, disorder, or injury.

[0185] One embodiment provides the use of an agent herein for the manufacture of a medicament for the treatment of a renal condition, disorder, or injury.

[0186] Heme is an essential prosthetic group in many proteins that broadly affect cellular function and metabolism. However, heme and heme-related products can be damaging to the kidney due to their biological reactivity and pro-oxidant effects. A key mechanism underlying the toxicity of heme and heme-related products is the induction and progression of oxidative stress, which in turn triggers various pathophysiological processes associated with kidney disease. Thus, in one embodiment herein, oxidative stress is induced and / or propagated by or associated with heme and / or heme-related products. In a further embodiment of the present disclosure, oxidative stress is induced and / or propagated by or associated with free heme.

[0187] Hematuria is a cardinal symptom of kidney disease. Pathological mechanisms involved in hematuria-associated kidney disease and / or injury include the release of heme and heme-related products from RBC degradation. The released heme and heme-related products are nephrotoxic and accumulate in various intracellular and extracellular compartments of the kidney, inducing and / or propagating oxidative stress, inflammation, cell death, and / or fibrosis, ultimately leading to a decline in renal function.

[0188] Thus, in further embodiments of the present disclosure, the renal disease, disorder, or injury is manifested by hematuria. In certain embodiments, the disease or disorder is pre-eclampsia. In further embodiments of the present disclosure, the renal disease, disorder, or injury is manifested by glomerular hematuria. In one embodiment of the present disclosure, the renal disease, disorder, or injury is caused, induced, and / or propagated by glomerulonephritis. In further embodiments of the present disclosure, the renal disease, disorder, or injury is caused, induced, and / or propagated by nephrotoxicity, e.g., a nephrotoxic drug. In further embodiments of the present disclosure, the renal disease, disorder, or injury is acute kidney injury (AKI). In further embodiments of the present disclosure, the acute kidney injury (AKI) is ischemia-reperfusion injury (IRI). In further embodiments of the present disclosure, the acute kidney injury (AKI) is caused, induced, and / or propagated by cisplatin. In further embodiments of the present disclosure, the acute kidney injury (AKI) is caused, induced, and / or propagated by administration of cisplatin to a subject in need thereof. In a further embodiment of the present disclosure, the kidney disease, disorder or injury is acute kidney disease (AKD).

[0189] One embodiment of the present disclosure provides a method of reducing oxidative stress in a cell, the method comprising contacting the cell with an agent of the present disclosure.One embodiment of the present disclosure provides a method of reducing oxidative stress in a subject, the method comprising administering to the subject an agent of the present disclosure.

[0190] In a further embodiment of the present disclosure, the renal disease, disorder, or injury is chronic kidney disease (CKD). In one embodiment of the present disclosure, the CKD is associated with and / or presents with hematuria. In one embodiment of the present disclosure, the CKD is selected from the group consisting of IgA nephropathy, primary focal segmental glomerulosclerosis (FSGS), Alport syndrome, thin basement membrane disease (TBMN), C3 glomerulonephritis (C3GN), lupus nephritis, ANCA-associated vasculitis, ANCA-associated glomerulonephritis, diabetic kidney disease (DKD), hypertensive nephrosclerosis, polycystic kidney disease, non-diabetic CKD, non-proteinuric CKD, interstitial nephritis, drug-induced CKD, CKD due to autoimmune disease, CKD due to unknown / non-specific causes, and CKD due to genetic abnormalities, including, but not limited to, APOL1 nephropathy. In one embodiment of the present disclosure, the CKD is caused by glomerulonephritis.

[0191] In further embodiments of the present disclosure, the kidney disease, disorder, or injury is classified according to the International Classification of Diseases 11 (ICD-11) codes MF8Y, GB4Z, GB60, and / or GB61.

[0192] In one embodiment, the disease or disorder is caused by or associated with bleeding, hemolysis, or anemia, e.g., hemolytic anemia. In one embodiment, the disease or disorder is hemolytic anemia or a systemic hemopathic disorder. In one embodiment, the disease or disorder is sickle cell anemia, hemolytic anemia, Diamond-Blackfan anemia, malaria, or sepsis. In one embodiment, the disease or disorder is a CNS disease or disorder, e.g., hemorrhagic stroke or ischemic stroke. In one embodiment, the disease or disorder is a vascular disease or disorder, e.g., a cardiovascular disease or disorder. In one embodiment, the vascular disease or disorder is atherosclerosis, vascular injury, peripheral arterial disease, ischemic heart disease, thrombotic vascular disease, heart failure, or myocardial infarction. In one embodiment, the disease or disorder is tissue or nerve damage due to hemolytic trauma or bleeding.

[0193] One embodiment provides a method of treating intraventricular hemorrhage (IVH) in a subject, the method comprising administering an agent of the present disclosure.

[0194] [Example]

[0195] Example 1: Cytochrome c reduction assay Materials and Methods This assay was modified from Allhorn et al. 2005 (Freead. Rad. Biol. Med 38:557-567) and adapted for use in 384-well plates. Cytochrome c assay substrate solution was prepared by mixing 150 μM cytochrome c (Sigma-Aldrich, C2506) and 150 μM NADH (from a 50 mM NADH stock solution in 0.01 M NaOH) in Dulbecco's phosphate-buffered saline (Gibco, 14190-094) and adding it to a 384-well plate immediately before starting the assay. RMC-035 reference protein solution (0.0085-70 μM) or peptide solution (concentration range 0.06-97 μM) was added in duplicate to the 384-well plate in 22 μL volumes. 33 μL of assay substrate solution was rapidly added to each well using a multichannel pipette. The 384-well plate was covered with plastic, mixed by shaking (linear, 900 rpm, 5 seconds), and incubated for 2 hours at room temperature, protected from light. After incubation, the plate was placed in a multiwell plate spectrophotometer (SpectroMax i3x, Molecular Devices) for the last 5 minutes of incubation to allow for temperature adjustment. The plate was mixed by shaking (linear, 900 rpm, 5 seconds), and the absorbance of the plate was measured at 550 nm. The absorbance values ​​at each concentration were plotted against RMC-035, GTI-11, GTI-12, or peptide concentration, and the EC values ​​for each peptide were calculated using a four-parameter logistic regression. 50Values ​​were calculated. Each experimental peptide was compared to a positive control, which was either RMC-035 (full-length recombinant modified A1M protein) or a peptide previously shown to be effective in the cytochrome C reduction assay (GTI-11 or GTI-12), as indicated. Efficacy was expressed as a percentage (%) of the positive control's maximal cytochrome C reduction activity, or the EC for cytochrome C reduction activity of each peptide. 50 was evaluated as a concentration.

[0196] All peptides tested had N-terminal acetylation and C-terminal amidation and were synthesized as trifluoroacetic acid (TFA) salts without removal of residual TFA.

[0197] GTI-1 to GTI-4 and GTI-6 to GTI-14 were designed to contain residues corresponding to C34 and / or C72 of A1M. GTI-5 was designed as a scrambled polypeptide, and GTI-15 was designed as a fragment of A1M without residues corresponding to C34 or C72.

[0198] GTI-16 to GTI-18 were designed by amino acid substitution and addition to enhance solubility.

[0199] GTI-24, GTI-25, GTI-29, and GTI-30 are initially attached on the resin with a C-terminal polyethylene glycol (PEG2, {2-[2-(Fmoc-amino)ethoxy]ethoxy}acetic acid, C21H23NO6) bond, designed to improve plasma / blood stability and pharmacokinetic profiles.

[0200] GTI-31 has a flexible C-terminal GS linker (GGGGGSAS; SEQ ID NO: 121) to a modified albumin binding domain peptide sequence (SDFYKRLINKAKTVEGVALKLHILAALP; SEQ ID NO: 119).

[0201] GTI-36 is the chloride salt of GTI-11. GTI-37 is a cysteine-cysteine ​​(SS) dimer of GTI-11. GTI-38 to GTI-40 are modifications of GTI-11 in which the cysteine ​​at position 4 is modified as iodoacetamide, homocysteine, or penacylamide-cysteine, respectively.

[0202] GTI-41 is a variant of GTI-11 in which the methionine amino acids at positions 11 and 14 are replaced with oxidized methionine amino acids.

[0203] GTI-42 and GTI-43 are truncated GTI-11 sequences with 4 and 10 amino acids, respectively.

[0204] GTI-44 to GTI-46 and GTI-50 chemically conjugate peptides to scaffolds C chemical Li nkage of P eptides onto S It is constructed using CLIPS technology, in which the CLIPS scaffold is attached to different amino acids within the peptide sequence, improving plasma / blood stability.

[0205] GTI-58 to 64 contain D-amino acid substitutions designed to enhance plasma / blood stability.

[0206] GTI-65 through GTI-72 contain side chain methylation and N-methylation variants to extend the plasma residence time of the peptides.

[0207] All peptides tested had N-terminal acetylation and C-terminal amidation and were synthesized as trifluoroacetic acid (TFA) salts without removal of residual TFA.

[0208] GTI-73 is a GTI-11 sequence containing a D-amino acid at position 2 (serine), G[S]TCPWLKKIMDRMTVSTLVLGEG.

[0209] The full-length albumin binding domain (ABD) is linked to the peptide via a linker to improve systemic residence time without compromising efficacy. The ABD is LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP (hereinafter referred to as ABD1; SEQ ID NO: 118). A linker is used to link ABD1 to the peptide (GGGGGSAS; SEQ ID NO: 121). Hereafter, this will be referred to as GSlink.

[0210] GTI-75 is a GTI-11 basic sequence with an ABD1 sequence and a GS link (SEQ ID NO: 131, ABD1-GSlink-GSTCPWLKKIMDRMTVSTLVLGEG) added to the N-terminus, and GTI-76 is a GTI-11 basic sequence with an ABD1 sequence and a GS link (SEQ ID NO: 132, GSTCPWLKKIMDRMTVSTLVLGEG-GSlink-ABD1) added to the C-terminus.

[0211] The GTI-87 and GTI-90 peptides were also designed with ABD1 and GSlink, which also contain repeat variants of the GSlink and GTI-82 to GTI-86 sequences. ● GTI-87:ABD1-GSlink-GTI-82x1 (sequence number 133). ● GTI-88:ABD-1-(GS-link-GTI-82 x3) (sequence number 134). ● GTI-89:ABD1-GSlink-GTI-86x1 (sequence number 135). ● GTI-90:ABD-1-(GS-link-GTI-86x3) (SEQ ID NO: 136).

[0212] Metabolic studies performed by incubating the peptide with hepatocytes and plasma suggested that threonine at position 15 may be a potential site of metabolism, and therefore the peptide was designed with modifications to improve stability in that region and to improve solubility.

[0213] GTI-77 to GTI-79 have the basic sequence of GTI-11, but threonine at position 15, valine at position 16, or both threonine at position 15 and valine at position 16 are substituted with D-amino acids.

[0214] ● GTI-77:GSTCPWLKKIMDRM[T]VSTLVLGEG (sequence number 123). ● GTI-78:GSTCPWLKKIMDRMT[V]STLVLGEG (sequence number 124). ● GTI-79:GSTCPWLKKIMDRM[T][V]STLVLGEG (sequence number 125).

[0215] GTI-82 and GTI-83 are GTI-11 sequences in which the valine at position 16 has been replaced with either glutamic acid or lysine. ● GTI-82:GSTCPWLKKIMDRMT[E]STLVLGEG (sequence number 126). ● GTI-83:GSTCPWLKKIMDRMT[K]STLVLGEG (SEQ ID NO: 127).

[0216] GTI-2 is a highly active peptide containing the DDDDK enterokinase cleavage site sequence, which has been removed and replaced with glutamic acid to improve water solubility, and the hydrophobic C-terminal region has also been removed: ● GTI-84 EEEEKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 128). ● GTI-85 EEKYNLAIGSTCPWLKKIMDRMTVSTLVLGE (sequence number 129). ● GTI-86: EEKYNLAIGSTCPWLKKIMDRMT[E]STLVLGE (valine at position 24 replaced with glutamic acid for solubility) (SEQ ID NO: 130).

[0217] result The absorbance values ​​and corresponding curve fits of GTI-1 to GTI-15 are shown in Figure 1. EC 50The values ​​(assessed by cytochrome c reduction assay) are shown in Table 1. Figure 2 shows an overview of the cytochrome c reducing ability of the polypeptides GTI-1 to GTI-15. Figure 3 shows the EC 50 The values ​​are summarized below. RMC-035 reduces oxidized cytochrome c with an EC50 of 0.8 μM. Polypeptides carrying A1M reductase showed EC50 values ​​in the low μM region. Polypeptides GTI-1 to GTI-4, GTI-5 to GTI-14, and GTI-16 to GTI-18 showed more than 30% of the efficacy of RMC-035. The negative controls GTI-5 and GTI-15 showed low efficacy, and their efficacy never exceeded 30% of that of RMC-035. Furthermore, the curves obtained were not sigmoidal, so the EC50 values ​​were not significant. 50 The fitting of the GTI-5 and GTI-15 data also showed low R-squared values ​​(R 2 value) was obtained.

[0218] TIFF2026507475000004.tif143165Activity (%) and EC of polypeptides GTI-24 to GTI-50 against GTI-11 evaluated by cytochrome c reduction assay 50 The values ​​are shown in Table 2. Polypeptides GTI-24 to GTI-36, GTI-39 to GTI-41, and GTI-44 to GTI-74 showed activities greater than 71% of that of GTI-11. GTI-37, GTI-38, and GTI-42 showed activities greater than 14% of that of GTI-11.

[0219] TIFF2026507475000005.tif236165TIFF2026507475000006.tif224165Activity (%) and EC of polypeptides GTI-54, GTI-57, GTI-75, GTI-77 to GTI-79, and GTI-82 to GTI-90 against GTI-12 as assessed by cytochrome c reduction assay 50The values ​​are shown in Table 3. Polypeptides GTI-54, GTI-57, GTI-75, GTI-77 to GTI-79, GTI-82 to GTI-90 showed at least 41% higher activity compared to GTI-12.

[0220] TIFF2026507475000007.tif171165

[0221] TIFF2026507475000008.tif39165TIFF2026507475000009.tif182165Efficacy (%) and EC of GTI-111 to GTI-119 polypeptides compared to GTI-11 as assessed by cytochrome c reduction assay 50 The values ​​are shown in Table 4. Polypeptides GTI-111, GTI-114, GTI-116, GTI-117, GTI-118, and GTI-119 showed efficacy at least as good as GTI-86.

[0222] conclusion RMC-035 reduces oxidized cytochrome C with an EC50 of 0.8 μM. Many polypeptides have reducing activity approaching the potency of RMC-035.

[0223] Polypeptides GTI-1 to GTI-4, GTI-5 to GTI-14, and GTI-16 to GTI-18 were all able to reduce cytochrome c in a dose-dependent manner, with at least 30% of the total activity of the positive control RMC-035. Negative control sequences, scrambled sequences lacking cysteine ​​amino acids (GTI-5 and GTI-15), showed no activity against RMC-035.

[0224] Peptides modified with PEG2 (GTI-24, 25, 29, and 30) and peptides containing the albumin-binding domain sequence attached via a GS linker (GTI-31, GTI-75, and GTI-89) retained dose-dependent cytochrome C reduction activity that was at least 41% of that of GTI-11 or GTI-12. Modifications made to GTI-11 and GTI-2 (GTI-82 to GTI-86) to introduce more soluble residues also maintained or improved reduction activity in the cytochrome C reduction assay.

[0225] The introduction of D-amino acid substitutions or methylated amino acids (GTI-58 to GTI-74 and GTI-77 to GTI-79) did not adversely affect the reducing activity of the peptides in the cytochrome C reduction assay.

[0226] Scrambled amino acid sequences containing cysteines showed cytochrome c reducing activity. GTI-37 (a cysteine-intercalated SS dimer), GTI-38 (iodoacetamidated cysteine), and 4- or 10-amino acid peptides (GTI-42 and 43) did not retain cytochrome c reducing activity compared to GTI-11.

[0227] A hybrid sequence of GTI-64 and GTI-83 (GTI-111) did not impair peptide reduction activity in the cytochrome c reduction assay. Hybrid sequences of GTI-64 and GTI-86 (GTI-117 and GTI-119), in which methionine was replaced with norleucine and N-methylserine to introduce increased stability due to methionine oxidation, retained 100% of the dose-dependent cytochrome c reduction activity of GTI-86.

[0228] The peptide GTI-120 was cyclized head-to-tail, with an additional cysteine ​​at the C-terminus to facilitate cyclization, to form GTI-125. The reducing activity was maintained, with an EC50 of 2.21 compared with 3.35 for GTI-120.

[0229] The fatty acid linkages (GTI-121 to GTI-124), side chain or N-terminal γGlu-2 × OEG-C20 diacid (octadecanedioic acid) linkages retained reducing activity relative to the unlinked peptide variants.

[0230] Example 2: Erythrocyte lysis assay Materials and Methods This assay was adapted from Kristiansson et al. 2020 (Freead. Rad. Biol. Med, "Human radical scavenger α1-microglobulin protects against hemolysis in vitro and α1-microglobulin knockout mice exhibit a macrocytic anemia phenotype") and adapted for use in 96-well plates. Blood was collected from Sprague-Dawley rats using K2EDTA-coated vacutainers. Red blood cells (RBCs) were isolated by centrifugation at 800g for 10 minutes to fractionate the blood. RBCs were then resuspended in phosphate-buffered saline (PBS) at pH 7.4 and washed five times by centrifugation at 800g for 10 minutes. Finally, RBCs were reconstituted and diluted to a stock concentration of 4% v / v. Heme (Hemin [Ferriprotoporphyrin IX chloride], Sigma-Aldrich 51280) was used to induce RBC lysis. A working stock solution of 0.2 mM hemin was prepared in PBS, pH 7.4. In a 96-well plate, a 250 μL mixture was prepared containing 148.5 μL of PBS, 62.5 μL of RBCs (final concentration 1% v / v), 25 μL of hemin (final concentration 20 μM), and 14 μL of RMC-035 reference protein solution (dilutions ranging from 0.273 to 35 μM) or peptide solution (concentrations ranging from 0.06 to 97 μM). The plate was incubated at room temperature for 3 hours with shaking (linear shaker, 300 rpm). After incubation, the plate was centrifuged at 500 g for 5 minutes, and the resulting supernatant was collected. Cell lysis was determined by measuring free hemoglobin by measuring the absorbance of 100 μL of the supernatant at 410 nm using a multiwell plate spectrophotometer (SpectroMax i3x, Molecular Devices). Additionally, lactate dehydrogenase (LDH) release was measured using the CytoTox96 Non-Radioactive Cytotoxicity Assay Kit (Promega).The assay was adapted for use in a 384-well plate. 12.5 μL of assay supernatant was mixed with 12.5 μL of CytoTox96 assay reagent, covered with aluminum foil to protect from light, and incubated for 30 minutes at room temperature. After incubation, 12.5 μL of stop solution was added to each well, and LDH was measured by measuring absorbance at 490 nm using a multiwell plate spectrophotometer (SpectroMax i3x, Molecular Devices). The absorbance of LDH and free hemoglobin measurements at each concentration was plotted against RMC-035 or peptide concentration, and the EC50 was calculated in both μM and μg / mL.

[0231] GTI-1 to GTI-4 and GTI-6 to GTI-14 were designed to contain residues corresponding to C34 and / or C72 of A1M. GTI-5 was designed as a scrambled polypeptide, and GTI-15 was designed as a fragment of A1M without residues corresponding to C34 or C72.

[0232] GTI-16 to GTI-18 were designed by amino acid substitution and addition to enhance solubility.

[0233] GTI-24, GTI-25, GTI-29, and GTI-30 are initially attached on the resin with a C-terminal polyethylene glycol (PEG2, {2-[2-(Fmoc-amino)ethoxy]ethoxy}acetic acid, C21H23NO6) bond, designed to improve plasma / blood stability and pharmacokinetic profiles.

[0234] GTI-31 has a flexible C-terminal GS linker (GGGGGSAS; SEQ ID NO: 121) to the albumin binding domain peptide sequence (SDFYKRLINKAKTVEGVALKLHILAALP; SEQ ID NO: 119).

[0235] GTI-36 is the chloride salt of GTI-11. GTI-37 is a cysteine-cysteine ​​(SS) dimer of GTI-11. GTI-38 to GTI-40 are modifications of GTI-11 in which the cysteine ​​at position 4 is modified as iodoacetamide, homocysteine, or penacylamide-cysteine, respectively.

[0236] GTI-41 is a variant of GTI-11 in which the methionine amino acids at positions 11 and 14 are replaced with oxidized methionine amino acids.

[0237] GTI-42 and GTI-43 are truncated GTI-11 sequences with 4 and 10 amino acids, respectively.

[0238] GTI-44 to GTI-46 and GTI-50 chemically conjugate peptides to scaffolds C chemical Li nkage of P It is constructed using peptides onto scaffolds (CLIPS) technology, in which the CLIIPS scaffold is attached to different amino acids within the peptide sequence, improving plasma / blood stability.

[0239] GTI-58 to 64 contain D-amino acid substitutions designed to enhance plasma / blood stability.

[0240] GTI-65 through GTI-72 contain side chain methylation and N-methylation variants to extend the plasma residence time of the peptides.

[0241] GTI-73 is a GTI-11 sequence containing a D-amino acid at position 2 (serine), G[S]TCPWLKKIMDRMTVSTLVLGEG.

[0242] The full-length albumin binding domain (ABD) is linked to the peptide via a linker to improve systemic residence time without compromising efficacy. The ABD is LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP (ABD1; SEQ ID NO: 118). The linker is used to link ABD1 to the peptide (GGGGGSAS; GSlink; SEQ ID NO: 121).

[0243] GTI-75 is a GTI-11 basic sequence with an ABD1 sequence and a GS link (SEQ ID NO: 131, ABD1-GSlink-GSTCPWLKKIMDRMTVSTLVLGEG) added to the N-terminus, and GTI-76 is a GTI-11 basic sequence with an ABD1 sequence and a GS link (SEQ ID NO: 132, GSTCPWLKKIMDRMTVSTLVLGEG-GSlink-ABD1) added to the C-terminus.

[0244] The GTI-87 and GTI-90 peptides were also designed with ABD1 and GSlink, which also contain repeat variants of the GSlink and GTI-82 to GTI-86 sequences. ● GTI-87:ABD1-GSlink-GTI-82x1 (sequence number 133). ● GTI-88: ABD-1-(GS-link-GTI-82x3) (sequence number 134). ● GTI-89:ABD1-GSlink-GTI-86x1 (sequence number 135). ● GTI-90:ABD-1-(GS-link-GTI-86x3) (SEQ ID NO: 136).

[0245] Metabolic studies performed by incubating the peptide with hepatocytes and plasma suggested that threonine at position 15 may be a potential site of metabolism, and therefore the peptide was designed with modifications to improve stability in that region and to improve solubility.

[0246] GTI-77 to GTI-79 have the basic sequence of GTI-11, but threonine at position 15, valine at position 16, or both threonine at position 15 and valine at position 16 are substituted with D-amino acids. ● GTI-77:GSTCPWLKKIMDRM[T]VSTLVLGEG (sequence number 123). ● GTI-78:GSTCPWLKKIMDRMT[V]STLVLGEG (sequence number 124). ● GTI-79:GSTCPWLKKIMDRM[T][V]STLVLGEG (sequence number 125).

[0247] GTI-82 and GTI-83 are GTI-11 sequences in which the valine at position 16 has been replaced with either glutamic acid or lysine.

[0248] ● GTI-82:GSTCPWLKKIMDRMT[E]STLVLGEG (sequence number 126). ● GTI-83:GSTCPWLKKIMDRMT[K]STLVLGEG (SEQ ID NO: 127).

[0249] GTI-2 is a potent peptide containing the DDDDK enterokinase cleavage site sequence, which has been removed and replaced by solubilizing glutamic acid, and the hydrophobic C-terminal region has been removed.

[0250] ● GTI-84 EEEEKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 128). ● GTI-85 EEKYNLAIGSTCPWLKKIMDRMTVSTLVLGE (SEQ ID NO: 129). - GTI-86 EEKYNLAIGSTCPWLKKIMDRMT[E]STLVLGE (valine at position 24 replaced with glutamic acid for solubility) (SEQ ID NO: 130).

[0251] result The LDH absorbance values ​​of polypeptides GTI-1 to GTI-15 are shown in Figure 4, and the EC 50 The values ​​are shown in Table 4. RMC-035, GTI-1 through GTI-4, and GTI-6 through GTI-12 all showed a decrease in absorbance as the polypeptide concentration increased. RMC-035, GTI-1 through GTI-4, and GTI-6 through GTI-12 all showed a decrease in absorbance as the polypeptide concentration increased. GTI-5 and GTI-15 showed no change in absorbance even at high polypeptide concentrations. The response curves for GTI-1 through GTI-4, GTI-6 through GTI-8, and GTI-10 through GTI-12 were sigmoidal, thereby indicating the EC 50 values ​​could be calculated.

[0252] TIFF2026507475000010.tif180165TIFF2026507475000011.tif201165The absorbance values ​​of free hemoglobin are shown in Figure 5, and the EC 50 The values ​​are shown in Table 5. RMC-035, GTI-1 through GTI-4, and GTI-6 through GTI-14 all showed a decrease in LDH absorbance with increasing polypeptide concentration, indicating that these polypeptides have the ability to prevent RBC lysis. GTI-5 and GTI-15 showed no change in absorbance even at high polypeptide concentrations. The response curves for GTI-1 through GTI-4, GTI-6 through GTI-8, and GTI-10 through GTI-12 were sigmoidal, thereby increasing the EC 50 values ​​could be calculated.

[0253] TIFF2026507475000012.tif209165TIFF2026507475000013.tif195165Conclusion GTI-1 to GTI-4, GTI-6 to GTI-14, and GTI-16 to GTI-90 all showed a decrease in LDH absorbance when the polypeptide concentration reached a certain threshold, indicating that these peptides reduced or prevented RBC lysis, i.e., exhibited a heme-binding effect. For GTI-1 to GTI-4, GTI-6 to GTI-8, and GTI-10 to GTI-12, EC 50 Values ​​were obtained based on data regarding LDH absorbance. Negative controls GTI-5 and GTI-15 showed no ability to prevent LDH-based RBC lysis.

[0254] Polypeptides GTI-1 to GTI-4 and GTI-6 to GTI-12 dose-dependently reduced or prevented RBC lysis, as assessed by the presence of free hemoglobin. For GTI-1 to GTI-4, GTI-6 to GTI-8, and GTI-10 to GTI-12, EC values ​​were calculated based on plots of hemoglobin absorbance. 50 The negative controls GTI-5 and GTI-15 showed no ability to prevent free hemoglobin-based RBC lysis.

[0255] Example 3: Cell viability assay with human kidney-2 (HK-2) proximal tubule cell line Materials and Methods The efficacy and potency of peptides in inhibiting heme-induced cell viability, as measured by lactate dehydrogenase (LDH release), was evaluated in a cell viability assay using human renal cortical proximal tubule epithelial cells (HK-2), ATCC® CRL-2190™. HK-2 cells were maintained in Keratinocyte SFM medium (Gibco) supplemented with 0.05 mg / mL bovine pituitary extract and 5 ng / mL epidermal growth factor. HK-2 cells were seeded at a density of 8,000 cells / well in 50 μL of culture medium into 384-well cell culture plates and incubated for 18 hours in a cell culture incubator.

[0256] Heme (hemin [Ferriprotoporphyrin IX chloride], Sigma-Aldrich 51280) was used to induce cytotoxicity by preparing a 1 mM hemin working stock solution in PBS (pH 7.4). In a 364-well plate containing HK-2 cells, 25 μL of medium was removed, and 20 μL of test peptide solution in assay medium (final well concentrations ranging from 0.068 to 70 μM) was added to the corresponding well of a 384-well cell culture plate containing HK-2 cells. Finally, 5 μL of either PBS pH 7.4 (negative control) or 20 μM hemin was added to each well, and the plates were incubated for 24 h.

[0257] After 24 hours, LDH release was assessed using the CytoTox96 non-radioactive cytotoxicity assay (Promega, G1780) according to the manufacturer's instructions. Five microliters of 10x lysis buffer provided by the LDH assay kit was added to wells designated as positive controls for LDH release. 12.5 μL of assay medium from each well was transferred to a new 384-well plate, and 12.5 μL of CytoTox96 assay reagent was added to each sample, which was then protected from light and incubated at room temperature for 30 minutes. After adding 12.5 μL of assay stop solution, the absorbance of each well was measured at 490 nm on a SpectraMax i3x (Molecular Devices) within 1 hour of adding the stop solution. The absorbance of the LDH measurements at each concentration was plotted against peptide concentration, and the EC50 was calculated in both μM and μg / mL.

[0258] result The absorbance values ​​of LDH are shown in Figure 12, and the corresponding EC 50 The values ​​are shown in Table 6. All tested peptides, except for RMC-035 and GTI-5, GTI-5, GTI-7, GTI-13, GTI-14, and GTI-15, showed a decrease in LDH absorbance with increasing polypeptide concentration. The peptide response curves, indicating inhibition of heme-mediated LDH release, were sigmoidal and the EC 50 values ​​could be calculated.

[0259] TIFF2026507475000014.tif139165TIFF2026507475000015.tif122165Conclusion Peptide EC 50 The values ​​ranged from 1.79 to 16.9 μM. RMC-035, GTI-1 to GTI-6, GTI-8, GTI-9, GTI-10 to GTI-12, GTI-24 to GTI-25, GTI-29 to GTI-31, GTI-46, GTI-50, GTI-61 to GTI-64, and GTI-74 all showed a decrease in LDH absorbance with increasing polypeptide concentration, indicating that these polypeptides have the ability to suppress heme-induced cell viability loss.

[0260] The scrambled random sequence (GTI-5) and the cysteine-free sequence (GTI-15) did not inhibit the hemin-induced decrease in HK-2 cell viability. PEGylation with 2 kDa PEG (GTI-29 and GTI-30), hydrophilic amino acid substitution (GTI-16), and addition of a hydrophilic amino acid to the C-terminus (GTI-18) did not adversely affect the peptide's ability to protect HK-2 cells from hemin toxicity.

[0261] Example 4: Heme oxygenase mRNA expression assay in human kidney-2 (HK-2) proximal tubule cell line Materials and Methods Human renal cortical proximal tubule epithelial cells (HK-2), ATCC® CRL--2190™, were used in Example 3. HK-2 cells were seeded into 24-well cell culture plates at a cell density of 0.2 million cells / well in 0.5 mL of keratinocyte SFM culture medium (Gibco) supplemented with 0.05 mg / mL bovine pituitary extract and 5 ng / mL epidermal growth factor, and cultured overnight in a cell culture incubator until 80-90% confluence was reached.

[0262] Heme (hemin [Ferriprotoporphyrin IX chloride], Sigma-Aldrich 51280) was used to induce cytotoxicity by preparing a 1 mM hemin working stock solution in PBS (pH 7.4). PBS, RMC-035 (final concentration 10 μM), or peptide GTI-11 (final concentration 35 μM) plus hemin (final concentration 20 μM) was added to the relevant wells of HK-2 cells in a 24-well plate, followed by a 4-hour incubation. After incubation, the medium was removed, and the cells were washed twice with Dulbecco's PBS (Gibco). RNA was extracted from the cells according to the PureLink™ RNA Mini Kit (Life Technologies, 12183025) protocol. Cells were lysed in 350 μL of PureLink RNA Extraction Kit lysis buffer with 1% 2-mercaptoethanol. The lysate was immediately frozen on dry ice and stored at -80°C until further processing. RNA was isolated according to the manufacturer's recommendations, including treatment with On-column PureLink DNase (Life Technologies, 12185-010). RNA was quantified using UV absorbance at 260 nm. cDNA was prepared from mRNA using iScript Reverse Transcription Supermix for RT-qPCR (BioRad, 1708841) according to the manufacturer's instructions. 15 ng of RNA was used per qPCR reaction in 20 μL reactions in a 384-well plate. Reverse transcription reactions were performed using a thermal cycler with priming at 25°C for 5 minutes, reverse transcription at 46°C for 20 minutes, and RT deactivation at 95°C for 1 minute.Real-time PCR was performed according to the manufacturer's instructions (iTaq Universal SYBR® Green Supermix, BioRad, 1725124) using primers for the heme oxygenase-1 (HO-1) gene sequence (forward: FH1_HMOX1, Sigma-Aldrich, 8816057690-10 / 0, CAACAAAGTGCAAGATTCTG; reverse: RH1_HMOX1, Sigma-Aldrich, 8816057690-10 / 1, TGCATTCACATGGCATAAAG). Each reaction contained 10 ng / μL cDNA, 5 μM forward and reverse primers, and 2.5 μL of 2x MasterMix in a 5 μL volume. The thermal cycler program was initial denaturation at 95°C for 1 minute, 39 cycles of denaturation at 95°C for 15 seconds, annealing at 55°C for 20 seconds, and extension at 60°C for 45 seconds.

[0263] result HO-1 mRNA expression levels, expressed as fold changes compared to PBS control treatment, are shown in Figure 13. A 4-hour incubation with hemin resulted in a 300-fold increase in HO-1 mRNA expression compared to control levels. This hemin-induced increase in HO-1 expression was completely inhibited by co-incubation with RMC-035 or the peptide GTI-11. Incubation with RMC-035 or GTI-11 alone did not increase HO-1 mRNA expression.

[0264] conclusion HO-1 expression is induced under oxidative stress. RMC-035 and peptide GTI-11 prevented the hemin-induced increase in HO-1 expression, indicating that RMC-035 and peptide GTI-11 are cytoprotective against oxidative stress and / or injury.

[0265] Example 5 In vivo efficacy of intravenously administered GTI-11 in rat ischemia-reperfusion injury (IRI)-acute kidney injury (AKI) Materials and Methods An ischemia-reperfusion injury (IRI) model of acute kidney injury (IRI-AKI) in rats was used to evaluate the in vivo activity of a peptide (GTI-11). Eight- to ten-week-old male Sprague Dawley rats (Charles River Laboratories, Italy) were anesthetized with isoflurane and placed on a heating pad to maintain body temperature. Under aseptic conditions, a midline abdominal incision was made to facilitate access to the renal pedicle. The right kidney was removed by blunt dissection, and the renal artery and renal vein were ligated. Ischemia of the left kidney was initiated by clamping the renal artery and renal vein with atraumatic clamps (Agnthos, Lidingo, Sweden) for 40 minutes. At the end of the ischemia-ischemic period, the clamps were removed, and the kidney was observed to ensure rapid resumption of blood flow. After completion of ischemia and reperfusion, the incision was sutured, and the animals were allowed to recover with free access to food and water.

[0266] Vehicle (10 mM Na-phosphate buffer, pH 7.4, 0.15 M NaCl, 2 mg / mL histidine) or GTI-11 (10 mg / mL) in vehicle was administered intravenously via the tail vein at a bolus dose of 0.5 mL / kg 0.5 h before ischemia and at 4, 8, 24, and 48 h after reperfusion. Vehicle and GTI-11 were administered intravenously via the tail vein at a dose of 0.5 mL / kg, for a final dose of 5 mg / kg. After surgery, rats were allowed to recover for 5 days. Blood was collected from the tail vein at baseline before surgery and on days 1 (24 h), 2 (48 h), 3 (72 h), and 5 (120 h) after surgery for evaluation of renal injury biomarkers. Serum creatinine (Biovision, Milpitas, CA, USA, product number K625) and blood urea nitrogen (BUN) (Thermo Fisher, MA, USA, EIABUN) were measured according to the manufacturer's instructions for each assay kit.

[0267] result Serum creatinine (mg / dL) and BUN (blood urea nitrogen, mg / dL) concentrations at baseline before surgery and on days 1 (24 hours), 2 (48 hours), 3 (72 hours), and 5 (120 hours) after surgery are shown in Figure 14.

[0268] At baseline, serum creatinine (mg / dL) and BUN (mg / dL) concentrations were comparable between the vehicle and GTI-11 groups. Serum creatinine (mg / dL) and BUN (mg / dL) concentrations were significantly reduced in the GTI-11 group compared with the vehicle group on days 1, 2, 3, and 5 after surgery.

[0269] conclusion These data indicate that intravenous administration of GTI-11 for 48 hours significantly inhibits IRI-induced renal injury by suppressing changes in systemic indices of renal function, such as serum creatinine and BUN, in a rat IRI model of AKI.

[0270] Example 6 In vivo efficacy of intravenous and subcutaneous administration of GTI-2 in a rat ischemia-reperfusion injury (IRI)-acute kidney injury (AKI) model Materials and Methods The in vivo activity of the peptide GTI-2 was evaluated in a study of ischemia-reperfusion injury (IRI) using a rat model of acute kidney injury (IRI-AKI). Eight- to ten-week-old male Sprague-Dawley rats (Charles River Laboratories, Italy) were anesthetized with isoflurane and placed on a heating pad to maintain body temperature. Under sterile conditions, a midline abdominal incision was made to facilitate access to the renal pedicle. The right kidney was removed by blunt dissection, and the renal artery and vein were ligated. Ischemia of the left kidney was initiated by clamping the renal artery and vein with atraumatic clamps (Agnthos, Lidingo, Sweden) for 40 minutes. At the end of the ischemic period, the clamps were removed, and the kidney was observed to ensure rapid resumption of blood flow. After completion of ischemia and reperfusion, the incision was sutured, and the animals were allowed to recover with free access to food and water.

[0271] Vehicle (sterile phosphate-buffered saline) or GTI-2 prepared in vehicle (concentration 10 mg / mL) was administered by intravenous bolus injection via the tail vein or subcutaneous administration 0.5 h before ischemia and 4, 24, and 48 h after reperfusion in the following manner.

[0272] IV dose: Peptide stock formulation at a concentration of 10 mg / mL in PBS, vehicle, or peptide was administered at a dose of 0.5 mL / kg for a total dose of 5 mg / kg. SC Dosage: Peptide stock formulation at a concentration of 15 mg / mL, vehicle or peptide was administered via SC injection at 666 μL / kg, resulting in a dose of 10 mg / kg.

[0273] After surgery, rats were allowed to recover for 3 days. Blood samples were collected from the tail vein at baseline and on days 1 (24 hours), 2 (48 hours), and 3 (72 hours) after surgery to assess renal injury biomarkers. Plasma creatinine (Biovision, Milpitas, CA, USA, K625) and blood urea nitrogen (BUN) (Thermo Fisher Scientific, Massachusetts, USA, EIABUN) were assessed according to the manufacturer's instructions. N = 7–8 per group.

[0274] Real-time GFR was measured at baseline (D0), postoperative day 1 (D1, 24 hours), and postoperative day 3 (D3, 72 hours). Real-time GFR was assessed in isoflurane-anesthetized rats by measuring the clearance of fluorescein isothiocyanate-labeled sinistrin (FITC-sinistrin). A MediBeacon transdermal detection system (MediBeacon Transdermal Mini GFR Monitor, MediBeacon, Mannheim, Germany) was attached to the shaved chest of the rat. A background measurement was obtained over 5 minutes before tail vein injection of FITC-sinistrin (5 mg per 100 g body weight). Blood FITC-sinistrin concentrations were recorded transdermally over 30–45 minutes, and FITC-sinistrin clearance was calculated using software provided by MediBeacon according to the manufacturer's instructions. The half-life of FITC-sinistrin was measured, and GFR was calculated using MediBeacon software using the following formula: GFR (mL / min / 100g body weight) = rat conversion factor 31.26 / FITC-sinistrin half-life (t 1 / 2 )(Ellery et al. (2015). Nephrology 20:117-123).

[0275] result Plasma creatinine and BUN concentrations (mg / dL) in rats 3 days after unilateral nephrectomy and ischemia-reperfusion injury, and after intravenous or subcutaneous administration of saline vehicle or peptide GTI-2, are shown in Figure 15. Plasma creatinine and BUN concentrations in rats treated with GTI-2 were significantly lower than those in rats treated with vehicle on both days 1 and 3 after surgery. This inductive effect of GTI-2 on plasma creatinine and BUN concentrations was observed with both IV and SC administration.

[0276] Real-time glomerular filtration rate (GFR) data from rats 3 days after unilateral nephrectomy and ischemia-reperfusion injury, and data from IV or SC administration of saline vehicle or peptide GTI-2, are shown in Figure 16. Three days after surgery, GFR was significantly higher in rats treated with GTI-2 compared with rats treated with vehicle. This GTI-2-induced effect on GFR was observed with both intravenous and subcutaneous administration.

[0277] conclusion These data demonstrate that GTI-2 significantly inhibits IRI-induced renal injury by inhibiting changes in glomerular filtration rate (GFR) and plasma creatinine and BUN levels, which are indicators of overall renal function, when administered either intravenously or subcutaneously for 48 hours in a rat IRI model of AKI.

[0278] Example 7 Pharmacokinetic Profiles of Intravenously and Subcutaneously Administered GTI-2 and GTI-11 in Rats Materials and Methods Male Sprague Dawley rats (250–275 g) were administered one of the following: intravenous (IV) injections of GTI-2 or GTI-11 in PBS at a single dose of 2 mL / kg for a total dose of 10 mg / kg; subcutaneous (SC) injections of GTI-2 or GTI-11 in PBS at a single dose of 5 mL / kg for a total dose of 25 mg / kg. Blood samples were collected from the sublingual vein into EDTA tubes at 1, 5, 10, 15, 30, 60, 120, 240, and 360 minutes after injection. Whole blood (50 μL + 150 μL MQ water) and plasma (prepared by centrifugation at 2000 g for 5 min at 4°C) samples were frozen at <-70°C before analysis by LC-MS / MS (Waters Acquity PREMIER UPLC, Waters Xevo TQ-XS triple quadrupole MS, Waters Acquity UPLC Premier Peptide CSH C18 column (2.1 x 100 mm, 1.7 μm) and pre-column filter) using MassLynx 4.2 software. Noncompartmental analysis (NCA) was performed on the data to calculate pharmacokinetic parameters. Parameters such as Cmax, Tmax, AUC, and T1 / 2 were calculated using PhoenixWinNonlin from Certara (Canterbury, UK).

[0279] result The plasma pharmacokinetic parameters of single doses of GTI-2 or GTI-11 administered subcutaneously (SC) or intravenously (IV) are shown in Table 8. IV administration of GTI-2 had a longer half-life (T 1 / 2 ) and Tmax were shortened, and Cmax and AUCinf_obs were increased. IV administration of GTI-11 significantly reduced Tmax compared with IV administration of GTI-2. 1 / 2 , Cmax and AUCinf_ob were shortened.

[0280] TIFF2026507475000016.tif71165T 1 / 2: Half-life Tmax: Time to reach maximum blood concentration Cmax: Maximum blood concentration AUCinf_obs: Area under the curve extrapolated to infinity based on the last observed concentration (_obs) from the time of administration.

[0281] conclusion Intravenous administration of peptides achieves rapid systemic exposure, but subcutaneous administration and manipulation of peptide length and sequence can extend terminal half-life.

[0282] Example 8 Plasma stability of peptides Materials and Methods The stability of the GTI peptide was determined in human, rat and dog plasma.

[0283] Plasma stability was assessed by incubating each peptide at a final concentration of 1 μM in a total plasma volume of 400 μL with a final DMSO concentration of 0.5% for 120 min. After 0, 20, 40, 60, and 120 min of incubation, 40 μL samples were withdrawn and two volumes of acetonitrile containing 0.1% trifluoroacetic acid and 6% DMSO were added. Samples were stored at -20°C until bioanalysis. Peptides were quantified by LC-MS / MS analysis using a Thermo Vanquish Horizon UHPLC / Thermo Q-Exactive Focus Orbitrap MS equipped with a Waters CSH C18 (2.1 × 50 mm, 1.7 μm particle size) column and pre-column filter. Analysis was performed using Thermo Xcalibur 4.1.31.9 software. Extrapolated half-lives (t 1 / 2 ) and in vitro intrinsic clearance (CL INT ) was calculated.

[0284] result Data showing the stability of GTI-2, GTI-11, GTI-65, GTI-79, GTI-83, and GTI-86 in rat, dog, and human plasma are presented in Table 9.

[0285] TIFF2026507475000017.tif127165T1 / 2, Half-life, CL INT , intrinsic clearance.

[0286] conclusion Substituting hydrophobic amino acids with hydrophilic amino acids or changing hydrophobic amino acid regions to hydrophilic amino acids to improve solubility did not adversely affect the plasma stability of the peptides.

[0287] Example 9 Hepatocyte metabolic stability of peptides Materials and Methods The metabolic profile and stability of the peptides were determined after incubation with rat, dog, or human hepatocytes. Hepatocytes (male SD rats, 66% viability; male beagle dogs, 55% viability; mixed-sex human, 79% viability) were incubated at a density of 1 million viable cells per mL in 300 μL of pH 7.4 Celsis IVT In Vitro GRO KHB medium, 0.5% DMSO, at a peptide concentration of 1 μM for up to 60 minutes at 37°C. At 0, 10, 20, 40, or 60 minutes of incubation, 40 μL of medium was removed and the incubation terminated by adding two volumes of chilled 75% acetonitrile containing 0.1% trifluoroacetic acid. Samples were immediately analyzed using LC-MS / MS (Waters Aquity UPLC + Thermo Q-Exactive Orbitrap MS equipped with a Waters HSS T3 (2.1 × 50 mm, 1.7 μm particle size) column) with Thermo Xcalibur 4.1.31.9 software. Hepatocyte metabolic stability of peptides was evaluated based on extrapolated half-lives (t) and in vitro intrinsic clearance (CL). INT ) and metabolic profiles were also determined by evaluation of relative peak profiles.

[0288] result The stability parameters of peptides GTI-11 and GTI-2 after incubation with rat, dog, or human hepatocytes are shown in Table 10. In rat, dog, and human hepatocytes, GTI-2 exhibited increased T1 / 2, CL, and CL2 compared to GTI-11. INT The relative abundance values ​​of the major metabolites of peptides GTI-11 and GTI-2 after incubation with rat, dog, or human hepatocytes are shown in Table 11. Incubation of hepatocytes with GTI-2 resulted in increased abundance of the original sequence and longer metabolites compared to hepatocytes incubated with GTI-11.

[0289] TIFF2026507475000018.tif57165T 1 / 2 , half-life, CL INT , intrinsic clearance.

[0290] TIFF2026507475000019.tif174165TIFF2026507475000020.tif144165Conclusion Metabolism of GTI peptides in hepatocytes occurs mainly from the C-terminus. Modification of the C-terminal sequence alters metabolism in hepatocytes, resulting in increased clearance (CL). INT ), and the half-life (t 1 / 2 ) and increased abundance of the native sequence and longer metabolites after hepatocyte incubation.

[0291] Example 10 In vivo efficacy of GTI-2 in LPS-induced acute kidney injury (AKI) Materials and Methods To evaluate the in vivo activity of the GTI-2 peptide, a mouse model of sepsis-induced renal injury was used, in which 10 mg / kg lipopolysaccharide (LPS) was administered intraperitoneally (IP). Male C57Bl 6J mice (Skanbuur, Germany) aged 8–10 weeks were IP injected with 10 mg / kg LPS (E. coli O111:B4, L2630, Sigma Aldrich, Solna, Sweden).

[0292] Vehicle (sterile phosphate-buffered saline) or GTI-2 prepared at 10 mg / mL in vehicle was administered by intravenous (IV) bolus injection through the tail vein 0.5 h before LPS administration and 4 and 8 h after LPS administration as follows: Peptide GTI-2 was administered in a stock formulation of 2 mg / mL in PBS at a dose of 5 μL / g with vehicle or peptide for a total dose of 10 mg / kg.

[0293] After LPS administration, mice were allowed to recover for 24 hours, after which blood samples were collected from the tail vein at baseline, 8 hours, and 24 hours after LPS administration to assess renal injury biomarkers (blood urea nitrogen and serum creatinine) and liver injury marker aspartate aminotransferase (AST). Plasma creatinine (pCreatinine) (Biovision, Milpitas, CA, USA, K625), blood urea nitrogen (BUN) (Thermo Fisher Scientific, MA, USA, EIABUN), and AST (MAK055-1KT, Sigma-Aldrich, Solna, Sweden) were measured according to the manufacturer's instructions.

[0294] result Plasma creatinine, BUN, and AST concentrations (AST in mg / dL and nmol / mL / min) in mice with 24-hour LPS-induced renal and liver injury and treatment with PBS vehicle or intravenous administration of peptide GTI-2 are shown in Figure 17. Plasma creatinine, BUN, and AST concentrations in mice treated with GTI-2 were significantly lower than those in vehicle-treated mice at 24 hours (BUN) and both 8 and 24 hours (plasma creatinine and AST) after LPS treatment.

[0295] conclusion These data indicate that in the mouse LPS model of kidney and liver injury, GTI-2 significantly inhibits LPS-mediated kidney and liver injury by inhibiting changes in systemic indices of kidney function (plasma creatinine and BUN) and AST, a biomarker of liver injury, after 24 hours of IV administration.

[0296] Example 11 In vivo efficacy of intravenously or subcutaneously administered GTI-2 or GTI-86 in a cisplatin-induced acute kidney injury (AKI) model Materials and Methods The in vivo activity of peptides GTI-2 and GTI-86 was evaluated using intraperitoneal (IP) injection of 20 mg / kg cisplatin in a chemotherapy-induced nephropathy model in mice.

[0297] Eight- to ten-week-old male C57BL / 6 mice (Scanbur, Germany) were administered a single dose of 20 mg / kg cisplatin (Sigma, PHR1624) prepared in PBS containing 10% DMSO, or vehicle, via IP injection at 1.25 mL / kg body weight per mouse. Thirty minutes before cisplatin treatment and daily thereafter (every 24 h), GTI-2 or IV vehicle control was administered via IV bolus injection using 2 mL / kg peptide in PBS (n = 6 per group). GTI-2 and GTI-86 were administered intravenously via the tail vein at a volume of 50 μL / 25 g body weight (2 mL / kg) from a 5 mg / mL stock solution, delivering 10 mg / kg. In a second study, GTI-86 was tested by subcutaneous (SC) injection of vehicle or GTI-86 (5, 10, or 20 mg / kg) 30 minutes before cisplatin administration and daily thereafter. Each SC injection was split into two injection sites, with a dose volume of 8 mL / kg, or 200 μL for a 25 g mouse. For the 20 mg / kg dose, a 2.5 mg / mL stock formulation was used. For the 10 mg / kg and 5 mg / kg doses, the stock was diluted to 1.25 mg / mL and 0.625 mg / mL in PBS, respectively. SC injections into the flanks of mice were alternated to reduce irritation at the injection site.

[0298] After cisplatin administration, mice were allowed to recover for 4 days. Blood samples were taken from the tail vein at baseline and on day 4 after administration to assess renal injury biomarkers, blood urea nitrogen and plasma creatinine. Plasma creatinine (Biovision, Milpitas, CA, USA, K625) and blood urea nitrogen (Thermo Fisher, MA, USA, EIABUN) were measured according to the manufacturer's instructions.

[0299] result Plasma creatinine and BUN concentrations (mg / dL) in mice treated with PBS vehicle or the peptides GTI-2 and GTI-86 via IV or SC administration after cisplatin-induced renal injury (up to 4 days) are shown in Figure 18. On day 4 after cisplatin treatment, plasma creatinine and BUN concentrations were significantly lower in mice receiving IV injections of GTI-2 and GTI-86 compared with vehicle-treated mice. When administered SC, GTI-86 produced a dose-dependent decrease in both BUN and plasma creatinine. The polypeptides GTI-115, GTI-117, GTI-120, GTI-125, and GTI-126 all demonstrated efficacy in a murine model of cisplatin-induced renal injury.

[0300] conclusion These data indicate that GTI-2 and GTI-86, administered either intravenously or subcutaneously, significantly inhibit cisplatin-induced renal injury by inhibiting changes in systemic indices of renal function (plasma creatinine and BUN). The polypeptides GTI-115, GTI-117, GTI-120, GTI-125, and GTI-126 all demonstrated efficacy in the murine cisplatin-induced renal injury model.

[0301] Example 12 In vivo efficacy of subcutaneously administered A1M peptide GTI-86 in murine diabetic kidney disease (DKD) Materials and Methods We used a mouse model of streptozotocin (STZ)-induced diabetes and subsequent diabetic kidney disease (DKD) to evaluate the potential of the A1M peptide (GTI-86) to prevent further renal damage in mice with pre-existing diabetes and impaired renal function. STZ destroys pancreatic islet cells, inducing a type 1 diabetes model in mice. Diabetes was induced in 7-8 week-old male DBA / 2J mice (Janvier, France) by intraperitoneal (IP) injection of STZ (streptozotocin, S0130, Sigma-Aldrich, Solna, Sweden) at 40 mg / kg for 5 consecutive days (0.2 mL of a 5 mg / mL stock solution in 0.1 M citric acid / sodium citrate buffer, pH 4.5) (n = 8 per group). Baseline blood and urine samples were collected the day before the first STZ injection. Non-fasting blood glucose levels and renal dysfunction biomarkers (plasma creatinine and BUN) were subsequently assessed every 7 days to determine the diabetic phenotype and the degree of renal dysfunction. On day 19 after STZ administration, elevated blood glucose levels and renal function biomarkers, plasma creatinine and BUN, were observed compared with the STZ vehicle group. Following this, PBS vehicle or GTI-86 was administered subcutaneously once daily at doses of 0.2, 0.6, or 2 mg / kg (prepared at 2 mg / mL from an 8 mg / mL stock solution in PBS). Blood and urine samples were collected at the start of GTI-86 intervention (day 0), and then on days 14 and 28, to assess the urinary albumin-to-creatinine ratio (UACR) and plasma creatinine. Albumin and creatinine were assessed using Creatinine (K625, BioVision, USA) and Albuwell M (Mouse Albumin ELISA) kits (1011, Ethos Biosciences, USA) according to the manufacturer's instructions. Blood urea nitrogen (Figure 19D) was assessed using a Urea Nitrogen (BUN) Colorimetric Detection Kit (Thermo Fisher, MA, USA, EIABUN).

[0302] result Plasma glucose (non-fasting) increased in STZ-treated mice from day 19 after STZ treatment (Figure 19A). Plasma creatinine (Figure 19B) and urinary albumin / creatinine ratio (UACR, Figure 19C) increased from baseline (day 0) to day 19 after STZ treatment. In STZ-treated mice, plasma creatinine (Figure 19B) and urinary albumin / creatinine ratio (UACR, Figure 19C) further increased from day 0 to days 14 and 28 after the start of vehicle (PBS) intervention. Daily administration of GTI-86 peptide significantly inhibited the STZ-induced increases in plasma creatinine, BUN, and UACR in a dose-dependent manner on both days 14 and 28 after the start of once-daily subcutaneous administration of GTI-86. The polypeptides GTI-111, GTI-115, and GTI-117 also protected mouse kidneys from renal dysfunction in an STZ-induced diabetic nephropathy model.The polypeptides GTI-111, GTI-115, and GTI-117 also protected mouse kidneys from renal dysfunction in an STZ-induced diabetic nephropathy model.

[0303] conclusion These data demonstrate that the A1M peptide GTI-86, administered once daily SC, significantly inhibited diabetic nephropathy in a mouse STZ model of diabetic kidney disease by inhibiting changes in systemic indices of renal function (plasma creatinine) and nephropathy (UACR).

[0304] Example 13 In vivo efficacy of subcutaneously administered A1M peptide GTI-86 in the adriamycin-induced focal segmental glomerulosclerosis (FSGS) model in mice Materials and Methods We evaluated the potential of the A1M peptide GTI-86 to prevent renal damage in a mouse model of adriamycin (ADR)-induced focal segmental glomerulosclerosis (FSGS). The structural and functional damage in adriamycin-induced nephropathy mimics human chronic kidney disease accompanied by proteinuria. Male 7-8 week-old Balb / c mice (Janvier, France) received a single IV injection (5 mL / kg) of either aqueous injectable vehicle or adriamycin (ADR, Sigma-Aldrich, St. Louis, USA; doxorubicin hydrochloride, D1515) at a dose of 10 mg / kg (2.5 mL / kg of a 4 mg / mL stock solution) (n = 8 per group). Treatment with PBS vehicle or GTI-86 at 0.2, 0.6, 2, or 6 mg / kg subcutaneously once daily (administered or prepared at 2 mg / mL from an 8 mg / mL stock in PBS) was initiated 30 minutes before adriamycin administration (day 0). Blood and urine samples were collected at the start of GTI-86 intervention (day 0) and then on days 9 and 14 to assess urinary albumin / creatinine ratio (UACR), plasma creatinine, and blood urea nitrogen (BUN). Albumin and creatinine were assessed using creatinine (K625, BioVision, USA) and Albuwell M (mouse albumin ELISA) kits (1011, Ethos Biosciences, USA) according to the manufacturer's instructions. Blood urea nitrogen was assessed using a Urea Nitrogen (BUN) Colorimetric Detection Kit (Thermo Fisher, MA, USA, EIABUN).

[0305] result Adriamycin treatment significantly increased plasma creatinine, BUN, and UACR levels in adriamycin-treated and vehicle-treated mice on both days 9 and 14 after adriamycin induction (Figure 20). The adriamycin-induced increases in plasma creatinine (Figure 20A), BUN (Figure 20B), and UACR (Figure 20C) were all significantly inhibited by once-daily treatment with GTI-86 in a dose-dependent manner. GTI-2, GTI-86, GTI-111, GTI-115, and GTI-117 all demonstrated inhibition of adriamycin-induced renal dysfunction.

[0306] conclusion These data indicate that the peptide GTI-86, administered once daily by SC, inhibited changes in systemic indices of renal function (plasma creatinine and BUN) and significantly inhibited renal injury as measured by UACR in the mouse adriamycin model of FSGS. The polypeptides GTI-111, GTI-115, and GTI-117 also protected mouse kidneys from renal dysfunction in the STZ-induced diabetic nephropathy model.

[0307] Example 14 - Peptide Design Using the amino acid sequence (Kaumeyer et al., 1986) and the three-dimensional crystal structure (Meining and Skerra, 2012) of human A1M, we designed A1M peptides covering the Cys34 position and various portions of the adjacent region. Two peptides were designed and chemically synthesized. The peptides were 36 and 46 amino acids long, respectively, and named using a three-letter combination representing the N-terminal three amino acids followed by the total residue number. The peptides are exact copies of the A1M sequence. The amino acid sequence and location of the peptides within the A1M polypeptide are shown in Figure 6, and the location of peptide ENF-46 within the three-dimensional A1M structure is shown in Figure 7. As can be seen from these figures, the peptides are derived from the N-terminal third of the protein and include most of β-strand 1, loop 1 containing the Cys34 position, and β-strand 2. The thiol group at Cys34 has been reported to be involved in the reductase, heme-binding, and radical-binding activities of A1M (Allhorn et al., 2005; Rutardottir et al., 2016; Akerstrom et al., 2007), and therefore is present in both peptides. For structural reasons, both peptides contain the sequence motif GKWY (positions 23-26), the first of three conserved lipocalin motifs (MOTIF1) proposed to be important for the β-barrel structure of the lipocalin fold (Flower, 1996). However, as shown throughout this study, this motif is not essential for A1M activity.

[0308] The physicochemical properties of the peptides are summarized in Figure 3 and compared with those of the full-length A1M variant, A1M-035. All parameters were predicted using bioinformatics, except for solubility, which was estimated experimentally in PBS (pH 7.4) at room temperature. High solubility: >1 mM, moderate solubility: 0.1–1 mM, and low solubility: <0.1 mM. As is evident from the figure, SRI-36 had limited solubility and may not be optimal for further testing.

[0309] Example 15 - Determination of size, aggregation, and thiol group activity The size and aggregation of the peptide ENF-46 were investigated by SDS-PAGE with and without β-mercaptoethanol (Figure 8). A strong band was observed at the expected molecular weight of 5 kDa. A weak band was observed at approximately 10 kDa, suggesting that the peptide exists in monomeric and dimeric forms, as seen in the full-length E. coli-derived A1M mutant (Figure 8) and also in A1M isolated from human urine (Ekstrom and Berggard, 1977). Addition of the thiol-reducing agent β-mercaptoethanol to the sample prior to electrophoresis resulted in a decrease in dimers and an increase in monomers (Figure 8, right panel). This indicates that dimer formation is mediated in part by a disulfide bond between the two peptide chains, consistent with the redox activity of the cysteine ​​residues.

[0310] Example 16 - Measurement of heme binding Heme binding of the ENF-46 peptide was investigated through mobility shift and tryptophan fluorescence quenching during native PAGE. As shown in Figure 9, the migration of ENF-46 became significantly faster in the presence of heme, suggesting an increased negative charge. This was also the case for the full-length A1M mutant, although the difference was less significant than for ENF-46. Furthermore, the tryptophan fluorescence of the full-length A1M and ENF-46 peptides was suppressed in the presence of heme (Figure 9, right panel). Previous studies have shown that the anodic mobility shift and tryptophan fluorescence quenching are specific features of recombinant E. coli-derived A1M (Karnaukhova et al., 2014; Rutardottir et al., 2016). Therefore, these results indicate that the ENF-46 peptide can bind to heme groups.

[0311] Heme binding of the polypeptides GTI-86, GTI-111, and GTI-115 compared to A1M-035 was investigated by determining the UV-spectrophotometric absorbance shift (Figure 21). Absorption spectra were measured in the UV-Vis region from 240 to 700 nm using an NP80 NanoPhotometer (Implen GmbH, Germany) at 22 °C. Heme (Applichem, Germany, Hemin porcine A0942) was added at a concentration of 40 μM in Tris buffer (10 mM Tris-HCl, pH 8.0), and A1M-035 was added at a concentration of 45 μM in the same Tris buffer and incubated for 120 min before UV spectroscopic measurements were performed in a 96-well plate. Similarly, 40 μM heme in phosphate-buffered saline (pH 7.4) was incubated with 45 μM peptide in phosphate-buffered saline (pH 7.4) for 120 min, followed by UV spectrophotometry in a 96-well plate. The absorbance spectra were evaluated for changes in the heme absorption curve, with the heme peak absorbance at 380-390 nm. 45 μM A1M-035 increased the heme absorption height from 1.78 to 1.98 nm, indicating heme binding, and further shifted the peak from 385 nm to 422 nm, indicating heme reduction (Figure 21A). Incubation of the polypeptides with heme shifted the peak absorbance of heme from 1.42 at 385 nm to 2.04 (GTI-86), 1.87 (GTI-111), and 2.09 (GTI-115) at 373 nm, respectively, indicating heme binding by the polypeptides (Figures ​(Figure21B–D). The heme-binding properties of GTI-86 were maintained by Nle substitution (GTI-115) and D-amino acid substitution (GTI-111).

[0312] When GTI-86 was incubated with 40 μM heme (both in PBS) at increasing concentrations of 45, 90, 180, and 278 μM for 2 h, peptide binding to heme was observed. This binding was evidenced by an increase in the absorbance peak at 383 nm upon incubation of the peptide GTI-86 with heme (Fig. 21E). As the concentration of GTI-86 increased, there was an increase in absorbance between 400 and 470 nm, corresponding to increased reductase activity with higher GTI-86 concentrations (Fig. 21E).

[0313] Example 17 - Measurement of cytochrome c reduction Reductase activity was examined by cytochrome c reduction. Enzymatic reduction of cytochrome c in the presence of NADH or NADPH has been reported for human A1M purified from urine and recombinantly produced in E. coli (Allhorn et al., 2005). In this study, reductase activity was completely dependent on the Cys34 thiol group; no reduction was observed when the free cysteine ​​was inhibited with iodoacetamide or when a mutant A1M in which the Cys34 residue was replaced with a serine residue (A1M-C34S) was used. As shown in Figure 10, the appearance of an absorption peak at 550 nm indicates that cytochrome c was reduced by both A1M-wt and the peptide ENF-46 in the presence of NADPH. Against a 100 μM cytochrome c / 100 μM NADPH mixture, 10 μM full-length A1M and 20 μM ENF-46 peptide showed similar reducing potency as estimated by the 550 nm peak height (FIG. 10).

[0314] Example 18 - Determination of cytoprotection The protective ability of free heme against cell damage was measured using three nucleated cell lines and human RBCs (Figure 11). First, K562 cells, a human erythroid cell line grown in suspension, were exposed to 100 μM heme for 1 h in the presence of full-length A1M, peptide SRI-36, or the control protein ovalbumin. Cell viability was measured by LDH leakage into the medium (Figure 11A). Heme alone induced massive LDH leakage, i.e., cell death. Addition of full-length A1M (wt and 035 mutant) prevented cell damage as expected, showing significant but slight inhibition at 3 μM and approximately 90% inhibition at 10 μM. This is consistent with previously published results (Olsson et al., 2008). Peptide SRI-36 showed slight but significant inhibition of LDH leakage at 10 μM and 20 μM, as well as 80–90% inhibition at 50 μM. The control protein ovalbumin showed no significant inhibition.

[0315] HK-2 cells, an adherent human renal (tubular) cell line, were exposed to different concentrations of heme for 2 hours in the presence of 10 μM A1M-035 or 30 μM ENF-46 peptide. Cell proliferation, measured by WST-1 uptake, was inhibited in a dose-dependent manner with increasing heme concentrations. At all concentrations, A1M-035 and ENF-46 reversed heme-induced inhibition of cell proliferation (Figure 11B). Using 30 μM heme, the effects of dilution series of A1M-035, ENF-46, and the non-A1M reference peptide, SWT-21, were measured after 2 hours of incubation. A1M-035 and ENF-46, but not SWT-21, produced significant dose-dependent inhibition (Figure 11C).

[0316] The protective effect of ENF-46 was also observed in the adherent human hepatocyte cell line HepG2, as measured by heme-induced LDH leakage after 1 hour of incubation. As shown in Figure 11D, 30 and 60 μM ENF-46, as well as 10 μM full-length A1M-035, produced significant, almost complete, inhibition of heme-induced LDH leakage. A slight but significant inhibition was observed with 7.5 and 15 μM ENF-46.

[0317] Recently, A1M was shown to protect RBCs from heme-induced cell damage, i.e., hemolysis (Kristiansson et al., 2020). Here, we were able to show that this was also achieved by the peptide ENF-46. Figure 11E shows the effect of a dilution series of ENF-46 (1.25–50 μM) on LDH leakage from RBCs incubated with 30 μM heme for 3 h. At concentrations of 25 μM and above, significant inhibition of heme-induced hemolysis was observed.

[0318] Array Overview [ka] TIFF2026507475000022.tif237165TIFF2026507475000023.tif238165TIFF202 6507475000024.tif242165TIFF2026507475000025.tif225165TIFF20265074750 00026.tif232165TIFF2026507475000027.tif253160TIFF2026507475000028.t if235165TIFF2026507475000029.tif233165TIFF2026507475000030.tif244170 TIFF2026507475000031.tif234165TIFF2026507475000032.tif237165TIFF202 6507475000033.tif234165TIFF2026507475000034.tif228165TIFF20265074750 00035.tif231165TIFF2026507475000036.tif236165TIFF2026507475000037.t if233165TIFF2026507475000038.tif252165TIFF2026507475000039.tif168165 References Allhorn M, Klapyta A, Akerstrom B. Redox properties of the lipocalin alpha1-microglobulin: reduction of cytochrome c, hemoglobin, and free iron. Free Radic. Biol. Med. 2005 Mar 1;38(5):557-67. Ekstrom B,Berggard I.Human alpha1-microglobulin. Purification procedure,chemical and physiochemical properties.J Biol. Chem.1977 Nov 25;252(22):8048-57. Flower,Biochem J.1996 Aug 15;318(Pt 1)(Pt 1):1-14. Karnaukhova,E.et al.,,Front Physiol,2014,5,p.465. Kaumeyer JF,Polazzi JO,Kotick MP.The mRNA for a proteinase inhibitor related to the HI-30 domain of inter-alpha-trypsin inhibitor also encodes alpha-1-microglobulin (protein HC).Nucleic Acids Res. 1986 Oct 24;14(20):7839-50. Kristiansson A, Bergwik J, Alattar AG, Flygare J,Gram M,Hansson SR,Olsson ML,Storry JR,Allhorn M,Akerstrom B.Human radical scavenger α1-microglobulin protects against hemolysis in vitro and α1-microglobulin knockout mice exhibit a macrocytic anemia phenotype.Free Radic Biol.Med.2020 Feb 21:S0891-5849(19)32350-0. Meining W,Skerra A.The crystal structure of human α(1)-microglobulin reveals a potential haem-binding site.Biochem.J.2012 Jul 15;445(2):175-82. Olsson, M. G.et al.,Free Radic Res,2008,42,p.725-36. Rutardottir,S.et al.,Biochim Biophys Acta, 2016,1864,p.29-41. Akerstrom B,Gram M. A1M, an extravascular tissue cleaning and housekeeping protein. Free Radic Biol. Med. 2014 Sep;74:274-82. Akerstrom,B.et al.,J Biol Chem,2007,282,p.31493-503. Yang PY,Zou H,Chao E,Sherwood L,Nunez V, Keeney M,Ghartey-Tagoe E, Ding Z, Quirino H, Luo X, Welzel G, Chen G, Singh P, Woods AK,Schultz PG, Shen W.Engineering a long-acting,potent GLP-1 analog for microstructure-based transdermal delivery. Proc Natl Acad Sci U S A.2016 Apr 12;113(15):4140-5. doi:10.1073 / pnas.1601653113.Epub 2016 Mar 28.PMID:27035989;PMCID:PMC4839405. Zhao F,Zhou Q,Cong Z,Hang K,Zou X,Zhang C,Chen Y,Dai A,Liang A,Ming Q,Wang M,Chen LN, Xu P,Chang R,Feng W,Xia T,Zhang Y,Wu B,Yang D,Zhao L,Xu HE,Wang MW.Structural insights into multiplexed pharmacological actions of tirzepatide and peptide 20 at the GIP,GLP-1 or glucagon receptors.Nat Commun.2022 Feb 25;13(1):1057.doi:10.1038 / s41467-022-28683-0.PMID:35217653;PMCID:PMC8881610.

Claims

1. A pharmaceutical agent comprising a polypeptide, the polypeptide comprising: i. consisting of 12 to 80 amino acid residues, said polypeptide having the amino acid sequence GSTCPWLKKIX １ (SEQ ID NO: 20), １ is M, K, R, or Nle, or ii. The polypeptide consists of 30 to 80 amino acid residues, and the polypeptide has the amino acid sequence GATEAEISMTSTX ２ WRKGVCEETSGAYEKTD (SEQ ID NO: 21), ２ is H, R, or K.

2. an agent comprising a polypeptide consisting of 10 to 80 amino acid residues, said polypeptide comprising a fragment consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29; X ３ is selected from D, N, or G; X ４ is selected from M, K, R, or Nle; X ５ is selected from H, R, or K; X ６ is selected from D, N, or E; X ７ is selected from M or Nle, the fragment has a length of at least 10 amino acid residues, The agent, wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO:

30.

3. 1. A pharmaceutical agent comprising a polypeptide consisting of at least 10 amino acid residues, said polypeptide comprising a fragment of alpha-1-microglobulin or a variant of alpha-1-microglobulin; The fragment is at least 10 amino acid residues in length. The agent, wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO:

30.

4. The polypeptide has the amino acid sequence GSTCPWLKKIX １ (SEQ ID NO: 20), １ 10. The method of claim 1, wherein is M, K, R, or Nle.

5. The polypeptide has the amino acid sequence GATEAEISMTSTX ２ WRKGVCEETSGAYEKTD (SEQ ID NO: 21), ２ 5. The method of claim 1, wherein is H, R, or K.

6. 2. The method of claim 1, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIM (SEQ ID NO: 22).

7. 2. The method of claim 1, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIK (SEQ ID NO: 23).

8. 2. The method of claim 1, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIR (SEQ ID NO: 24).

9. 10. The method of claim 1, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKINle (SEQ ID NO: 145).

10. 10. The method of claim 1, wherein the polypeptide comprises the amino acid sequence GATEAEISMTSTHWRKGVCEETSGAYEKTD (SEQ ID NO: 25).

11. 10. The method of claim 1, wherein the polypeptide comprises the amino acid sequence GATEAEISMTSTRWRKGVCEETSGAYEKTD (SEQ ID NO: 26).

12. 10. The method of claim 1, wherein the polypeptide comprises the amino acid sequence GATEAEISMTSTKWRKGVCEETSGAYEKTD (SEQ ID NO: 27).

13. 2. The method of claim 1, wherein the polypeptide comprises a fragment of any one of SEQ ID NO:28 or SEQ ID NO:

29.

14. 2. The agent of any one of the preceding claims, wherein the polypeptide comprises a fragment of any one of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:

19.

15. 2. The method of claim 1, wherein the polypeptide is a fragment of any one of SEQ ID NO:28 or SEQ ID NO:

29.

16. 2. The agent of any one of the preceding claims, wherein the polypeptide is a fragment of any one of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:

19.

17. 10. The method of claim 1, wherein the polypeptide consists of at least 13 amino acid residues, such as at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, such as at least 24 amino acid residues.

18. 10. The agent of any one of the preceding claims, wherein the polypeptide consists of 79 or fewer amino acid residues, such as 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56 or fewer, for example 55 or fewer amino acid residues.

19. 2. The method of claim 1, wherein the polypeptide comprises amino acid residues corresponding to C34 or C72 of SEQ ID NO:

30.

20. 2. The method of claim 1, wherein the polypeptide comprises an amino acid residue corresponding to C34 of SEQ ID NO:

30.

21. 2. The method of claim 1, wherein the polypeptide comprises an amino acid residue corresponding to C72 of SEQ ID NO:

30.

22. the polypeptide i. DDDDKQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 1); ii. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 2); iii. DDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIM (SEQ ID NO: 3), iv. KGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 4), v. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHRKGVCEETSGAYEK (SEQ ID NO: 6), vi. DDDDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRM (SEQ ID NO: 7), vii. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEET (SEQ ID NO: 8); viii. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRK (SEQ ID NO: 9); ix. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMT (SEQ ID NO: 10), x. GSTCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 11), xi. NLAIGSTCPWLKKIMDR (SEQ ID NO: 12), xii. GEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDG (SEQ ID NO: 13), xiii. GATEAEISMTSTHWRKGVCEETSGAYEKTD (SEQ ID NO: 14), xiv. GSTCPWLKKIMDRMTVSTLDLGEG (SEQ ID NO: 31), xv. GSTCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 32), and xvi. The agent of any one of the preceding claims, comprising an amino acid sequence selected from the group consisting of: GSTCPWLKKIMDRMTVSTLVLGEGATE (SEQ ID NO: 33).

23. the polypeptide i. DDDDKQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 1); ii. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 2); iii. DDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIM (SEQ ID NO: 3), iv. KGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 4), v. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHRKGVCEETSGAYEK (SEQ ID NO: 6), vi. DDDDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRM (SEQ ID NO: 7), vii. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEET (SEQ ID NO: 8); viii. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRK (SEQ ID NO: 9); ix. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMT (SEQ ID NO: 10), x. GSTCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 11), xi. NLAIGSTCPWLKKIMDR (SEQ ID NO: 12), xii. GEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDG (SEQ ID NO: 13), xiii. GATEAEISMTSTHWRKGVCEETSGAYEKTD (SEQ ID NO: 14), xiv. GSTCPWLKKIMDRMTVSTLDLGEG (SEQ ID NO: 31), xv. GSTCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 32), and xvi. The agent of any one of the preceding claims, selected from the group consisting of: GSTCPWLKKIMDRMTVSTLVLGEGATE (SEQ ID NO: 33).

24. 10. The method of claim 1, wherein the polypeptide is effective in reducing cytochrome c.

25. 10. The agent of any one of the preceding claims, wherein the polypeptide has at least 35% of the cytochrome c reducing activity of SEQ ID NO: 16, such as at least 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the cytochrome c reducing activity of SEQ ID NO:

16.

26. The polypeptide is an EC ５０ 10. An agent according to any one of the preceding claims, having a value of 500 μM or less, such as 400 μM, 300 μM, 200 μM, 100 μM or 50 μM or less.

27. 10. The agent of any one of the preceding claims, wherein the polypeptide binds heme.

28. 10. The method of claim 1, wherein the polypeptide is an antioxidant.

29. 10. An agent according to any one of the preceding claims, wherein the agent further comprises a moiety capable of altering a biophysical property of the polypeptide.

30. A pharmaceutical agent comprising a biologically active polypeptide, said biologically active polypeptide comprising: i. consisting of 12 to 80 amino acid residues, said polypeptide having the amino acid sequence GSTCPWLKKIX １ (SEQ ID NO: 20), １ is M, K, R, or Nle, or ii. The polypeptide consists of 30 to 80 amino acid residues, and the polypeptide has the amino acid sequence GATEAEISMTSTX ２ WRKGVCEETSGAYEKTD (SEQ ID NO: 21), ２ is H, R, or K.

31. 1. An agent comprising a biologically active polypeptide consisting of 10 to 80 amino acid residues, said polypeptide comprising a fragment consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29; X ３ is selected from D, N, or G; X ４ is selected from M, K, R, or Nle; X ５ is selected from H, R, or K; X ６ is selected from D, N, or E; X ７ is selected from M or Nle, the fragment is at least 10 amino acid residues in length; The agent, wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO:

30.

32. 1. An agent comprising a biologically active polypeptide consisting of at least 10 amino acid residues, said polypeptide comprising a fragment consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, or SEQ ID NO:29; X ３ is selected from D, N, or G; X ４ is selected from M, K, R, or Nle; X ５ is selected from H, R, or K; X ６ is selected from D, N, or E; X ７ is selected from M or Nle, the fragment is at least 10 amino acid residues in length; The agent, wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO:

30.

34. 1. A pharmaceutical agent comprising a biologically active polypeptide having a length of 10 to 92 amino acid residues, said polypeptide being human wild-type alpha-1-microglobulin, A1M, SEQ ID NO: 83: GPVPTPPDNI QVQENFNISR IYGKWYNLAI GSTCPWLKKI MDRMTVSTLV LGEGATEAEI SMTSTRWRKG VCEETSGAYE KTDTDGKFLY HK, The agent, wherein the fragment has a length of 10 to 92 amino acid residues, the fragment comprises one or more of Y22, C34, K69, or K92, and the position refers to a position in human wild-type A1M (SEQ ID NO: 30).

35. 10. The agent of any one of the preceding claims, wherein the fragment is selected from positions 1 to 92 of human wild-type A1M, and wherein the fragment has at least 50% identity with the corresponding fragment of human wild-type A1M (SEQ ID NO: 30).

36. 10. The method of claim 1, wherein the fragment has a length of 15 to 80 amino acid residues.

37. 10. The method of claim 1, wherein the fragment has a length of 20 to 75 amino acid residues.

38. 10. The agent of any one of the preceding claims, wherein the fragment comprises C34 and the position refers to a position in human wild-type A1M (SEQ ID NO: 30).

39. 10. The agent of any one of the preceding claims, wherein the fragment comprises Y22 and the position refers to the position in human wild-type A1M (SEQ ID NO: 30).

40. 10. The agent of any one of the preceding claims, wherein the fragment comprises K92, and the position refers to the position in human wild-type A1M (SEQ ID NO: 30).

41. The fragment is human wild-type A1M (SEQ ID NO: 84): YGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKG VCEETSGAYEKTDTDGKFLYHK, wherein the fragment has a length of 10 to 71 amino acid residues.

42. The fragment is located at any of the following positions in human wild-type A1M (SEQ ID NO: 30): i) positions 22 to 62 (SEQ ID NO: 85); ii) positions 26 to 62 (SEQ ID NO: 86); iii) positions 30 to 62 (SEQ ID NO: 87); iv) positions 31 to 56 (SEQ ID NO: 88); v) positions 26 to 56 (SEQ ID NO: 89); vi) positions 26 to 92 (SEQ ID NO: 90); vii) positions 30 to 92 (SEQ ID NO: 91), or viii) An agent according to any one of the preceding claims, selected from one of positions 31 to 92 (SEQ ID NO: 92).

43. The following human wild-type A1M sequence (SEQ ID NO: 30): i) positions 22 to 62 (SEQ ID NO: 85); ii) positions 26 to 62 (SEQ ID NO: 86); iii) positions 30 to 62 (SEQ ID NO: 87); iv) positions 31 to 56 (SEQ ID NO: 88); v) positions 26 to 56 (SEQ ID NO: 89); vi) positions 26 to 92 (SEQ ID NO: 90); vii) positions 30 to 92 (SEQ ID NO: 91); viii) positions 31 to 92 (SEQ ID NO: 92), an amino acid sequence having 50% or more sequence identity with one of the following:

44. The following human wild-type A1M sequence: i) positions 22 to 62 (SEQ ID NO: 85); ii) positions 26 to 62 (SEQ ID NO: 86); iii) positions 30 to 62 (SEQ ID NO: 87); iv) positions 31 to 56 (SEQ ID NO: 88); v) positions 26 to 56 (SEQ ID NO: 89); vi) positions 26 to 92 (SEQ ID NO: 90); vii) positions 30 to 92 (SEQ ID NO: 91); viii) An agent according to any one of the preceding claims, comprising an amino acid sequence corresponding to one of positions 31 to 92 (SEQ ID NO: 92).

45. 10. The method of claim 9, wherein the polypeptide at the N-terminus and / or C-terminus comprises an amino acid sequence Z having a length of 1 to 20 amino acid residues.

46. 10. The agent of any one of the preceding claims, wherein Z comprises one or more lysine, K, residue(s), one or more glutamic acid, E, residue(s), one or more arginine, R, residue(s) and / or one or more aspartic acid, D, residue(s).

47. 10. An agent according to any one of the preceding claims, wherein Z comprises one or more hydrophobic amino acid residue(s).

48. 10. The agent of any one of the preceding claims, wherein Z comprises one or more histidine, H, residue(s), one or more glutamine, G, residue(s), and / or one or more isoleucine, I, residue(s).

49. Y-Π1-K, Y-Π2-C, or C-Π3-K, Π1 has at least 50% sequence identity with SEQ ID NO: 100: GKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYH (SEQ ID NO: 100); Π2 has at least 50% sequence identity with SEQ ID NO:101: GKWYNLAIGST) (SEQ ID NO:101); 10. The method of claim 1, wherein Π3 has at least 50% sequence identity with SEQ ID NO: 102: PWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYH (SEQ ID NO: 102).

50. 10. An agent according to any one of the preceding claims, having at least 50% sequence identity with SEQ ID NO:

83.

51. 1. An agent having a length of 10 to 100 amino acid residues, wherein the polypeptide comprises a fragment selected from human wild-type alpha-1-microglobulin, A1M, positions 1 to 183 of SEQ ID NO: 30, wherein the fragment has a length of 10 to 100 amino acid residues and comprises one or more of Y22, C34, K69, K92, K118, H122, Y132, L180, I181, P182, and R183.

52. 10. The agent of any one of the preceding claims, wherein the fragment is selected from positions 1 to 183 of human wild-type A1M (SEQ ID NO: 30), and wherein the fragment has at least 50% identity with the corresponding fragment of human wild-type A1M (SEQ ID NO: 30).

53. 10. The method of claim 1, wherein the fragment has a length of 15 to 80 amino acid residues.

54. 10. The method of claim 1, wherein the fragment has a length of 20 to 75 amino acid residues.

55. 10. The agent of any one of the preceding claims, wherein the fragment comprises one or more of Y22, C34, K69, K92, K118, H122, and Y132.

56. 10. The method of claim 1, wherein the fragment comprises one or more of K92, K118, H122, Y132, L180, I181, P182 and R183.

57. 10. The method of claim 1, wherein the fragment comprises one or more of L180, I181, P182 and R183.

58. The method of any one of the preceding claims, wherein the fragment is selected from positions 22 to 183 of human wild-type A1M (SEQ ID NO: 105).

59. The method of any one of the preceding claims, wherein the fragment is selected from positions 22 to 160 of human wild-type A1M (SEQ ID NO: 106).

60. 2. The method of claim 1, wherein the fragment is selected from positions 51 to 155 of human wild-type A1M (SEQ ID NO: 107).

61. The method of any one of the preceding claims, wherein the fragment is selected from positions 59 to 135 of human wild-type A1M (SEQ ID NO: 108).

62. The method of any one of the preceding claims, wherein the fragment is selected from positions 92 to 183 of human wild-type A1M (SEQ ID NO: 109).

63. The method of any one of the preceding claims, wherein the fragment is selected from positions 92 to 135 of human wild-type A1M (SEQ ID NO: 110).

64. 10. An agent according to any one of the preceding claims, having a length corresponding to between 10 and 75 amino acid residues, such as between 10 and 60 amino acid residues.

65. The fragment has the following range of human wild-type A1M: i) positions 22 to 135 (SEQ ID NO: 110); ii) positions 26 to 135 (SEQ ID NO: 111); iii) positions 30 to 135 (SEQ ID NO: 112); iv) positions 31 to 135 (SEQ ID NO: 113); v) positions 61 to 135 (SEQ ID NO: 114); vi) positions 66 to 135 (SEQ ID NO: 115); vii) positions 87 to 135 (SEQ ID NO: 116); viii) positions 92 to 135 (SEQ ID NO: 117).

66. The following human wild-type A1M sequence (SEQ ID NO: 30): i) positions 22 to 135 (SEQ ID NO: 110); ii) positions 26 to 135 (SEQ ID NO: 111); iii) positions 30 to 135 (SEQ ID NO: 112); iv) positions 31 to 135 (SEQ ID NO: 113); v) positions 59 to 135 (SEQ ID NO: 108); vi) positions 66 to 135 (SEQ ID NO: 115); vii) positions 87 to 135 (SEQ ID NO: 116); viii) The agent according to any one of the preceding claims, comprising an amino acid sequence having 50% or more sequence identity with any one of positions 92 to 135 (SEQ ID NO: 117).

67. The following human wild-type A1M sequence (SEQ ID NO: 30): i) positions 59 to 135 (SEQ ID NO: 108); ii) positions 66 to 135 (SEQ ID NO: 115); iii) positions 87 to 135 (SEQ ID NO: 116); iv) positions 92 to 135 (SEQ ID NO: 117).

68. 10. The method of claim 9, wherein the polypeptide at the N-terminus and / or C-terminus comprises an amino acid sequence Z having a length of 1 to 20 amino acid residues.

69. 10. The agent of any one of the preceding claims, wherein Z comprises one or more lysine, K, residue(s), one or more glutamic acid, E, residue(s), one or more arginine, R, residue(s) and / or one or more aspartic acid, D, residue(s).

70. 10. An agent according to any one of the preceding claims, wherein Z comprises one or more hydrophobic amino acid residue(s).

71. 10. The agent of any one of the preceding claims, wherein Z comprises one or more histidine, H, residue(s), one or more glutamine, G, residue(s), and / or one or more isoleucine, I, residue(s).

72. The N-terminal polypeptide has the following sequence: i) HHHHHHHHGGGGGGIEGR (8H5GIEGR) (SEQ ID NO: 93); ii) HHHHHHHHDDDDDK (8H4DK) (SEQ ID NO: 94); iii) HHHHHHDDDDDK (6H4DK) (SEQ ID NO: 95), or iv) An agent according to any one of the preceding claims, comprising Z which is or comprises one or more of: HHHHHHHH(8H) (SEQ ID NO: 96).

73. The C-terminal polypeptide has the following sequence: i) RGIEGGGGGGHHHHHHHH (RGIE5G8H) (SEQ ID NO: 97) ii) KDDDDDHHHHHHHH (K4D8H) (SEQ ID NO: 98) iii) KDDDDDHHHHHH (K4D6H) (SEQ ID NO: 99); iv) An agent according to any one of the preceding claims, comprising Z which is or comprises one or more of: HHHHHHHH(8H) (SEQ ID NO: 96).

72. 10. The method of any one of the preceding claims, comprising an amino acid sequence having at least 50% sequence identity to SRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 103).

73. 10. The agent of any one of the preceding claims, having at least 50% sequence identity with ENFNISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAE (SEQ ID NO: 104).

74. 10. The agent according to any one of the preceding claims, wherein the polypeptide comprises one or more further modifications.

75. 10. The agent according to any one of the preceding claims, wherein the further modification is a non-peptide modification.

76. 10. The agent according to any one of the preceding claims, wherein the further modification is a substitution of one or more atoms or one or more moieties.

77. 10. The agent according to any one of the preceding claims, wherein the further modification is a peptide-based modification.

78. 10. The method of claim 1, wherein the polypeptide is further modified at the N-terminus.

79. 10. The method of claim 1, wherein the polypeptide is further modified at the C-terminus.

80. 10. The method of claim 1, wherein the polypeptide is further modified at the side chain.

81. 2. The method of claim 1, wherein the polypeptide is acetylated at the N-terminus.

82. 10. The method of claim 1, wherein the polypeptide is amidated at the C-terminus.

83. 10. The method of claim 1, wherein the polypeptide comprises a substitution with a non-natural amino acid.

84. 10. The method of claim 1, wherein the polypeptide comprises substitutions with D-amino acids.

85. 10. An agent according to any one of the preceding claims, wherein a residue in the polypeptide is replaced with the same amino acid in the D-configuration.

86. 10. The method of claim 1, wherein the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions with D-amino acids.

87. the polypeptide a. NLAIGSTCP[D-Trp]LKKIMDR (SEQ ID NO: 66); b. NLAIGSTCPW[D-Leu]KKIMDR (SEQ ID NO: 67); c. NLAIGSTCPWLK[D-Lys]IMDR (SEQ ID NO: 68); d. GSTCPWLK[D-Lys]IMDRMTVSTLVLGEG (SEQ ID NO: 69); e. GSTCPWLKKIMD[D-Arg]MTVSTLVLGEG (SEQ ID NO: 70); f. GSTCPWLKKIMDRMTVSTLV[D-Leu]GEG (SEQ ID NO: 71); g. GSTCPWLKKIMDRMTVSTL[D-Asp]LGEG (SEQ ID NO: 72); h. G[D-Ser]STCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 81), and i. a sequence selected from the group consisting of: [D-Asn]NLAIGSTCPWLKKIMDR (SEQ ID NO: 82); or a sequence having at least 70% sequence identity thereto, such as at least 75%, at least 80%, at least 85%, at least 90%, for example at least 95% sequence identity thereto.

88. the polypeptide a. NLAIGSTCP[D-Trp]LKKIMDR (SEQ ID NO: 66); b. NLAIGSTCPW[D-Leu]KKIMDR (SEQ ID NO: 67); c. NLAIGSTCPWLK[D-Lys]IMDR (SEQ ID NO: 68); d. GSTCPWLK[D-Lys]IMDRMTVSTLVLGEG (SEQ ID NO: 69); e. GSTCPWLKKIMD[D-Arg]MTVSTLVLGEG (SEQ ID NO: 70); f. GSTCPWLKKIMDRMTVSTLV[D-Leu]GEG (SEQ ID NO: 71); g. GSTCPWLKKIMDRMTVSTL[D-Asp]LGEG (SEQ ID NO: 72); h. G[D-Ser]STCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 81), and i. [D-Asn]LAIGSTCPWLKKIMDR (SEQ ID NO: 82).

89. 10. The method of claim 1, wherein one, two or more methionine residues of the polypeptide are substituted with norleucine.

90. 10. An agent according to any one of the preceding claims, wherein the methionine residue corresponding to M41 of A1M is substituted with norleucine in said polypeptide.

91. 10. An agent according to any one of the preceding claims, wherein the methionine residue corresponding to M44 of A1M is substituted with norleucine in said polypeptide.

92. 10. An agent according to any one of the preceding claims, wherein both of the methionine residues corresponding to M41 and M44 of A1M are substituted with norleucine in the polypeptide.

93. the polypeptide i. GSTCPWLKKI[Nle]DR[Nle]TVSTLVLGEG (SEQ ID NO: 139); ii. GSTCPWLKKI[Nle]DR[Nle]TVSTL[D-Asp]LGEG (SEQ ID NO: 140); GSTCPWLKKI[Nle]DR[Nle]TVSTLVLGEG[D-Ala][D-Thr][D-Glu] (SEQ ID NO: 141), iv. GSTCPWLKKI[Nle]DR[Nle]TKSTLVLGEG (SEQ ID NO: 142), v. EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TVSTLVLGE (SEQ ID NO: 143), and vi. The agent of any one of the preceding claims, selected from the group consisting of: GSTCPWLKKI[Nle]DR[Nle]TVSTL[D-Asp]LGEG (SEQ ID NO: 144).

94. 10. The method of claim 1, wherein the polypeptide is PEGylated.

95. 10. The agent of any one of the preceding claims, wherein the PEGylation is at the N-terminus, C-terminus, or on a side chain.

96. 2. The agent of any one of the preceding claims, wherein the PEGylation is at the C-terminus.

97. 10. The agent according to any one of the preceding claims, wherein the PEGylation is with PEG having a molecular weight of 60 kDa or less, such as with PEG having a molecular weight of 50 kDa or less, for example 40 kDa or less, such as 30 kDa or less, for example 20 kDa or less, such as 15 kDa or less, for example 10 kDa or less, such as 5 kDa or less.

98. 2. The method of claim 1, wherein the PEGylation is by any one of PEG1 to PEG50.

99. 2. The method of claim 1, wherein the PEGylation is by any one of PEG1 to PEG30.

100. 2. The method of claim 1, wherein the PEGylation is by any one of PEG2 to PEG20.

101. 2. The agent of any one of the preceding claims, wherein the PEGylation is with PEG2, PEG5, PEG10, or PEG20.

102. the PEGylated polypeptide is a. GSTCPWLKKIMDRMTVSTLVLGEG-PEG2-CONH ２ (SEQ ID NO: 34), b. GSTCPWLKKIMDRMTVSTLDLGEG-PEG2-CONH ２ (SEQ ID NO: 35), c. Ac-NLAIGSTCPWLKKIMDR-CONH ２ -PEG2 (SEQ ID NO: 39), d. Ac-DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT-PEG2 (SEQ ID NO: 40); e. NLAIGSTCPWLKKIMDR-PEG5 (SEQ ID NO: 60); f. NLAIGSTCPWLKKIMDR-PEG10 (SEQ ID NO: 61); g. GSTCPWLKKIMDRMTVSTLVLGEG-PEG5 (SEQ ID NO: 62); h. GSTCPWLKKIMDRMTVSTLVLGEG-PEG10 (SEQ ID NO: 63), i. NLAIGSTCPWLKKIMDR-PEG20 (SEQ ID NO: 64), and j. GSTCPWLKKIMDRMTVSTLVLGEG-PEG20 (SEQ ID NO: 65); or a sequence having at least 70% sequence identity thereto, such as at least 75%, at least 80%, at least 85%, at least 90%, for example at least 95% sequence identity thereto.

103. the PEGylated polypeptide is a. GSTCPWLKKIMDRMTVSTLVLGEG-PEG2-CONH ２ (SEQ ID NO: 34) b. GSTCPWLKKIMDRMTVSTLDLGEG-PEG2-CONH ２ (SEQ ID NO: 35) c. Ac-NLAIGSTCPWLKKIMDR-CONH ２ -PEG2 (SEQ ID NO: 39) d. Ac-DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT-PEG2 (SEQ ID NO: 40) e. NLAIGSTCPWLKKIMDR-PEG5 (SEQ ID NO: 60) f. NLAIGSTCPWLKKIMDR-PEG10 (SEQ ID NO: 61) g. GSTCPWLKKIMDRMTVSTLVLGEG-PEG5 (SEQ ID NO: 62) h. GSTCPWLKKIMDRMTVSTLVLGEG-PEG10 (SEQ ID NO: 63) i. NLAIGSTCPWLKKIMDR-PEG20 (SEQ ID NO: 64), and j. The agent of any one of the preceding claims, selected from the group consisting of: GSTCPWLKKIMDRMTVSTLVLGEG-PEG20 (SEQ ID NO: 65).

104. 10. The method of claim 1, wherein the polypeptide further comprises an albumin binding domain or a fragment of an albumin binding domain.

105. 10. The agent of any one of the preceding claims, wherein the albumin binding domain has the amino acid sequence of SEQ ID NO:

118.

106. The method according to any one of the preceding claims, wherein the albumin binding domain fragment consists of 10 to 50 amino acid residues.

107. 10. The agent of any one of the preceding claims, wherein the albumin binding domain or fragment thereof is attached to the N-terminus of the polypeptide, the C-terminus of the polypeptide, or a side chain of a peptide.

108. 10. The agent of any one of the preceding claims, wherein the albumin binding domain or fragment thereof is attached to the polypeptide via a linking moiety.

109. 10. The method of claim 1, wherein the linker is a PEGylated linker.

110. 10. The agent of any one of the preceding claims, wherein the linking moiety is a peptide linker.

111. The connecting portion is a. GGGGGSAS (SEQ ID NO: 121), and GGGGSGGGGSGGGGGSAS (SEQ ID NO: 122).

112. 10. The method of any one of the preceding claims, wherein the albumin domain binding fragment comprises or consists of the amino acid sequence LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVALKLHILAALP (SEQ ID NO: 118) or SDFYKRLINKAKTVEGVialKLHILAALP (SEQ ID NO: 119).

113. the polypeptide GSTCPWLKKIMDRMTVSTLVLGEGSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 36), b. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 37); c. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSGGGGGSGGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 38), d. NLAIGSTCPWLKKIMDRGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 41), e. NLAIGSTCPWLKKIMDRGGGGSGGGGGSGGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 42), f. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 43), g. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSGGGGGSGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 44) and h. EEKYNLAIGSTCPWLKKIMDRMTESTLVLGEGGGGGSASLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVALKLHILAALP (SEQ ID NO: 163); or a sequence having at least 70% sequence identity thereto, such as at least 75%, at least 80%, at least 85%, at least 90%, for example at least 95% sequence identity thereto.

114. the polypeptide GSTCPWLKKIMDRMTVSTLVLGEGSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 36), b. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 37); c. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSGGGGGSGGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 38), d. NLAIGSTCPWLKKIMDRGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 41), e. NLAIGSTCPWLKKIMDRGGGGSGGGGGSGGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 42), f. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 43), and DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSGGGGGSGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 44).

115. 10. The method of claim 1, wherein the polypeptide comprises modifications of one or more amino acid residues.

116. 10. The agent according to any one of the preceding claims, wherein the modification is selected from methylation, such as N-methylation, dimethylation, such as N- or C-dimethylation, side chain methylation, oxidation, such as oxidation of cysteine ​​or methionine, and dimerization, such as dimerization between two cysteine ​​moieties.

117. 10. The method of claim 1, wherein the modification is carbamidomethylation of cysteine ​​(cysteine ​​CAM).

118. 10. The method of claim 1, wherein the polypeptide comprises or consists of the amino acid sequence GSTC(Cam)PWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 48), wherein Cam indicates carbamidomethylation.

119. 10. The method of claim 1, wherein the polypeptide comprises homocysteine.

120. 2. The method of claim 1, wherein a cysteine ​​moiety of the polypeptide is substituted by homocysteine.

121. 10. The method of claim 1, wherein the polypeptide comprises or consists of the amino acid sequence GST{hC}PWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 49), wherein hC is homocysteine.

122. 10. The method of claim 1, wherein the polypeptide comprises a penicillamine residue.

123. 2. The method of claim 1, wherein a cysteine ​​moiety of the polypeptide is substituted with penicillamine.

124. 10. The method of claim 1, wherein the polypeptide comprises or consists of the amino acid sequence GST{Penc}PWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 50), wherein Penc is a penicillamine residue.

125. 10. The method of claim 1, wherein the polypeptide comprises one or more methionine sulfoxides.

126. 10. The method of claim 9, wherein the polypeptide comprises or consists of the amino acid sequence GSTCPWLKKIM(ox)DRM(ox)TVSTLVLGEG (SEQ ID NO: 51), wherein M(ox) represents methionine sulfoxide.

127. 10. The method of claim 1, wherein the polypeptide is provided as a TFA salt or a chloride salt.

128. 10. The method of claim 1, wherein the polypeptide is methylated or dimethylated.

129. the polypeptide a. arginine side chain methylation, b. Lysine methylation, c. lysine dimethylation, d. N-methylation of threonine, and e. N-methylation of isoleucine.

130. the polypeptide a. GSTCPWLKKIMD[Arg methylated side chain]MTVSTLVEG (SEQ ID NO: 73), b. GSTCPWLK[Lys dimethylated]IMDRMTVSTLVEG (SEQ ID NO: 74); c. GSTCPWLKKIMDRM[Thr N-methylated]VSLVLGEG (SEQ ID NO: 75); d. GSTCPWLKK[Ile N-methylated]MDRMTVSTLGLVEG (SEQ ID NO: 76); e. NLAIGSTCPWLK[Lys dimethylated]IMDR (SEQ ID NO: 77); f. NLAIGS[Thr N-methylated]CPWLKKIMDR (SEQ ID NO: 78); g. NLA[Ile N-methylated]GSTCPWLKKIMDR (SEQ ID NO: 79), and h. an amino acid sequence selected from the group consisting of: NLAIGSTCPWLKKIMD[Arg methylated side chain] (SEQ ID NO: 80); or comprising or consisting of a sequence having at least 70% sequence identity thereto, such as at least 75%, at least 80%, at least 85%, at least 90%, for example at least 95% sequence identity thereto.

131. 10. An agent according to any one of the preceding claims, wherein the polypeptide is cyclised, for example cyclised from the N-terminus to the C-terminus.

132. 10. The agent of any one of the preceding claims, wherein the polypeptide comprises a linker moiety connecting two amino acid residues of the polypeptide.

133. The linking moiety has formula (I): 【Chemistry 1】 and In the formula, R is H, F, Cl, Br, I, NO ２ ,NO,SH,SOH,,SO ２ H, SO ３ H, alkyl, alkenyl, alkynyl, COOH, COOR ２ , CHO, OH, R ２ , and OR ２ and R may be 1, 2, 3, or 4 substitutions independently selected from the group consisting of ２ is alkyl, alkenyl, or alkynyl; 10. An agent according to any one of the preceding claims, wherein the dashed line indicates the point of attachment to an amino acid residue of the polypeptide.

134. The linking moiety has formula (Ia): 【Chemistry 2】 and 10. An agent according to any one of the preceding claims, wherein the dashed line indicates the point of attachment to an amino acid residue of the polypeptide.

135. 2. The method of claim 1, wherein the polypeptide comprises a linker moiety connecting two amino acid residues, the linker moiety being prepared using 1,3-bis(bromomethyl)benzene.

136. 10. The agent of any one of the preceding claims, wherein the two linked amino acid residues are linked via groups independently selected from an N-terminal amine, a side chain heteroatom selected from S, N, and O, a C-terminal acid, and a C-terminal amide.

137. the polypeptide a. C1-GSTCPWLKKIMDR-C1-TVSTLVLGEG (SEQ ID NO: 54); b. C1-NLAIGSTCPWLKKIMDRMTVS-C1 (SEQ ID NO: 55); c. C1-AIGSTCPWLKKIMDRMT-C1 (SEQ ID NO: 56), and d. a structure selected from the group consisting of: C1-NLAIGSTCPWLKKIMDR-C1 (SEQ ID NO:59); or comprising or consisting of a sequence having at least 70% sequence identity thereto, e.g., at least 75%, at least 80%, at least 85%, at least 90%, e.g., at least 95% sequence identity thereto; 10. An agent according to any one of the preceding claims, wherein C1 indicates the point of attachment of the linker moiety.

138. the polypeptide a. C1-GSTCPWLKKIMDR-C1-TVSTLVLGEG (SEQ ID NO: 54); b. C1-NLAIGSTCPWLKKIMDRMTVS-C1 (SEQ ID NO: 55); c. C1-AIGSTCPWLKKIMDRMT-C1 (SEQ ID NO: 56), and d. C1-NLAIGSTCPWLKKIMDR-C1 (SEQ ID NO:59); 10. An agent according to any one of the preceding claims, wherein C1 indicates the point of attachment of the linker moiety.

139. the polypeptide a. cyclo(ClAc-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-Cys)-NH2 (SEQ ID NO: 160), and b. The agent of any one of the preceding claims, selected from the group consisting of cyclo(ClAc-EEKYNLAIGSTCPWLKKIMDRMTESTLVLGE-Cys)-NH2 (SEQ ID NO: 162).

140. 10. The method of claim 1, wherein the polypeptide comprises a second polypeptide.

141. 2. The agent of any one of the preceding claims, wherein the second polypeptide is not a fragment of A1M (SEQ ID NO: 120).

142. The agent of any one of the preceding claims, wherein the agent comprises 80 or fewer consecutive amino acid residues of human A1M (SEQ ID NO: 120) or 80 or fewer consecutive amino acid residues of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to human A1M (SEQ ID NO: 120).

143. 10. The method of claim 1, wherein the second polypeptide is an albumin binding domain (ABD1, SEQ ID NO: 118).

144. 10. The method of claim 1, wherein the second polypeptide is an albumin binding domain (SEQ ID NO: 119).

145. 10. The agent of any one of the preceding claims, wherein the second polypeptide is a human Fc fragment, such as a human IgG Fc fragment, such as a human IgG1 Fc fragment, such as a human IgG1 Fc fragment having SEQ ID NO:

138.

146. the polypeptide a. GSTCPWLKKIMDRMTKSTL[d-D]LGEG (SEQ ID NO: 146) b. EEKYNLAIGSTCPWLKKIMDRMTESTLDLGE (SEQ ID NO: 147) c. EEKYNLAKGSTCPWLKKIMDRMTESTLDLGE (SEQ ID NO: 148) d. GSTCPWLKKI[NLe]DR[NLe]TKSTL[d-D]LGEG (SEQ ID NO: 149) e. EEKYNLAIGSTCPWLKKI[NLe]DR[NLe]TESTLDLGE (SEQ ID NO: 150) f. EEKYNLAKGSTCPWLKKI[NLe]DR[NLe]TESTLDLGE (SEQ ID NO: 151) g. GSTCPWLKKI[NLe]DR[NLe]TK[N-Meth S]TL[D]LGEG (SEQ ID NO: 152) h. EEKYNLAIGSTCPWLKKI[NLe]DR[NLe]TE[N-Meth S]TLDLGE (SEQ ID NO: 153) i. EEKYNLAKGSTCPWLKKI[NLe]DR[NLe]TE[N-Meth S]TLDLGE (SEQ ID NO: 154) j. Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-NH2 (SEQ ID NO: 155) k. The agent of any one of the preceding claims, selected from the group consisting of Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TESTLVLGE-NH2 (SEQ ID NO: 161).

147. 10. The method of claim 1, wherein the polypeptide further comprises a fatty acid.

148. 10. The method of claim 1, wherein the polypeptide binds to a fatty acid.

149. 2. The method of claim 1, wherein the fatty acid is attached to the N-terminus of the polypeptide, the C-terminus of the polypeptide, or a side chain of the peptide.

150. 10. The method of claim 1, wherein the fatty acid is attached to the polypeptide via a linking moiety.

151. 10. The method of claim 9, wherein the linking moiety is γGlu-2xOEG.

152. 10. The method of claim 1, wherein the linking moiety is OEG.

153. 10. The method of claim 1, wherein the fatty acid is a C20 fatty diacid moiety (C20DA).

154. 10. The method of claim 1, wherein the polypeptide binds to γGlu-2xOEG-C20 diacid.

155. the polypeptide Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]T[K([C20DA]-[yGlu]-[OEG]-[OEG]-)]STLVLGE-NH2 (SEQ ID NO: 156 and SEQ ID NO: 164) b. [C20DA]-[yGlu]-[OEG]-[OEG]-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-NH2 (SEQ ID NO: 157) c. Ac-GSTCPWLK[K([C20DA]-[yGlu]-[OEG]-[OEG]-)]I[NLe]DR[NLe]TK[NMeSer]TL[D]LGEG-NH2 (SEQ ID NO: 158 and SEQ ID NO: 165) d. The agent of any one of the preceding claims, selected from the group consisting of: [C20DA]-[yGlu]-[OEG]-[OEG]-GSTCPWLKKI[NLe]DR[NLe]TK[NMeSer]TL[D]LGEG-NH2 (SEQ ID NO: 159).

156. A polynucleotide encoding a polypeptide or agent according to any one of the preceding claims.

157. A vector comprising a polynucleotide according to any one of the preceding claims.

158. 10. A cell comprising a polynucleotide according to any one of the preceding claims or a vector according to any one of the preceding claims.

159. A pharmaceutical composition comprising an agent according to any one of the preceding claims and at least one pharmaceutically acceptable carrier, excipient or diluent.

160. 10. A pharmaceutical composition according to any one of the preceding claims, wherein the composition is formulated for oral administration.

161. 10. An agent according to any one of the preceding claims for use in medicine.

162. 10. A medicament as defined in any one of the preceding claims for use in the treatment of intraventricular hemorrhage (IVH).

163. 10. A medicament according to any one of the preceding claims for use in the treatment of renal disease, disorder or injury.

164. 10. A medicament for use according to any one of the preceding claims, wherein the renal disease, disorder or damage is induced, propagated and / or associated with oxidative stress.

165. 10. The agent for use according to any one of the preceding claims, wherein the oxidative stress is induced, propagated and / or associated with heme and / or heme-related products.

166. 10. The agent for use according to any one of the preceding claims, wherein the oxidative stress is induced, propagated and / or associated with free heme.

167. 10. The method of claim 1, wherein the renal disease, disorder or injury is characterized by hematuria.

168. 10. The method of claim 1, wherein the renal disease, disorder or injury is characterized by glomerular hematuria.

169. 10. A medicament for use according to any one of the preceding claims, wherein the kidney disease, disorder or damage is caused, precipitated and / or propagated by glomerulonephritis.

170. 10. A medicament for use according to any one of the preceding claims, wherein the kidney disease, disorder or damage is caused, induced and / or propagated by nephrotoxicity.

171. 10. The method of claim 1, wherein the renal disease, disorder or injury is acute kidney injury (AKI).

172. 10. The method of claim 1, wherein the acute kidney injury (AKI) is ischemia-reperfusion injury (IRI).

173. 10. The medicament for use according to any one of the preceding claims, wherein the acute kidney injury (AKI) is caused, precipitated and / or propagated by sepsis.

174. 10. The method of claim 1, wherein the acute kidney injury (AKI) is caused, induced and / or propagated by cisplatin.

175. 10. The method of claim 1, wherein the renal disease, disorder or injury is acute kidney disease (AKD).

176. 10. The method of claim 1, wherein the renal disease, disorder or injury is chronic kidney disease (CKD).

177. 10. The medicament for use according to any one of the preceding claims, wherein the CKD is associated with and / or manifests with hematuria.

178. 10. The medicament for use according to any one of the preceding claims, wherein the CKD is selected from the group consisting of IgA nephropathy, primary focal segmental glomerulosclerosis (FSGS), Alport syndrome, thin basement membrane disease (TBMN), C3 glomerulonephritis (C3GN), lupus nephritis, ANCA-associated vasculitis, ANCA-associated glomerulonephritis, diabetic kidney disease (DKD), hypertensive nephrosclerosis, polycystic kidney disease, non-diabetic CKD, non-proteinuric CKD, interstitial nephritis, drug-induced CKD, CKD due to autoimmune disease, CKD due to unknown / non-specific causes, and CKD due to genetic abnormalities, including genetic abnormalities of APOL1 nephropathy.

179. 10. The method of claim 1, wherein the CKD is caused by glomerulonephritis.

180. 10. The method of claim 1, wherein the CKD is selected from the group consisting of IgA nephropathy, primary focal segmental glomerulosclerosis (FSGS), Alport syndrome, thin basement membrane disease (TBMN), C3 glomerulonephritis (C3GN), lupus nephritis, ANCA-associated vasculitis, and ANCA-associated glomerulonephritis.

181. 10. A medicament according to any one of the preceding claims for use in the treatment of a disease or disorder selected from the group consisting of a disease or disorder caused by or associated with bleeding, hemolysis, or anemia, systemic heme disease, sickle cell anemia, hemolytic anemia, Diamond-Blackfan anemia, malaria, sepsis, vascular injury, peripheral arterial disease, vascular diseases or disorders such as, e.g., cardiovascular diseases or disorders, atherosclerosis, ischemic heart disease, thrombotic vascular disease, heart failure, myocardial infarction, diseases or disorders of the CNS such as, e.g., hemorrhagic stroke or ischemic stroke, tissue or nerve damage due to hemolytic trauma or bleeding.

182. 10. A method of treating a disease or disorder comprising administering a therapeutically effective amount of an agent according to any one of the preceding claims.

183. 10. A method of reducing oxidative stress in a subject, the method comprising administering to the subject an agent according to any one of the preceding claims.

184. A method for reducing oxidative stress in a cell, the method comprising contacting the cell with an agent according to any one of the preceding claims.

185. 10. Use of an agent according to any one of the preceding claims in the manufacture of a medicament for the treatment of a disease or disorder.

Images