Stable peptides with renalase agonist activity - Patents.com

Abstract

Stable peptides are disclosed that have renalase agonist activity and are useful for treating diseases such as AKI and AP, including those associated with SARS-CoV-2.

Description

[Technical field]

[0001] Cross-referencing of sequence listings submitted via EFS-Web The contents of the ASCII text file of the sequence listing entitled "8712-0001WO SEQUENCE LISTING_seq_ST25", created on May 21, 2021, and submitted electronically contemporaneously with the filing via EFS-Web, 70kb in size, are hereby incorporated by reference in their entirety.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 032055, filed May 29, 2020, which is incorporated by reference in its entirety.

[0003] The present disclosure relates to stable peptides that exhibit tissue protective activity and are useful for treating kidney or pancreatic diseases, such as acute kidney injury or acute pancreatic injury, in particular kidney disease or pancreatic injury associated with or exacerbated by SARS-CoV-2. [Background technology]

[0004] The following discussion is provided merely to aid the reader in comprehending the present disclosure and is not admitted to describe or constitute prior art thereof.

[0005] Renalase (RNLS) is secreted by the kidney and has multiple biological functions. See, for example, US Patent No. 5,339,436; US Patent No. 5,339,437; US Patent No. 5,339,437; and US Patent No. 5,339,437 (International Patent Application US18 / 67608), which are incorporated herein in their entirety as if fully reproduced herein. Administration of recombinant or biologically isolated renalase has been shown to treat certain diseases and conditions, as described in the above-mentioned US Patent No. 5,339,437 and Non-Patent Document 1. RNLS has been shown to prevent acute kidney injury independent of its oxidase function by cell signaling mechanisms (Non-Patent Document 1).

[0006] Acute kidney injury (AKI) and acute pancreatitis (AP) are seen in at least one-third and one-fifth of hospitalized COVID-19 patients, respectively, and occur more frequently in severe disease. Renalase (RNLS) is a unique circulating protein that potently increases cell survival and reduces inflammation to treat kidney disease such as AKI and / or AP.

[0007] However, the synthesis and administration of the entire renalase chain A is cumbersome and expensive, and may result in complex pharmacological properties due to both oxidase and cell signaling functions (Non-Patent Document 1). Stable synthetic peptides that exhibit renalase agonist activity, especially with cell signaling and tissue repair functions, are highly desirable. The present disclosure provides such peptides. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 7,700,095 [Patent Document 2] U.S. Patent No. 7,858,084 [Patent Document 3] U.S. Patent No. 7,932,067 [Patent Document 4] U.S. Patent No. 10066025 [Patent Document 5] International Publication No. 2019 / 133665 [Non-patent literature]

[0009] [Non-Patent Document 1] Wang et al., J.Am.Soc.Nephrol., June 2014, 25(6):1226-1235 Summary of the Invention

[0010] The present invention relates to a novel peptide derived from renalase chain A (1-342) (SEQ ID NO: 1) shown in FIG. 1. The peptide comprises: <Chemical formula 1> [ka] and, <Chemical formula 2> [ka] and Cys by an amino acid residue selected from 220 at least residues 220-229 of renalase A(1-342) in which In formula 1, R 1 and R 2 are independently H, C1-C8 n-alkyl optionally substituted with a hydroxyl group, C3-C8 branched alkyl optionally substituted with a hydroxyl group, C4-C8 bibranched alkyl optionally substituted with a hydroxyl group, C3-C6 cycloalkyl optionally substituted at one or more optional positions with a hydroxyl group or a methyl group or both, including all structurally feasible stereoisomers, CH2-C3-C6 cycloalkyl optionally substituted at one or more optional positions with a hydroxyl group or a methyl group or both, and R 1 and R 2 is optionally substituted at any position by a methyl group or a hydroxyl group or both (CH2) n where n is 2, 3, 4, or 5; In formula 2, Y is optionally substituted at any one or more positions with a methyl or hydroxyl group or both, with the proviso that the carbon attached to the amino group may only be substituted with a methyl group, and n is 2, 3, 4, or 5 (CH2). n, optionally substituted at any one or more positions with a methyl group or a hydroxyl group, or both, with the proviso that the carbon bonded to the amino group may only be substituted by a methyl group, and including all structurally feasible diastereoisomers.

[0011] This modification improves the biological potency of the peptides and stabilizes them by potential in situ dimerization and / or oligomerization. 220 is selected from Ser, Ala, Leu, Val, Ile, Nle, β-Ala, Aib, cyclopropyl-glycine and (cyclopropylmethyl)-glycine.

[0012] The additional amino acid residues correspond to a part or all of the amino acid residues at positions 205 to 219 of the sequence of renalase A shown in the corresponding positions in FIG. 220 or Lys corresponding to some or all of the amino acid residues at positions 230 to 253 of the sequence of renalase A shown at the corresponding positions in FIG. 229 Additionally, long chains of poly(ethylene)glycol [PEG] or bis-poly(ethylene)glycol (bis-PEG) of varying average molecular weights (e.g., 5,000-20,000 amu), or similar polymers known in the art (as described below), can be added to the COOH-terminus of X. 220 or at the NH2-terminus of the long fragment, or at Lys 229 or to the COOH-terminus of a long fragment.

[0013] The expression "corresponding to some or all of the amino acid residues in the sequence from positions 205 to 219 of renalase A" means that the added amino acid residues may be included in the sequence from positions 205 to 219, such as from positions 219, from positions 218 and 219, from positions 217 to 219, from positions 216 to 219, etc. Similarly, the expression "corresponding to some or all of the amino acid residues in the sequence from positions 230 to 253 of renalase A" means that the added amino acid residues may be included in the sequence from positions 230 to 253, such as from positions 230, from positions 230 to 231, from positions 230 to 232, etc.

[0014] The present disclosure also provides methods of treating renal disease, including AKI and AP, as well as renal disease caused by SARS-CoV-2. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows renalase chain A (1-342). [Diagram 2] FIG. 1 shows that renalase agonist peptide 10 (BP-1002) reduces damage in a mouse model of acute pancreatitis. [Diagram 3] FIG. 1 shows the activity of renalase agonists against cisplatin lethality. [Figure 4] FIG. 1 shows that renalase agonist peptide 81 reduced cisplatin-induced kidney injury. [Diagram 5] FIG. 1 shows that renalase agonist BP-1002 reduces inflammatory responses to SARS-CoV-2 peptides. [Figure 6] FIG. 1 shows the anti-inflammatory effect of peptide 10 (BP-1002). [Figure 7] FIG. 2 shows another demonstration of the anti-inflammatory effect of peptide 10. [Figure 8] FIG. 1 shows the anti-inflammatory effect of peptide 81. [Figure 9] 1 shows the comparative anti-inflammatory effect of peptide 10 (BP-1002). [Figure 10]FIG. 1 shows the correlation between reduced renalase levels and mortality in COVID-19 patients. [Figure 11] FIG. 1 shows that renalase agonist peptide 10 (BP-1002) improves survival in COVID-19 model mice. [Figure 12] FIG. 1 shows that treatment with renalase agonist peptide 10 (BP-1002) protects renalase-deficient mice in a model of severe viral infection. [Figure 13] FIG. 1 shows that peptide 10 (BP-1002) was effective in reducing necrosis in a model of severe caerulein-induced pancreatitis. [Figure 14] FIG. 1 shows that peptide 10 (BP-1002) reduces the levels of cronin1, a marker for inflammatory cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Synthetic peptides, pharmaceutical compositions comprising the peptides, and methods of treating human diseases using the synthetic peptides and pharmaceutical compositions are provided by the present disclosure.

[0017] The inventors discovered that certain peptide fragments within the sequence of renalase chain A exhibit renalase agonist activity. However, they also discovered that these peptides are unstable and therefore not suitable for use as therapeutic agents. The inventors discovered that the instability of the peptide fragments can be eliminated by replacing the cysteine ​​residue at position 220 of renalase chain A (1-342) by a different amino acid selected appropriately. Furthermore, the modified peptide fragments have increased potency compared to the unmodified fragments.

[0018] The amino acid at position 220 is <Chemical formula 3> [ka] and, <Chemical formula 4> [ka] and, In formula 3, R 1 and R 2 are independently H, C1-C8 n-alkyl optionally substituted with a hydroxyl group, C3-C8 branched alkyl optionally substituted with a hydroxyl group, C4-C8 bibranched alkyl optionally substituted with a hydroxyl group, C3-C6 cycloalkyl optionally substituted at one or more optional positions with a hydroxyl group or a methyl group or both, including all structurally feasible stereoisomers, CH2-C3-C6 cycloalkyl optionally substituted at one or more optional positions with a hydroxyl group or a methyl group or both, and R 1 and R 2 is optionally substituted at any position by a methyl group or a hydroxyl group or both (CH2) n where n is 2, 3, 4, or 5; In formula 4, Y is optionally substituted at any one or more positions with a methyl group or a hydroxyl group, with the proviso that the carbon attached to the amino group may be substituted only with a methyl group, and n is 2, 3, 4, or 5 (CH2). n, optionally substituted at any one or more positions with a methyl group or a hydroxyl group, or both, with the proviso that the carbon bonded to the amino group may only be substituted by methyl, and including all structurally feasible diastereoisomers.

[0019] The peptide of the present invention is a peptide having X 220 and including a renalase A fragment having positions 220 to 229 and additional amino acids at appropriate positions in renalase A on either or both sides of positions 220 to 229, the peptides generally vary in length from about 49 amino acids to about 20 amino acid residues and may be longer if necessary.

[0020] Furthermore, as is known in the pharmaceutical art, poly(ethylene) glycols (PEGs) or bis-poly(ethylene) glycols (bis-PEGs) of various average molecular weights (e.g., 500-20,000 amu) can be attached to either end of a peptide to promote long-term activity of the peptide. Poly(ethylene) glycols are amphiphilic polymers consisting of repeating units of ethylene oxide that can associate into linear or branched structures to give a variety of PEGs with different architectures and molecular weights. PEGs need to be activated in order to be covalently attached to the appropriate sites of biopharmaceutical compounds (including peptides and proteins) and thereby improve their pharmacological and pharmaceutical properties. In addition to improving solubility, conjugation with PEG can protect biopharmaceutical compounds from the host's immune system, thereby reducing immunogenicity and antigenicity and extending biological half-life. The resulting PEGylated pharmaceuticals can be used at reduced dosages and frequency without compromising efficacy. PEGs are available in various average molecular weights and can be functionalized at one end (the other end is usually protected as methoxy). Alternatively, both ends of the polymer can be functionalized to provide homobifunctional or heterobifunctional derivatives that can be used to link two things together. A review of first and second generation PEG derivatives with more diverse and efficient functional groups for conjugation to peptides and proteins is given in Roberts, Adv. Drug Deliv. Rev. 54, 459 (2002). Third generation PEGylation agents, in which the polymer is branched, have been developed and may offer the additional advantage of protecting the protein from proteolysis and further reducing immunogenicity and antigenicity. [PEG] is commonly used for conjugation to peptides and proteins, but other polymers with suitable flexibility and appropriately functionalized, including but not limited to carbohydrate moieties, can also be used and are reviewed by Sola and Griebenow, J. Pharm. Sci. 98, 1223 (2009) and Witteloostuijn, Pedersen and Jensen, ChemMedChem, 11, 4 (2016), both of which are incorporated herein by reference.

[0021] The amino acids selected for substitution at position 220 of the peptides of the invention include glycine, serine, alanine, leucine, valine, isoleucine, norleucine, beta-alanine, cyclopropyl-glycine, (cyclopropylmethyl)-glycine and other hydrophobic amino acids as well as the D-amino acid enantiomers of the listed amino acids. Additionally, Lys 229 Lys may be substituted with its D-isomer to provide additional stability. 205 , Arg 222 , Lys 230 and / or Arg 231 Other amino acids, including those listed above, may also be substituted with the corresponding D-amino acids for similar enhanced stability against enzymatic degradation.

[0022] In one embodiment, R 220 -Ile 221 -Arg 222 -Phe 223 -Val 234 -Ser 225 -Ile 226 -Asp 227 -Asn 228 -Lys 229 A peptide comprising -R', where the superscript represents a position in the renalase A chain (1-342), R is selected from Ac-Ala-Gly-Thr-, Ac-Gly-Thr-, Ac-Thr-, Ac-, Ac-Z, H-, HZ, BZ- and B, Z is selected from one or more of the amino acid residues at positions 205-219 of the renalase A chain, R' is selected from -NH2, Z'-NH2, -B and Z'-B, Z' is selected from one or more of the amino acid residues at positions 230-253 of the renalase A chain, B is PEG or bis-PEG, and X is <Chemical formula 5> [ka] and, <Chemical formula 6> [ka] and, In formula 5, R 1and R 2 are independently H, C1-C8 n-alkyl optionally substituted with a hydroxyl group, C3-C8 branched alkyl optionally substituted with a hydroxyl group, C4-C8 bibranched alkyl optionally substituted with a hydroxyl group, C3-C6 cycloalkyl optionally substituted at one or more optional positions with a hydroxyl group or a methyl group or both, CH2-C3-C6 cycloalkyl optionally substituted at one or more optional positions with a hydroxyl group or a methyl group or both, and R 1 and R 2 is optionally substituted at any position by a methyl group or a hydroxyl group or both (CH2) n where n is 2, 3, 4, or 5; Y is optionally substituted at any one or more positions with a methyl group or a hydroxyl group or both, with the proviso that the carbon attached to the amino group may be substituted only with a methyl group, and n is 2, 3, 4, or 5 (CH2). n and optionally substituted at any one or more positions with methyl or hydroxyl groups or both, with the proviso that the carbon attached to the amino group may only be substituted with a methyl group and with the proviso that the peptide comprises at least 20 amino acid residues.

[0023] In another embodiment, the sequence R-Lys 205 -Ile 206 -Asp 207 -Val 208 -Pro 209 -Trp 210 -Ala 211 -Gly 212 -Gln 213 -Tyr 214 -Ile 215 -Thr216 -Ser 217 -Asn 218 -Pro 219 -X 220 -Ile 221 -Arg 222 -Phe 223 -Val 234 -Ser 225 -Ile 226 -Asp 227 -Asn 228 -Lys 229 -Lys 230 -Arg 231 -Asn 232 -Ile 233 -Glu 234 -Ser 235 -Ser 236 -Glu 237 -Ile 238 -Gly 239 -Pro 240 -Ser 241 -Leu 242 -Val 243 -Ile 244 -His 245 -Thr 246 -Thr 247 -Val 248 -Pro 249 -Phe 250 -Gly 251 -Val 252 -Thr 253 -R' is provided, wherein R is selected from Ac-Ala-Gly-Thr-, Ac-Gly-Thr-, Ac-Thr-, Ac-, H- and PEG, and R' is selected from -Tyr-Leu-Glu-NH2, Tyr-Leu-NH2, -Tyr-NH2, -NH2, -OH and PEG; and X 220 is selected from Gly, Ser, Ala, Leu, Val, Ile, Nle, β-Ala, cyclopropyl-Gyl, (cyclopropylmethyl)-Gly, and Aib.

[0024] Thus, the peptide of the present invention is 220 ]-Ac-renalase A(205-240)-NH2 (SEQ ID NO: 2), [X 220]-Ac-renalase A(214-253)-NH2 (SEQ ID NO: 3), [X 220 ]-Ac-renalase A(214-240)-NH2 (SEQ ID NO: 4) and [X 220 ]-Ac-renalase A(205-253)-NH2 (SEQ ID NO: 5), wherein X is selected from glycine, serine, alanine, leucine, valine, isoleucine, norleucine, cyclopropyl-glycine, (cyclopropylmethyl)-glycine and beta-alanine.

[0025] Representative compounds of the present invention include, but are not limited to, [Ala 220 ]-Ac-renalase A(205-240)-NH2 (SEQ ID NO: 6), [Ala 220 ]-Ac-renalase A(214-253)-NH2 (SEQ ID NO: 7), [Ala 220 ]-Ac-renalase A(214-240)-NH2 (SEQ ID NO: 8), [Ala 220 ]-Ac-renalase A(205-253)-NH2 (SEQ ID NO: 9), [Val 220 ]-Ac-renalase A(214-240)-NH2 (SEQ ID NO: 10), [Ser 220 ]-Ac-renalase A(214-253)-NH2 (SEQ ID NO: 11), [Ala 220 ,D-Lys 229 ]-Ac-renalase A(205-240)-NH2 (SEQ ID NO: 12), [Ser 220 ]-Ac-renalase A(205-240)-NH2 (SEQ ID NO: 13), [Ala 220 ]-Ac-renalase A(214-234)-NH2 (SEQ ID NO: 14), [Ala 220 ]-Ac-renalase A(220-239)-NH2 (SEQ ID NO: 15), [Gly 220 ]-Ac-renalase A(205-240)-NH2 (SEQ ID NO: 16), [Gly 220 ]-Ac-renalase A(214-253)-NH2 (SEQ ID NO: 17), [Cyclopropyl-Gly 220 ]-Ac-renalase A(205-253)-NH2 (SEQ ID NO: 18), [(Cyclopropylmethyl)-Gly 220 ]-Ac-Renalase A(214-240)-NH2 (SEQ ID NO: 19) Includes.

[0026] A representative example of the PEGylated peptide of the present invention is [bis-PEG 5000 ]-Lys 205 -Ile 206 -Asp 207 -Val 208 -Pro 209 -Trp 210 -Ala 211 -Gly 212 -Gln 213 -Tyr 214 -Ile 215 -Thr 216 -Ser 217 -Asn 218 -Pro 219 -Ala 220 -Ile 221 -Arg 222 -Phe 223 -Val 234 -Ser 225 -Ile 226 -Asp 227 -Asn 228 -Lys 229 -Lys 230 -Arg 231 -Asn 232 -Ile 233 -Glu 234 -Ser 235 -Ser 236 -Glu 237 -Ile 238 -Gly 239 -Pro 240 -NH2 (SEQ ID NO: 20).

[0027] Peptides are generally prepared using solid-phase synthesis as described by Merrifield, J. Am. Chem. Soc. 85, 2149 (1963), although other equivalent chemical syntheses, including liquid-phase synthesis or biological production using recombinant techniques, well known to those skilled in the art, may be used. Solid-phase synthesis is initiated from the C-terminus of the peptide by coupling an NH2-protected amino acid to a suitable resin. The starting material is an N アルファ The peptides were prepared by attaching the COOH terminus of 9-fluorenylmethoxycarbonyl (Fmoc) amino acids to a commercially available 4,4'-dimethoxybenzhydryl-amine, (Mbh)-handle linked to a solid-phase resin. Solid-phase synthesis and coupling with Fmoc-amino acids (containing appropriately protected side chains for trifunctional amino acids) proceeded with stepwise elongation of the desired peptide chain using carbodiimide / HOBt-mediated reactions. Final cleavage of the side chain protecting groups and liberation of the C-terminal amide moiety was achieved by treatment with trifluoroacetic acid in the presence of scavengers. The peptides were purified (>95% purity) by preparative high-performance liquid chromatography and characterized by amino acid analysis and mass spectrometry. Specific details regarding the synthesis of the peptides are provided below.

[0028] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding any other elements of essential importance to the composition or method. "Consisting of" is intended to mean excluding more than trace elements of other ingredients for the claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. Thus, it is intended that the methods and compositions may include (comprising / comprising) additional steps and ingredients, or may alternatively include (consist essentially of) insignificant steps and ingredients, or may alternatively contemplate (consist of) only the described method steps or compositions.

[0029] As used herein, "about" means ±10%.

[0030] As used herein, "optional" or "optionally" means that a subsequently described event or circumstance may or may not occur, and that the description includes instances in which the aforementioned event or circumstance occurs and instances in which it does not occur.

[0031] As used herein, the terms "individual," "patient," or "subject" can refer to an individual organism, a vertebrate, a mammal (e.g., a cow, a dog, a cat, or a horse), or a human. In a preferred embodiment, the individual, patient, or subject is a human.

[0032] As used herein, the phrases "therapeutically effective amount" and "therapeutic level" refer to a peptide dose or plasma concentration, respectively, in a subject that provides a particular pharmacological effect when the peptide is administered to a subject in need of such treatment, i.e., treatment to reduce, alleviate or eliminate the effects or symptoms of renal disease. It is emphasized that a therapeutically effective amount or therapeutic level of a drug will not always be effective in treating the conditions / diseases described herein, even if such an administration amount would be considered a therapeutically effective amount by one of skill in the art. A therapeutically effective amount may vary based on the condition of the subject, including, among other factors, the route and form of administration, the age and weight of the subject, and / or the type and stage of amyloidosis at the time treatment is initiated.

[0033] As used herein in reference to renal disease, the term "treat" or "treating" refers to reducing, alleviating or eliminating one or more symptoms or effects of the disease or condition.

[0034] "Therapeutic response" means an improvement in at least one measure of renal disease.

[0035] As used herein, the term "pharmaceutical acceptable carrier" refers to a material for mixing with a pharmaceutical compound (e.g., a chimeric peptide) for administration to a patient, e.g., as described in Ansel's Pharmaceutical Dosage Forms and Delivery Systems, 10th Edition (2014).

[0036] Abbreviation The following abbreviations are used herein: "Ac" is acetyl, -NH2 is amide (-CO -NH 2), where AKI is acute kidney injury, AP is acute pancreatitis, PMCA4b is plasma membrane ATPase4b, and RNLS is renalase.

[0037] Amino acids are represented by the IUPAC abbreviations as follows: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), valine (Val; V), norleucine (Nle), and 2-aminobutyric acid (Aib).

[0038] The expression “[X 220 ]-Ac-renalase A(205-240)-NH2" means a peptide containing the amino acids at positions 205 to 240 of renalase A, the amino acid "X" at position 220, an acyl group at the NH2 terminus of the peptide, and an amide group at the COOH terminus of the peptide. Thus, this peptide is Ac-Lys 205 -Ile 206 -Asp 207 -Val 208 -Pro 209 -Trp 210 -Ala 211 -Gly 212 -Gln213 -Tyr 214 -Ile 215 -Thr 216 -Ser 217 -Asn 218 -Pro 219 -X 220 -Ile 221 -Arg 222 -Phe 223 -Val 234 -Ser 225 -Ile 226 -Asp 227 -Asn 228 -Lys 229 -Lys 230 -Arg 231 -Asn 232 -Ile 233 -Glu 234 -Ser 235 -Ser 236 -Glu 237 -Ile 238 -Gly 239 -Pro 240 -NH2 (SEQ ID NO: 6). When X is Ala, the compound is sometimes referred to herein as "Peptide 10" or "BP-1002."

[0039] Similar expressions for other peptides of the invention have corresponding meanings.

[0040] Pharmaceutical preparations Pharmaceutical compositions suitable for use in the methods described herein can include one or more of the disclosed peptides and a pharma- ceutically acceptable carrier or diluent.

[0041] The compositions may be formulated for intravenous, subcutaneous, intraperitoneal, intramuscular, topical, oral, buccal, nasal, pulmonary or inhalation, ocular, vaginal, or rectal administration. In certain embodiments, the peptides are formulated for intravenous, subcutaneous, intraperitoneal, intramuscular administration, or target tissue delivery, such as in a solution, suspension, emulsion, liposomal formulation, etc. The pharmaceutical compositions may be formulated to be immediate release compositions, sustained release compositions, delayed release compositions, etc., using techniques known in the art.

[0042] Pharmacologically acceptable carriers for various administration forms are known in the art.For example, excipients, lubricants, binders and disintegrants for solid preparations are known, and solvents, solubilizers, suspending agents, isotonicity agents, buffers and emollients for liquid preparations are known.In some embodiments, the pharmaceutical composition comprises one or more additional components, such as one or more preservatives, antioxidants, stabilizers, etc.

[0043] Further, the disclosed pharmaceutical compositions can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, the composition preferably includes an isotonic agent, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of the injectable composition can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts or gelatin.

[0044] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of the above-listed ingredients, and then performing sterilization microfiltration as necessary.Generally, dispersion is prepared by incorporating the active compound in a sterile vehicle that contains the basic dispersion medium and other ingredients required from the above-listed ingredients.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which obtains the powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0045] The pharmaceutical composition of the present disclosure can be administered in combination with other therapeutic agents that are part of the current standard treatment for kidney disease, pancreatic disease or tissue damage, particularly those that can benefit from the regulation of excessive immune inflammation.For example, fenoldopam, which is found as a selective dopamine 1 receptor agonist, is utilized in acute kidney injury and can be used together with the subject peptide in treatment.

[0046] Treatment In the present invention, at least one peptide is administered to a patient (e.g., a human patient) suffering from renal disease, including AKI, and AP. In one embodiment, a therapeutically effective amount of the peptide is administered together with a pharma- ceutically acceptable carrier. Suitable pharma-ceutically acceptable carriers are known in the art, as described below. A common route of administration is parenteral (e.g., intravenous, subcutaneous, or intramuscular), as is well understood by those skilled in the medical field. Other routes of administration are of course possible. Administration may be by single or multiple doses. The amount of peptide administered and the frequency of dosing may be optimized by a physician for a particular patient.

[0047] SARS-CoV-2 viral infection produces morbidity and mortality and shows selective patterns of tissue injury. Acute inflammatory injury generated in certain tissues can disseminate and seed the entire body, resulting in multiple organ failure and death. For example, a key feature of severe acute pancreatitis is the development of multiorgan injury in the lungs and kidneys, the primary tissues affected secondarily. Renal injury also occurs in less severe bouts of acute pancreatitis, but recovers rapidly in this setting. Relevant to numerous types of acute injury and this proposal that injury in one organ leads to functional impairment in others. Some critical COVID-19 patients, especially those with severe disease, are expected to show distinct patterns of tissue injury. SARS-CoV-2 infection can also injure the kidney, with the most prominent effect being on the proximal tubules, the site of renalase (RNLS) synthesis. SARS-CoV-2 infection of the kidney is expected to reduce circulating RNLS levels, which sensitizes target tissues to injury from the virus and other factors. We predict that the kidney and pancreas will be important pathological targets and that reduced plasma RNLS levels will increase renal injury and also sensitize the pancreas to AP. Injury to these organs will further reduce plasma RNLS levels, driving a negative feedback loop.

[0048] Clinical studies indicate that both kidney injury and acute pancreatitis occur frequently in SARS-CoV-2 infection. Although clinical studies of the natural history of SARS-CoV-2 infection are just beginning, clear patterns of injury are emerging. In one limited study of 55 patients, evidence of acute pancreatitis was observed in 17% and there was an 8% incidence of renal dysfunction. This hospitalized cohort appeared to have mostly moderate and not severe disease. Other studies have reported that blood creatinine levels in COVID-19 patients correspond to disease severity, and the incidence of kidney injury in COVID-19 patients is reported to be up to one-third of hospitalized patients. In 52 patients with severe SARS-CoV-2 infection, 17% required dialysis. The presence of underlying kidney disease, a condition associated with reduced plasma RNLS levels, significantly increases the risk of death from SARS-CoV-2. SARS-COV-2-induced damage to proximal tubule cells, the site of RNLS generation, was reported in pathology studies.

[0049] Inflammatory response to SARS-CoV-2 infection Based on clinical findings, SARS-CoV-2 pathology is divided into the following stages: (I) early viral response, (II) pulmonary disease phase, and (III) hyperinflammation. One study documenting the time course of these responses in patients with mild versus severe SARS-CoV-2 pathology found a significant increase in serum IL6 levels throughout the entire 2-week period only in patients with severe disease. It is relevant that elevated plasma IL6 may be a marker of AP severity and may damage the kidney. IL6 and its precursor IL1 have been investigated in therapeutic trials for SARS-COV-2 infection. Another observation is that interferon-gamma (IFN-gamma) levels, which are thought to be important in suppressing viral infection, were suppressed in both mild and severe disease.

[0050] Renalase functions as a pro-survival factor in AKI and AP models. RNLS is a 37 kD secreted protein made primarily by renal proximal tubule cells but also in other tissues. Its main intracellular target is the widely distributed plasma membrane calcium efflux transporter, plasma membrane calcium ATPase 4b (PMCA4b). Activation of PMCA4b is required for the protective function of RNLS in cellular AP models and other tissues. Through the use of selective PMCA4b inhibitors and genetic deletion models, we found that PMCA4b is required for RNLS to have protective cellular effects.

[0051] Provided herein are methods of treating kidney disease, including kidney disease associated with SARS-CoV-2, AKI, and AP in a patient (e.g., a human patient) in need of such treatment, comprising administering to the patient one or more of the disclosed peptides in an amount effective to treat the disease, together with a pharma- ceutically acceptable carrier.

[0052] Therapeutically Effective Doses and Dosing Regimens In certain embodiments, a therapeutically effective dose of the peptide may be administered 1, 2, 3, or no more than 4 times within a 3 month period.

[0053] The therapeutically effective dose and dosing regimen of the aforementioned method may vary as would be readily understood by one skilled in the art. Dosage regimens may be adjusted to provide the optimum desired response. For example, in some embodiments, a single dose of peptide may be administered, while in some embodiments, several divided doses may be administered over time, and the dose may be proportionally reduced or increased in subsequent doses as indicated by circumstances. For example, in some embodiments, the disclosed peptides may be administered once or twice a week by subcutaneous, intravenous, or intramuscular injection. In some embodiments, the disclosed peptides may be administered once or twice a month by subcutaneous, intravenous, or intramuscular injection. In some embodiments, the disclosed peptides may be administered once or twice a year by subcutaneous, intravenous, or intramuscular injection. In certain embodiments, the disclosed peptides may be administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, twice a year, or once a year, as the patient's condition or status may indicate.

[0054] The therapeutically effective dose of the peptide administered to the patient (whether administered in a single dose or multiple doses) should be sufficient to treat the renal disease or AP. Such a therapeutically effective amount can be determined by assessing symptomatic changes in the patient.

[0055] Exemplary doses may vary depending on the size and health of the individual being treated and the condition being treated. In some embodiments, an effective amount of the disclosed peptides will be about 2200 mg, although in some circumstances the dose may be higher or lower. In some embodiments, a therapeutically effective amount may be 50-5000 mg, 60-4500 mg, 70-4000 mg, 80-3500 mg, 90-3000 mg, 100-2500 mg, 150-2000 mg, 200-1500 mg, 250-1000 mg, or any dose therebetween. For example, in certain embodiments, a therapeutically effective amount is about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 200 0, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3100, about 3200, about 3300, about 3400, about 3500, about 3600, about 3700, about 3800, about 3900, about 4000, about 4100, about 4200, about 4300, about 4400, about 4500, about 4600, about 4700, about 4800, about 4900, about 5000 mg or more.

[0056] Similarly, in some embodiments, an effective amount of peptide will be about 25 mg / kg, although in some embodiments, concentrations can be higher or lower. In some embodiments, an effective amount can be about 1-50 mg / kg, about 5-40 mg / kg, about 10-30 mg / kg, or about 15-25 mg / kg, or any value therebetween. For example, in some embodiments, an effective amount can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg / kg or more.

[0057] The disclosed methods of treatment may also be combined with other known methods of treatment as the situation may require. For example, renalase agonists may counteract the potential "cytokine storm" that leads to excessive tissue damage and death in SARS-CoV-2 and potentially other respiratory viral diseases. For example, the H1N1 pandemic has resulted in widespread kidney damage. The following examples reflect SARS-CoV-2 according to the current literature.

[0058] Potential Drug Combinations [Table 1-1] [Table 1-2]

[0059] The therapeutically effective dose and dosing regimen of the aforementioned method may vary as readily understood by those skilled in the art. Dosage regimens may be adjusted to provide the optimal desired response. For example, in some embodiments, a single bolus dose of peptide may be administered, while in some embodiments, several divided doses may be administered over time, and the dose may be proportionally reduced or increased in subsequent doses as indicated by the circumstances. For example, in some embodiments, the disclosed peptides may be administered once or twice a week by subcutaneous, intravenous, or intramuscular injection. In some embodiments, the disclosed peptides may be administered once or twice a month by subcutaneous, intravenous, or intramuscular injection. In some embodiments, the disclosed peptides may be administered once or twice a year by subcutaneous, intravenous, or intramuscular injection. In certain embodiments, the disclosed peptides may be administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, twice a year, or once a year, as the patient's condition or status may indicate.

[0060] Example Peptide synthesis Peptides were synthesized on ChemMatrix Rink Amide resin using standard Fmoc-synthesis protocols on an APEX396 automated synthesizer. For removal of the Fmoc protecting group, the resin was swollen in N,N-dimethylformamide (DMF) for 30 min, treated with 20% piperidine-DMF for 8 min at 50° C., and washed three times with DMF. For the coupling reaction, Fmoc-protected amino acid, 6-chloro-1-hydroxybenzotriazole (Cl-HOBt), diisopropyl-carbodiimide (DCI) and N-methyl-2-pyrrolidine (NMP) were added to the resin. The mixture was vortexed for 20 min at 50° C. Afterwards, the resin was washed once with DMF. The cycle of Fmoc deprotection and coupling steps was repeated until the last amino acid residue was attached. After the final removal of the Fmoc protecting group, the resin was treated with 20% acetic anhydride-NMP for 20 min, then washed with DMF, methylene chloride (DCM), and air-dried. The peptide was cleaved from the resin by treatment with a trifluoroacetic acid (TFA) cocktail [95% TFA, 2.5% water, and 2.5% triisopropylsilane (TIS)] for 3 h. The crude peptide was precipitated by adding ice-cold anhydrous ethyl ether, washed three times with anhydrous ethyl ether, and dried under vacuum. Some specific representative synthesis examples are given below.

[0061] [Ala 220 ]-Ac-Renalase A(205-240)-CONH2 (Peptide 10): Synthesized by solid-phase synthesis as outlined above, confirmed to be >95% pure by high-performance liquid chromatography (HPLC) and confirmed by mass spectrometry and amino acid analysis.

[0062] [Ser 220 ]-Ac-Renalase A(205-240)-CONH2: Synthesized by solid-phase synthesis as outlined above. Confirmed to be >95% pure by high-performance liquid chromatography (HPLC) and confirmed by mass spectrometry and amino acid analysis.

[0063] [Ala 220 ]-Ac-Renalase A(214-240)-CONH2: Synthesized by solid-phase synthesis as outlined above, confirmed to be >95% pure by high-performance liquid chromatography (HPLC) and confirmed by mass spectrometry and amino acid analysis.

[0064] Ac-Renalase A(205-240)-CONH2: Synthesized by solid-phase synthesis as outlined above. Confirmed to be >95% pure by high-performance liquid chromatography (HPLC) and confirmed by mass spectrometry and amino acid analysis.

[0065] [Ala 220 ]-Ac-Renalase A(205-253)-CONH2: Synthesized by solid-phase synthesis as outlined above. Confirmed to be >95% pure by high-performance liquid chromatography (HPLC) and confirmed by mass spectrometry and amino acid analysis.

[0066] Ac-Renalase A(205-240)-OH (Peptide 81): Synthesized by solid-phase synthesis as outlined above, using 2-chlorotrityl-resin instead of Rink-Amide resin, and confirmed to be >95% pure by high-performance liquid chromatography (HPLC) and confirmed by mass spectrometry and amino acid analysis.

[0067] Example Test Reninase agonists reduce injury in experimental AP: RNLS agonist [Ala 220In vivo studies with ]-Ac-Renalase A(205-240)-NH2 (peptide 10) show promising and dramatic effects on the AP response (Figure 2). While we found no effect on the earliest response involving acinar cells (not shown), the later response (6 h), which is mostly mediated by inflammatory cells, showed that peptide 10 reduced active trypsin levels (Figure 2A) and dramatically reduced tissue neutrophil susceptibility (Figure 2B), and reduced mitochondrial damage as determined by OPA-1 and Parkin levels (Figure 2C, D). This suggests that peptide 10 has a very profound effect on reducing pancreatitis damage for the first 1-2 h events of AP. This likely represents inhibition of a central inflammatory pathway in AP pathogenesis, with inflammatory activation being the likely target (Figure 5). Since the majority of AP patients are present long after the onset of the disease and subsequent responses to the disease are of utmost importance in defining the severity of AP, the beneficial effects of peptide 10 should be therapeutically valuable even at later stages of the disease. In this context, we showed that an RNLS agonist reduced damage when administered 2 h after the onset of caerulein AP and 12 h after induction of arginine AP.

[0068] RNLS peptide agonists reduce injury in renal models: We found that RNLS peptide agonists were also protective in models of renal injury. 220 We have shown that RNLS agonist peptide RNLS-Ac(205-240)-NH2 eliminates cisplatin-induced injury in cultured kidney cells. In Figure 3, we show that this peptide reduces injury in a cultured proximal tubule cell line. We also show that this RNLS agonist peptide reduces renal ischemia-reperfusion injury in a preclinical mouse model (Figure 3). For such RNLS agonists, data are presented below.

[0069] Figure 4 shows that the RNLS agonist, renalase A(205-240)-OH (peptide 81), reduces kidney injury after cisplatin-induced treatment. A key measure of this toxicity is the loss of kidney mass, which is limited when renalase A(205-240)-OH is given. The effect of the peptide on the decline in renal function seen in cisplatin-induced kidney injury is more dramatic. As seen in the right panel of Figure 4, plasma creatinine levels increased four-fold 17 days after cisplatin treatment, and the RNLS agonist reduced this toward the normal range. This preliminary data suggests that RNLS agonists can reduce injury to renal cells and may also be effective in an in vivo AKI model.

[0070] RNLS agonist, [Ala 220 ]-Ac-Renalase A(205-240)-NH2 attenuates COVID19-induced innate immune responses (Figure 5): We found that RNLS agonists can have anti-inflammatory effects. Amplification of the inflammatory response mediates severe pancreatitis, kidney damage and SARS-CoV-2 infection. In the case of SARS-CoV-2, these can be triggered by a response to viral antigens. To address this issue, we exposed blood from healthy donors to a mixture of peptides contained in the three major SARS-CoV-2 capsid proteins (SARS-CoV-2 proteins S, M or N) from Miltenyi Biotec at a concentration of 0.19 nM. To this, we added buffer sham control or RNLS peptide at 50 μg / ml. After 3 hours of incubation, samples were assayed for cytokine production by ELISA and compared by 2-way ANOVA / Tukey's comparison. As shown in Figure 5, the SARS-CoV-2 peptides stimulated cytokine responses to different degrees, with peptides S and M giving robust responses for each response (P<0.01), whereas peptide N produced little response (not shown). Both SARS-CoV-2 peptides S and M increased levels of interferon-gamma, a response that was enhanced by the RNLS agonist [Ala 220 ]-Ac-renalase A(205-240)-NH2 was dramatically reduced by RNLS agonists. Because interferon-gamma is thought to be important in suppressing viral growth, the effects of this RNLS agonist should be avoided, as described below. However, other potentially beneficial effects were observed. Thus, [Ala 220 ]-Ac-Renalase A(205-240)-NH2, peptide 10 inhibited SARS-CoV-2 peptide-induced increases in TNF-alpha, as well as IL1-beta and IL6 (p<0.01). These innate immune responses are involved in causing both renal and pancreatic damage. They are also considered central to the hyperinflammatory state that characterizes severe SARS-CoV-2 infection and is responsible for many of the deaths.

[0071] RNLS agonist [Ala 220]-Ac-Renalase A(205-240)-NH2 exhibits anti-inflammatory activity as determined by carrageenan-induced footpad edema.

[0072] This test provides a rapid in vivo model for the evaluation of anti-inflammatory efficacy of test substances. The CPE model in mice quantitatively evaluates the inhibition of edema induced by intraplantar injection of carrageenan. The standard test duration is 6 hours, with edema measured at 0, 2, 4 and 6 hours, and the results presented here are taken at a 10 hour observation time. Carrageenan-induced inflammation is accompanied by migration of polymorphonuclear leukocytes from the circulation to the site of inflammation, followed by release of myeloperoxidase and other cytokines in the interstitial tissue and plasma exudation to the site of inflammation. Increased footpad volume is measured by the water displacement method. Anti-inflammatory drugs reduce footpad edema. Results shown in Figures 6 and 7 (identical data in two different formats) show that peptide 10, [Ala 220 ]-Ac-Renalase A(205-240)-NH2 has anti-inflammatory effects in this model.

[0073] procedure 1. Receive (SOP1910, SOP1920) and quarantine (SOP560) 125 CD-1 mice (Charles River Laboratories, male, 5-6 weeks old). 2. Animals are ear tagged (SOP810), weighed and sorted into 9 groups of 10 animals / group and 5 groups of 7 animals / group based on average weight. A 3.3% carrageenan solution is prepared. 1) Prepare at least 2 weeks before use. 2) Weigh 600 mg of λ-carrageenan into a glass beaker. 3) Add 20 ml of deionized water. 4) Heat gently and stir until the carrageenan is completely dissolved. 5) Cool the solution to room temperature. 6)Store at 4~8℃. 4. Store the renalase peptide as a dry powder at -20°C until the day of use. Day 5.0 a. Test substances are prepared in sterile saline (vehicle). b. On the day of dosing, dissolve the renalase peptide. c. The day before administration, check the carrageenan solution for clarity (no visible signs of contamination). 1) Equilibrate the carrageenan to room temperature. d. Record the mouse's weight in your lab notebook. e. The initial volume of the footpad is measured and recorded. 1) Place a beaker of water on the balance and set the tare to zero. 2) The right hind paw is placed in a beaker of water on a balance with the top of the ankle joint in the center of the meniscus of the water. The reading displayed on the balance is recorded. 3) Wet feet will alter the readings, so be sure to dry your feet after each measurement. 4) Tare the scale to zero between measurements to account for the loss in water volume. f. Vehicle and test substances are administered at 10 mg / kg by subcutaneous (SC, SOP1610) injection 30 minutes prior to administration of carrageenan, as shown in Table 1. [Table 2]

[0074] A study was conducted to compare the inhibition of inflammation by peptide 10 as compared to the corresponding peptide fragment without the alanine substitution at position 220 (peptide 81). The results are shown in Figures 8 and 9 and in Table 2 below. Values ​​represent percent change from control and * denotes <<0.05 statistical significance. [Table 3]

[0075] The results show that peptide 10, [Ala 220]-Ac-Renalase A(205-240)-NH2 was more active and more sustained active than peptide 81, Ac-Renalase A(205-240)-OH, consistent with peptide 10 being more stable than peptide 81. Note that peptide 10 had activity close to that of standards dexamethasone and indomethacin. The results are from comparable but different experiments performed by the same group with the same protocol but different drugs. Note, for example, that at 2 hours both peptide 10 and peptide 81 were active, but only peptide 10 was active for a longer period of time.

[0076] Furthermore, as shown in Figure 10, reduced renalase activity correlates with increased mortality in COVID-19, while treatment with the renalase agonist BP-1002 has beneficial effects on COVID-19, other infections and inflammation, as shown in Figures 11-14.

[0077] All references cited herein are hereby incorporated by reference as if included in their entirety.

[0078] In the description and claims of this specification, the word "comprise" and variations of that word such as "comprises" and "comprising" are not intended to exclude other features, additions, components, integers or steps; rather, unless otherwise expressly stated, the scope of these words should be interpreted broadly to have an inclusive rather than exclusive meaning.

[0079] While the compositions and methods of the present invention are described in this disclosure as illustrative examples, it should be understood that the invention is not limited thereto and that variations thereof can be made, as known to those skilled in the art, without departing from the teachings of the present invention as defined by the appended claims.

Claims

1. [Ala ２２０ ]-Ac-Renalase A(205-240)-NH ２ A peptide consisting of

2. A therapeutic agent for acute kidney injury or acute pancreatitis, comprising the peptide according to claim 1 as an active ingredient.

3. A pharmaceutical composition comprising the peptide of claim 1 in admixture with a pharma- ceutically acceptable carrier.

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