Relaxin analogs and methods of use thereof

Abstract

Relaxin (RLN) analogs are disclosed that include modifications that increase half-life compared to native human RLN, maintain selectivity for the RXFP1 receptor, and provide stability in vitro and in vivo, improving druggability and reducing immunogenicity. Pharmaceutical compositions are also disclosed that include one or more of the RLN analogs described herein in a pharmaceutically acceptable carrier. Methods of making and using the RLN analogs are also disclosed, particularly for treating cardiovascular, pulmonary, and / or renal conditions, diseases, or disorders.

Description

[Technical Field]

[0001] The present disclosure relates generally to biology and medicine, and more particularly to relaxin (RLN) analogs, particularly long-acting, single-chain RLN analogs that can function as RLN / insulin-like family peptide (RXFP) receptor agonists by binding to RLN / RXFP receptors, such as the RXFP1 receptor. The disclosure further relates to compositions comprising same and their use in treating cardiovascular, pulmonary, and / or renal conditions, diseases, or disorders. [Background technology]

[0002] Relaxin (RLN) is part of the insulin superfamily, which in humans contains seven peptides with high structural similarity but low sequence homology: RLN1 (H1RLX, RLXH1, or H1), RLN2 (H2RLX, RLXH2, or H2), RLN3 (RXN3, ZINS4, or H3), insulin-like (INSL) peptide 3 (INSL3), INSL4, INSL5, and INSL6. Of particular interest here is RLN2, which is a heterodimer of two peptide chains (chain A and chain B) of 24 and 29 amino acids, respectively, linked by two disulfide bonds, with the A chain having an additional intramolecular disulfide bond (see Schwabe & McDonald (1977) Science 197:914-915). RLN2 is produced from its precursor hormone, prorelaxin, by cleaving the C peptide.

[0003] Physiologically, RLN2, originally recognized as a pregnancy hormone, exhibits diverse functions, including vasodilatory, antifibrotic, and angiogenic activities, regulating cardiovascular, hepatic, neuronal, pancreatic, pulmonary, and renal adaptations. RLN2 signaling occurs through two distinct classes of G protein-coupled receptors (GPCRs): LGR7 and LGR8, leucine-rich repeat-containing GPCRs now termed RXFP1 and RXFP2 receptors, respectively. Two other receptors in this family include RXFP3 and RXFP4 receptors. RLN2 has a short half-life (t1 / 2), which presents challenges when using RLN2 as a therapeutic agent. Indeed, the in vivo t1 / 2 of native RLN2 is only a few minutes. As a result, clinicians administer RLN2 via continuous intravenous infusion, which often results in inconvenience for individuals receiving RLN compounds and short-term efficacy.

[0004] There are several RLN2 analogs with improved tl / 2. For example, International Patent Application Publication No. 2018 / 148419 describes analogs containing unnatural amino acid residues, such as paraacetylphenylalanine, to which linkers, polymers, and / or pharmacokinetic enhancers can be attached to improve tl / 2. International Patent Application Publication No. 2018 / 138170 describes analogs that are fusions of A and B chains with a linker of at least 15 amino acids and a half-life extending moiety to improve tl / 2. International Patent Application Publication No. 2017 / 201340 describes analogs that are fusions of A and B chains with a variable light chain fragment to improve tl / 2. International Patent Application Publication No. 2015 / 067791 describes carrier-linked prodrug analogs, particularly PEG-based carriers, to improve tl / 2 (see also WO2012 / 024452 for additional PEG-linked analogs). International Patent Application Publication No. WO 2014 / 102179 describes analogs that are fusions of A and B chains with the Fc portion of IgG2 or IgG4 to improve t1 / 2. International Patent Application Publication No. WO 2013 / 004607 describes analogs that are fusions of A and B chains with a linker of at least 5 but less than 15 amino acids, or with the Fc domain of an antibody to improve t1 / 2.

[0005] Given the increasing understanding of the various physiological roles of RLN, there remains a need for long-acting RLN analogs with improved t1 / 2.

[0006] To address this need, the present disclosure first describes single-chain RLN analogs that have primary activity at the RXFP1 receptor (i.e., act as RXFP1 receptor agonists). Such RXFP1 receptor agonists include those having the basic structure from the amino terminus (N-terminus) to the carboxy terminus (C-terminus): VHH-L1-A-L2-B, VHH-L1-B-L2-A, A-L2-B-L1-VHH, or B-L2-A-L1-VHH, VHH is a moiety that can act as a pharmacokinetic enhancer, A is the RLN A chain, B is the RLN B chain, L1 is the first linker, and L2 is the second linker.

[0007] In some cases, the VHH portion may have the amino acid sequence of SEQ ID NO: 10, 11, 12, or 13, particularly SEQ ID NO: 10 or 12. In other cases, the VHH portion may have one or more additions, deletions, insertions, or substitutions such that the VHH portion has an amino acid sequence with at least about 90% to about 99% sequence identity to any one of SEQ ID NOs: 10, 11, 12, or 13 (see, e.g., SEQ ID NOs: 45-66).

[0008] In some cases, the A chain can have the amino acid sequence of SEQ ID NO: 2, 5, or 8, particularly SEQ ID NO: 5. In other cases, the A chain can have one or more additions, deletions, insertions, or substitutions such that the A chain has an amino acid sequence with at least about 90% to about 99% sequence identity to any one of SEQ ID NOs: 2, 5, or 8. For example, the A chain can be des1 to 4 of SEQ ID NO: 5.

[0009] In some cases, the B chain can have the amino acid sequence of SEQ ID NO: 3, 6, or 9, particularly SEQ ID NO: 6. In other cases, the B chain can have one or more additions, deletions, insertions, or substitutions such that the B chain has an amino acid sequence with at least about 90% to about 99% sequence identity to any one of SEQ ID NOs: 3, 6, or 9. For example, the B chain can be des1 of SEQ ID NO: 6.

[0010] In some cases, L1 is (GGGGQ) n (SEQ ID NO: 14), (GGGQ) n (SEQ ID NO: 15), (GGGGS) n (SEQ ID NO: 16), (PGPQ) n (SEQ ID NO: 17), or (PGPA) n(SEQ ID NO: 18), where n is 1 to 10, particularly about 5 to about 8. In other cases, L1 may have the amino acid sequence of SEQ ID NO: 19, 20, or 21. In still other cases, L1 may have one or more additions, deletions, insertions, or substitutions such that L1 has an amino acid sequence having at least about 90% to about 99% sequence identity to any one of SEQ ID NOs: 14 to 21.

[0011] In some cases, L2 may have the amino acid sequence of SEQ ID NO: 22, 23, or 67. In other cases, L2 may have one or more additions, deletions, insertions, or substitutions.

[0012] In certain cases, the RLN analogs may have an amino acid sequence that includes a VHH of SEQ ID NO: 10, 11, 12, or 13, an A chain of SEQ ID NO: 2, 5, or 8, a B chain of SEQ ID NO: 3, 6, or 9, an L1 of SEQ ID NO: 19, 20, or 21, and an L2 of SEQ ID NO: 22, 23, or 67. Alternatively, the RLN analogs may have an amino acid sequence that has at least about 90% to about 99% sequence identity to an amino acid sequence that includes an amino acid sequence that includes a VHH of SEQ ID NO: 10, 11, 12, or 13, an A chain of SEQ ID NO: 2, 5, or 8, a B chain of SEQ ID NO: 3, 6, or 9, an L1 of SEQ ID NO: 19, 20, or 21, and an L2 of SEQ ID NO: 22, 23, or 67.

[0013] In certain cases, the RLN analogs can have the amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39, particularly SEQ ID NO: 26, 27, 30, 31, 34, or 35. Alternatively, the RLN analogs can have an amino acid sequence having at least about 90% to about 99% sequence identity to the amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39, particularly SEQ ID NO: 26, 27, 30, 31, 34, or 35.

[0014] In some cases, the RLN analogs have a binding affinity at the RXFP1 receptor comparable to that of native human RLN2 (SEQ ID NOs: 5 and 6). In other cases, the RLN analogs have a binding affinity at the RXFP1 receptor that is greater than that of native human RLN2 (SEQ ID NOs: 5 and 6). In still other cases, the RLN analogs have a binding affinity at the RXFP1 receptor that is less than that of native human RLN2 (SEQ ID NOs: 5 and 6).

[0015] In some cases, the RLN analogs have a t1 / 2 longer than that of native human RLN2 (SEQ ID NOs: 5 and 6), including up to about 20 to about 30 days longer when administered to humans.

[0016] The compositions described above may alternatively be vectors and host cells comprising nucleic acid sequences encoding the amino acid sequences described herein, as well as nucleic acid sequences for expressing the RLN analogs described herein.

[0017] Second, the present invention describes a pharmaceutical composition comprising at least one RLN analog or a pharmaceutically acceptable salt thereof (e.g., trifluoroacetate, acetate, or hydrochloride) and a pharmaceutically acceptable carrier. In some cases, the pharmaceutically acceptable carrier is a buffer solution such as saline, phosphate-buffered saline, citrate-buffered saline, or histidine-buffered saline. In certain cases, the buffer solution is histidine, a histidine buffer, or a histidine-buffered saline. In other cases, the pharmaceutical composition may further comprise a carrier, a diluent, and / or an excipient.

[0018] Additionally, the pharmaceutical composition may include at least one additional therapeutic agent, such as, for example, an agent used as a standard of care in cardiovascular, pulmonary, and / or renal conditions, diseases, or disorders. In some cases, the at least one additional therapeutic agent may be an anticoagulant, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), an ARB-neprilysin inhibitor (ARNI), a beta-blocker, a diuretic, digitalis, digoxin, hydralazine nitrate / isorbide, a mineralocorticoid receptor antagonist (MRA, or aldosterone antagonist), a sodium-glucose cotransporter-2 (SGLT2) inhibitor, a statin, and / or an antiglycemic agent.

[0019] Third, methods for using the RLN analogs herein, particularly methods for using the RLN analogs to treat cardiovascular, pulmonary, and / or renal conditions, diseases, or disorders, are described, which include, at least, administering to an individual in need thereof, an effective amount of at least one RLN analog described herein, or a pharmaceutically acceptable salt thereof.

[0020] In some cases, the RLN analogs can be administered via any standard route of administration, such as intramuscularly, intravenously, parenterally, subcutaneously, or transdermally. In certain cases, the RLN analogs are administered subcutaneously (SQ), intramuscularly (IM), or intravenously (IV). In certain cases, the RLN analogs can be administered to an individual SQ or IV.

[0021] Similarly, in some cases, the RLN analogs can be administered daily, every other day, three times a week, twice a week, once a week (i.e., every week), every other week (i.e., every other week), once a month (i.e., every month), every other month (i.e., every other month), or even every three months. In certain cases, the RLN analogs can be administered SQ every other day, three times a week, twice a week, once a week, every other week, or once a month. In certain cases, the RLN analogs are administered SQ weekly (QW).

[0022] Alternatively, the RLN analog can be administered to an individual via IV. As described above, the RLN analog can be administered daily, every other day, three times a week, twice a week, once a week (i.e., every week), every other week (i.e., every other week), or monthly. In certain cases, the RLN analog can be administered via IV every other day, three times a week, twice a week, once a week, every other week, or monthly. In certain cases, the RLN analog is administered via IV once a week.

[0023] The method may also include administering the RLN analog in combination with an effective amount of at least one additional therapeutic agent. Briefly, standard of care for many of the conditions / diseases / disorders herein includes anticoagulants, ACE inhibitors, ARBs, ARNIs, beta-blockers, diuretics, digitalis, digoxin, hydralazine nitrate / isorbide, MRAs or other aldosterone antagonists), SGLT2 inhibitors, statins, and / or antiglycemic agents, as well as other therapeutic agents for controlling co-morbidities, including, but not limited to, high cholesterol, high blood pressure, atrial fibrillation, and diabetes. In some cases, the additional therapeutic agent may be administered simultaneously, separately, or sequentially with the RLN analog.

[0024] For example, the additional therapeutic agent may be administered at the same frequency as the RLN analog (i.e., every other day, twice a week, weekly, or even monthly). In other cases, the additional therapeutic agent may be administered at a different frequency than the RLN analog. In other cases, the additional therapeutic agent may be administered SQ or IV. In still other cases, the RLN analog may be administered SQ and the additional therapeutic agent may be administered orally or IV. Alternatively, the RLN analog is administered IV and the additional therapeutic agent is administered SQ.

[0025] In some cases, the individual in need has diabetic hypertension accompanied by renal dysfunction and / or obesity.

[0026] The method may also include steps such as measuring or obtaining blood pressure and comparing such obtained values ​​to one or more baseline or previously obtained values ​​to assess the effectiveness of the treatment / therapy.

[0027] These methods may also be combined with diet and exercise, and / or may be combined with additional therapeutic agents other than those discussed above.

[0028] Fourth, uses comprising at least one of the RLN analogs herein are described. For example, the RLN analogs herein can be provided for use in therapy, particularly in the treatment of cardiovascular, pulmonary, and / or renal conditions, diseases, or disorders. The RLN analogs can optionally be administered simultaneously, separately, or sequentially (i.e., in combination) with at least one additional therapeutic agent. Similarly, uses of the RLN analogs herein are provided in the manufacture of a medicament for treating cardiovascular, pulmonary, and / or renal conditions, diseases, or disorders, which optionally can further comprise one or more additional therapeutic agents as described above.

[0029] Fifth, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 10). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO: 10.

[0030] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 11). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO: 11.

[0031] Alternatively, a compound is provided that includes the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 12). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO: 12.

[0032] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 13). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO: 13.

[0033] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGTLVTVSS (SEQ ID NO:45). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:45.

[0034] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDSTAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSRVANLYPYWGQGTLVTVSS (SEQ ID NO:46). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:46.

[0035] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASYRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:47). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:47.

[0036] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGAYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:48). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:48.

[0037] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDETYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:49). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:49.

[0038] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDQTYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:50). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:50.

[0039] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITAYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:51). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:51.

[0040] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITEYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:52). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:52.

[0041] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITQYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:53). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:53.

[0042] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:54). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:54.

[0043] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITTYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:55). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:55.

[0044] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGKPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:56). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:56.

[0045] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGQPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:57). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:57.

[0046] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGSPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:58). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:58.

[0047] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRELITSKVADLYPYWGQGTLVTVSS (SEQ ID NO:59). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:59.

[0048] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRQLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 60). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO: 60.

[0049] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRSLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 61). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO: 61.

[0050] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPEITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 62). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO: 62.

[0051] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPGITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 63). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO: 63.

[0052] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPQITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 64). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO: 64.

[0053] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPTITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 65). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO: 65.

[0054] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITEKVADLYPYWGQGTLVTVSS (SEQ ID NO:66). In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence identity to SEQ ID NO:66.

[0055] The advantage of the RLN analogs herein is that they can be chemically or recombinantly synthesized as single-chain polypeptides (i.e., monomers), and therefore do not require intracellular proteolytic processing for biological activity. However, in some cases, it is contemplated that the VHH moiety can be conjugated not only to single-chain RLNs but also to double-chain RLNs (e.g., native). In the case of the VHH moiety, it can be conjugated not only to the N- and C-termini but also to any surface-exposed amino acids of the VHH (although such conjugation does not completely abolish albumin binding).

[0056] An advantage of the RLN analogs herein is that the VHH moieties can be used not only with the native A and B chain sequences, but also with modified versions thereof. Furthermore, the VHH moieties may be further modified to have enhanced or additional functions via other peptide / protein fusions or small molecules attached to the VHH moiety.

[0057] An advantage of the RLN analogs herein is that the VHH portion provides an extended duration of action in mammals, such as humans, and can have a t1 / 2 of about 20 to about 30 days, which can improve compliance by allowing for at least weekly or biweekly administration when compared to native human RLN, particularly native human RLN2 (SEQ ID NOs: 5 and 6).

[0058] An advantage of the RLN analogs herein is that they have similar or better selectivity, affinity, and / or potency for the RXFP1 over the RXFP2 receptor when compared to native human RLN2 (SEQ ID NOs: 5 and 6). Alternatively stated, the RLN analogs herein have full activity at the RXFP1 receptor and reduced or insufficient activity at one or more of the RXFP2, RXFP3, and RXFP4 receptors.

[0059] An advantage of the RLN analogues herein is that they have tunable pharmacokinetics, achieved by altering the albumin affinity of the VHH moiety.

[0060] An advantage of the RLN analogs herein is that they have improved stability in stored formulations when compared to native human RLN2 (SEQ ID NOs: 5 and 6) or RLN analogs with Fc fusions.

[0061] Furthermore, an advantage of VHH moieties is that they bind equally well to human serum albumin as well as to serum albumin from dogs, monkeys, mice, pigs, and rats, allowing pharmacodynamic, pharmacokinetic, and toxicology studies to be more easily translated from these species to humans.

[0062] An advantage of the VHH moieties is that they can be used not only to improve the t1 / 2 of the RLN analogs herein when compared to native human RLN2 (SEQ ID NOs: 5 and 6), but also to improve the t1 / 2 of other biologically active peptides and proteins, such as, for example, insulin, growth differentiation factor 15 (GDF-15), or glucose-dependent insulinotropic peptide 1 (GLP-1).

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the RLN analogs, pharmaceutical compositions, and methods, the preferred methods and materials are described herein.

[0064] Also, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there be only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0065] definition As used herein, "about" means within a statistically significant range of a value or values, such as, for example, a specified concentration, length, molecular weight, pH, sequence identity, time frame, temperature, volume, etc. Such values ​​or ranges may be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" depends on the particular system under study and can be readily understood by one of ordinary skill in the art.

[0066] As used herein, with respect to one or more of the RXFP receptors, "activity," "activate," "activating," and the like, refer to the ability of a compound, such as an RLN analogue herein, to bind to that receptor and induce a response thereat, as measured using assays known in the art, such as the in vitro assays described below.

[0067] As used herein, "amino acid" refers to a molecule that, from a chemical standpoint, is characterized by the presence of one or more amine groups and one or more carboxylic acid groups, and may contain other functional groups. As is known in the art, there is a set of 20 amino acids that are designated as standard amino acids and can be used as building blocks for the majority of peptides / proteins produced by any living organism. The amino acid sequences of the present disclosure include the standard one-letter or three-letter codes for the 20 standard amino acids.

[0068] As used herein, "analog" means a compound, such as a synthetic peptide or polypeptide, that activates a target receptor and elicits at least one in vivo or in vitro effect elicited by the receptor's natural agonist.

[0069] As used herein, "conservative substitution" refers to a variant of a reference peptide or polypeptide that is identical to the reference molecule except for one or more conservative amino acid substitutions in its amino acid sequence. Generally, conservatively modified variants contain an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference amino acid sequence. More specifically, conservative substitution refers to the substitution of an amino acid with an amino acid that has similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone structure, and rigidity) and has minimal effect on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitution of functionally similar amino acids is well known in the art and does not need to be described in detail herein.

[0070] As used herein, "effective amount" means an amount or dose of one or more RLN analogs herein, or pharmaceutically acceptable salts thereof, that, upon single or multiple administration to an individual in need thereof, provides a desired effect (i.e., that can produce a clinically measurable difference in the individual's condition, such as, for example, increased angiogenesis, increased vascular compliance, increased cardiac blood flow, increased hepatic blood flow, increased pulmonary blood flow, increased renal blood flow, increased glomerular filtration rate, lowered blood pressure, reduced (or prevented) inflammation, and / or reduced (or prevented) cardiac, renal, hepatic, or pulmonary fibrosis) in such individual during diagnosis or treatment. An effective amount can be readily determined by one of ordinary skill in the art by the use of known techniques and by observing results obtained under similar circumstances. In determining an effective amount for an individual, numerous factors are taken into consideration, including, but not limited to, the species of mammal, its size, age, and general health, the particular disease or disorder involved, the extent of the disease or disorder or its involvement or severity, the individual's response, the particular RLN analog administered, the mode of administration, the bioavailability characteristics of the administered preparation, the selected dosing regimen, the use of concomitant medications, and other relevant circumstances.

[0071] As used herein, "long-lasting action" means that the binding affinity and activity of the RLN analogs herein lasts for a longer period than native RLN, particularly native human RLN2 (SEQ ID NOs: 5 and 6), thereby allowing for dosing at least once daily or even as frequently as three times weekly, twice weekly, or even once weekly. The time-action profile of the RLN analogs may be measured using known pharmacokinetic testing methods, such as those utilized in the Examples below.

[0072] As used herein, "half-life" or "t1 / 2" refers to the time it takes for half of the amount of a compound, such as native RLN or an RLN analog herein, to be removed by biological processes from a fluid, such as serum or plasma, or other physiological space of an individual. Alternatively, t1 / 2 can refer to the time it takes for an amount of such a compound to lose half of its pharmacological, physiological, or radiological activity.

[0073] As used herein, "median effective concentration" or "EC 50 " refers to the concentration of compound that results in 50% activation / stimulation of an assay endpoint such as a dose-response curve (e.g., cAMP, PI3K-Akt, NFκβ, VEGF, and / or nitric oxide (NO) signaling pathway).

[0074] As used herein, "in combination with" means that at least one of the RLN analogs herein is administered simultaneously, sequentially, or in a single combined formulation with one or more additional therapeutic agents.

[0075] As used herein, "an individual in need thereof" means a mammal, such as a human, having a condition, disease, disorder, or symptom that requires treatment or therapy, including, for example, those listed herein. In particular, the individual to be treated is a human.

[0076] As used herein, "long-acting" means that the binding affinity and activity of the RLN analogs herein lasts for a longer period than native human RLN2 (SEQ ID NOs: 5 and 6), thereby allowing for dosing at least once daily, or even as frequently as three times weekly, twice weekly, once weekly, or even once monthly. The time-action profile of the RLN analogs may be measured using known pharmacokinetic testing methods, such as those described in the Examples below.

[0077] As used herein, "non-standard amino acid" refers to an amino acid that may occur naturally in cells but is not involved in peptide synthesis. Non-standard amino acids may be building blocks of peptides and are often produced by modification of standard amino acids in peptides (i.e., via post-translational modification). Non-standard amino acids may include D-amino acids that have the opposite absolute chirality to the standard amino acids listed above.

[0078] As used herein, "pharmaceutically acceptable buffer" means any of the standard pharmaceutical buffers known to those of skill in the art.

[0079] As used herein, "RLN" refers to relaxin obtained or derived from any species, including mammalian species, particularly humans, where the native form is a heterodimeric peptide consisting of two peptide chains (e.g., A and B chains) connected via two disulfide bonds, with the A chain further possessing a single intramolecular disulfide bond. RLN includes both native RLN (i.e., full-length) and its variants (i.e., additions, deletions, insertions, and / or substitutions of native RLN). In humans, there are three native RLN isoforms: RLN1, RLN2, and RLN3. RLN processing begins with preprorelaxin, which is then processed into prorelaxin (containing A chain, B chain, and C peptide; native RLN has the structure BCA). The sequence of native human proRLN1 is set forth in SEQ ID NO: 1 (see also UniProt / SwissProt database accession number P04808), the sequence of native human proRLN2 is set forth in SEQ ID NO: 4 (see also UniProt / SwissProt database accession number P04090), and the sequence of native human proRLN3 is set forth in SEQ ID NO: 7 (see also UniProt / SwissProt database accession number Q8WXF3). Prorelaxin undergoes further processing, in which the C-peptide is cleaved to arrive at RLN. The sequences of the A chains of native human RLN1, RLN2, and RLN3 are set forth in SEQ ID NOs: 2, 5, and 8, respectively. Similarly, the sequences of the B chains of native human RLN1, RLN2, and RLN3 are set forth in SEQ ID NOs: 3, 6, and 9, respectively.

[0080] In humans, there are four RLN receptors that act as GPCRs: RXFP1 (SEQ ID NO: 40; also see UniProt / SwissProt database accession number Q9HBX9), RXFP2 (SEQ ID NO: 41; also see UniProt / SwissProt database accession number Q8WXD0), RXFP3 (SEQ ID NO: 42; also see UniProt / SwissProt database accession number Q9NSD7), and RXFP4 (SEQ ID NO: 43; also see UniProt / SwissProt database accession number Q8TDU9) (see Halls et al. (2007) Br. J. Pharmacol. 150: 677-691). Of interest here are the RXFP1 and RXFP2 receptors, both of which can bind to RLN1 and RLN2. RXFP1 receptors are found in the brain, blood cells, bone, heart, kidney, lung, liver, and vasculature, whereas RXFP2 receptors are much more restricted, found in bone and the conductance band. Stimulation of RXFP1 and RXFP2 receptors activates signaling networks involving adenylate cyclase, protein kinase A, protein kinase C, phosphatidylinositol 3-kinase, and / or extracellular signal-regulated kinase (Erk1 / 2).

[0081] As used herein, "RLN analogs" and the like refer to compounds, such as peptides or polypeptides, that elicit one or more of the effects of native RLN at one or more RXFP receptors but differ in some way in amino acid sequence from native RLN. RLN analogs can also include variants of these compounds, which are functionally equivalent to RLN but are fragments, or have the complete sequence but contain additions, deletions, insertions, and / or substitutions. All references to amino acid positions in unmodified or modified RLN described herein are based on the corresponding positions in the A chain of native human RLN2, SEQ ID NO: 5, or the B chain of native human RLN2, SEQ ID NO: 6, unless otherwise specified. In some cases, RLN analogs herein may bind to RXFP with higher or lower affinity but exhibit a longer t1 / 2 in vivo or in vitro when compared to native RLN, particularly native human RLN2 (SEQ ID NOs: 5 and 6). Thus, RLN analogs herein are synthetic compounds that act as RXFP receptor agonists.

[0082] As used herein, "sequence homology" refers to a quantitative characteristic of two or more nucleic acid or amino acid sequences of biological compounds, such as the match over the entire length or comparison window of two or more sequences. Sequence homology can be measured by (1) percent identity or (2) percent similarity. Percent identity measures the percentage of identical residues between two biological compounds divided by the length of the shortest sequence, while percent similarity measures identity and also includes sequence gaps and residue similarity in the assessment. Methods and algorithms for determining sequence homology are well known in the art and need not be described exhaustively herein. A specific percentage of identical nucleotide or amino acid positions is at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0083] As used herein, "single-chain relaxin," "scRLN," and the like refer to an RLN polypeptide in which the A and B chains are connected to each other by a linker (i.e., L2), such as A-L2-B or B-L2-A. Additionally, scRLN may contain at least one of the native inter- and / or intra-chain disulfide bonds to maintain correct structural folding.

[0084] As used herein, "two-chain relaxin," "tcRLN," and the like refer to an RLN polypeptide in which the A and B chains are connected to each other by one or more inter- and / or intra-chain disulfide bonds, rather than by any linker, to maintain the correct structural folding, such as native RLN.

[0085] As used herein, "treating" or "to treat" means managing or caring for an individual having a condition, disease, disorder, or condition for which administration of an RLN analog is indicated for the purpose of alleviating, inhibiting, reversing, slowing, or halting the progression or severity of the condition, disease, disorder, or condition. Treating includes administering an RLN analog herein or a composition comprising an RLN analog herein to an individual to prevent the onset of a condition or complication, alleviate a condition or complication, or eliminate the condition, disease, disorder, or condition. Treating includes administering an RLN analog herein or a composition comprising an RLN analog herein to an individual to produce, for example, increased angiogenesis, increased vascular compliance, increased cardiac blood flow, increased hepatic blood flow, increased pulmonary blood flow, increased renal blood flow, increased glomerular filtration rate, lowered blood pressure, reduced (or prevented) inflammation, and / or reduced (or prevented) fibrosis of the heart, kidney, liver, or lung. The individual to be treated is a mammal, particularly a human.

[0086] As used herein, "individual," "patient," and "subject" are used interchangeably and refer to a mammal, particularly a human. In certain instances, the individual is further characterized by a condition, disease, disorder, and / or symptom that would benefit from administering an RLN analog herein.

[0087] As used herein, "VHH" or "VHH moiety" refers to an antibody fragment in the form of a single domain antibody, particularly a single monomeric variable region of a heavy-chain-only antibody (HcAb), which is very small in size, approximately 15 kDa. It has now been discovered that VHH moieties can be used as pharmacokinetic enhancers to extend the duration of action and / or improve the t1 / 2 of the RLN analogs herein. While the VHH moieties herein bind to serum albumin, they can also be used to bind to IgG (including the Fc domain), neonatal Fc receptor (FcRn), or other long-lasting serum proteins. While the VHH moieties herein are used to improve the t1 / 2 of RLN, they can similarly be used to improve the t1 / 2 of other biologically active peptides / proteins, such as insulin, GDF-15, or GLP-1.

[0088] Certain abbreviations are defined as follows: "ACR" refers to urinary albumin / urinary creatinine ratio, "amu" refers to atomic mass unit, "AUC" refers to area under the curve, "Boc" refers to tert-butoxycarbonyl, "cAMP" refers to cyclic adenosine monophosphate, "CMV" refers to cytomegalovirus, "CV" refers to column volume, "DNA" refers to deoxyribonucleic acid, "DMF" refers to dimethylformamide, "DMSO" refers to dimethyl sulfoxide, "EDC" refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, "EDTA" refers to ethylenediaminetetraacetic acid, "EIA / RIA" refers to enzyme immunoassay / radioimmunoassay, "ETA" refers to ethanolamine, "GS" refers to glutamine synthetase, "HIC" refers to hydrophobic interaction chromatography, "hr" refers to hour(s), "HTRF" refers to homogeneous time-resolved fluorescence, and "IV" refers to intravenous. where "IP" refers to intraperitoneal, "kDa" refers to kilodaltons, "LC / MS" refers to liquid chromatography mass spectrometry, "min" refers to minute(s), "MS" refers to mass spectrometry, "MSX" refers to methionine sulfoximine, "NaOAc" refers to sodium acetate, "NHS" refers to N-hydroxysuccinimide, "OtBu" refers to O-tert-butyl ether, "Pbf" refers to N-G-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl ether, "PEI" refers to polyethyleneimine, "RP-HPLC" refers to reversed-phase high-performance liquid chromatography, "RU" means resonance units, "sec" refers to second(s), "SPR" refers to surface plasmon resonance, "SQ" refers to subcutaneous, "SEC" refers to size exclusion chromatography, "SEM" refers to standard error of the mean, "TFA" refers to trifluoroacetic acid, and "Trt" refers to trityl ether.

[0089] RLN analog The RLN analogs herein are structurally similar to native human RLN, but have many structural differences. For example, when compared to native human RLN2 (SEQ ID NOs: 5 and 6), the RLN analogs lack one or more amino acids present in native human RLN2, contain a peptide linker between the A and B chains, and contain an albumin-binding VHH moiety. The RLN analogs exhibit full activity at the RXFP1 receptor and reduced or insufficient activity at one or more of the RXFP2, RXFP3, and RXFP4 receptors. Similarly, the RLN analogs possess beneficial attributes related to their potential development as therapeutic treatments, including improved solubility in aqueous solutions, improved chemical and physical formulation stability, an enhanced pharmacokinetic profile (which can be tailored based on VHH affinity for serum albumin), and / or minimized immunogenicity potential.

[0090] Briefly, the RLN analogs herein comprise an amino acid sequence from N-terminus to C-terminus having one of the following structures: VHH-L1-A-L2-B, VHH-L1-B-L2-A, A-L2-B-L1-VHH, or B-L2-A-L1-VHH, VHH is a moiety that acts as a pharmacokinetic enhancer, A is the RLN A chain, B is the RLN B chain, L1 is a first peptide linker, and L2 is a second peptide linker, where L1 and L2 are different from each other (i.e., each has an amino acid sequence that is not the same).

[0091] With respect to the A chain, it can be a native RLN A chain, such as native human RLN1 A chain (SEQ ID NO:2), native human RLN2 A chain (SEQ ID NO:5), or native human RLN3 A chain (SEQ ID NO:8). Alternatively, the A chain can be a variant thereof. For example, one A chain variant can have an amino acid sequence lacking residues 1-4 of SEQ ID NO:5 (i.e., des1-4 human RLN2 A chain or desA1-4).

[0092] Similarly, with respect to the B chain, this can be a native RLN B chain, e.g., native human RLN1 B chain (SEQ ID NO:3), native human RLN2 B chain (SEQ ID NO:6), or native human RLN3 B chain (SEQ ID NO:9). Alternatively, the B chain can be a variant thereof. For example, one B chain variant can have an amino acid sequence lacking residue 1 of SEQ ID NO:6 (i.e., des1 human RLN2 B chain or desB1).

[0093] In some cases, the A chain can be a native human RLN1 A chain (SEQ ID NO:2) and the B chain can be a native human RLN1 B chain (SEQ ID NO:3); the A chain can be a native human RLN2 A chain (SEQ ID NO:5) and the B chain can be a native human RLN2 B chain (SEQ ID NO:6); the A chain can be a native human RLN3 A chain (SEQ ID NO:8) and the B chain can be a native human RLN3 B chain (SEQ ID NO:9); the A chain can be a native human RLN1 A chain (SEQ ID NO:2) and the B chain can be a native human RLN2 B chain (SEQ ID NO:6); the A chain can be a native human RLN1 A chain (SEQ ID NO:2) and the B chain can be a native human RLN3 B chain (SEQ ID NO:9); the A chain can be a native human RLN2 A chain (SEQ ID NO:5) and the B chain can be a native human RLN1 B chain (SEQ ID NO:3); the A chain can be a native human RLN2 A chain (SEQ ID NO:5) and the B chain can be a native human RLN3 The A chain may be the natural human RLN3 A chain (SEQ ID NO: 8) and the B chain may be the natural human RLN1 B chain (SEQ ID NO: 3), or the A chain may be the natural human RLN3 A chain (SEQ ID NO: 8) and the B chain may be the natural human RLN2 B chain (SEQ ID NO: 6).

[0094] In some cases, the A chain can be an RLN2 A chain variant lacking residues 1-4 (desA1-4), and the B chain can be any naturally occurring B chain. In other cases, the A chain can be any naturally occurring A chain, and the B chain can be an RLN2 B chain variant lacking residue 1 (desB1). In yet other cases, the A chain can be an RLN2 A chain variant lacking residues 1-4 (desA1-4), and the B chain can be an RLN2 B chain variant lacking residue 1 (desB1). In certain cases, the A chain is a desA1-4 variant. In certain cases, the B chain is a desB1 variant.

[0095] Other A and B chains that can be used in the RLN analogs herein are described, for example, in International Patent Application Publication Nos. 2018 / 148419, 2018 / 138170, 2017 / 201340, 2016 / 149501, 2015 / 157829, 2015 / 067791, 2015 / 067113, 2014 / 102179, 2013 / 177529, 2013 / 007563, 2013 / 004607, 2012 / 031326, and 2012 / 024452, and U.S. Patent Application Publication No. 2011 / 0243942. Chan et al. (2012) J.Biol.Chem.287:41152-41164, Claasz et al. (2002) Eur.J.Biochem.269:6287-6293, Hossain et al. (2015) Org.Biomol.Chem.13:10895-10890, Hossain et See also Park et al. (2008) J. Biol. Chem. 283:32099-32109, and Wilkinson et al. (2005) BMC Evol. Biol. 5:14.

[0096] With respect to L1, it can be a peptide of about 1 amino acid to about 50 amino acids. Alternatively, L1 can be about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 amino acids. Further alternatively, L1 can be about 5 amino acids to about 10 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 45 amino acids, or about 45 amino acids to about 50 amino acids. In some cases, L1 can be omitted, allowing the A or B chain to be directly conjugated to the VHH moiety. In some cases, L1 can be selected from the group consisting of (GGGGQ) n(SEQ ID NO: 14), where n can be from about 1 to about 10, particularly 5 (i.e., (GGGGQ)5, SEQ ID NO: 19). In other cases, L1 can include a repeat sequence of (PGPQ) n (SEQ ID NO: 17), where n can be from about 1 to about 10, particularly 8 (i.e., (PGPQ)8, SEQ ID NO: 20). In still other cases, L1 can include a repeat sequence of (PGPA) n (SEQ ID NO: 18), where n can be about 1 to about 10, particularly 8 (ie, (PGPA) 8 , SEQ ID NO: 21).

[0097] Other linkers that can be used in RLN analogs as L1 include (GGGQ) n (SEQ ID NO: 15) or (GGGGS) n (SEQ ID NO: 16).

[0098] With respect to L2, it can be a peptide of about 1 to about 15 amino acids. Alternatively, L2 can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 amino acids. Further alternatively, L2 can be about 1 to about 5 amino acids, about 5 to about 10 amino acids, about 10 to about 15 amino acids, particularly 10 to 15 amino acids. In some cases, L2 can contain a mixture of Ala / A, Gln / Q, Gly / G, Pro / P, and Ser / S residues. In other cases, L2 can be SEQ ID NO: 22, 23, or 67.

[0099] With respect to the VHH, it can be a polypeptide of about 50 to about 200 amino acids, particularly about 125 to about 150 amino acids, capable of binding to serum albumin or another serum protein with a long t. In some cases, the VHH can be any one of SEQ ID NOS: 10-13. Alternatively, the VHH can be any one of SEQ ID NOS: 45-66. The structural features of these VHH moieties result in RLN analogs with longer t when compared to native RLN, particularly native human RLN2 (SEQ ID NOS: 5 and 6). Given that the VHH moieties herein target serum albumin, the t of the RLN analog can therefore be expected to be similar to the t of serum albumin of the species to which the RLN analog is administered (taking into account target-mediated drug disposition).

[0100] In addition to the modifications described in this disclosure, the RLN analogs herein may include one or more additional amino acid modifications, particularly conservative substitutions, provided that the RLN analogs are still capable of binding to and activating the RXFP1 receptor.

[0101] In summary, exemplary RLN analogs are: RLN analog 1, which contains a VHH portion (underlined), (G4Q)5L1 (italicized), the A chain of RLN2, 10 residues of L2 (bold), and the B chain of RLN2 (desB1), has the following amino acid sequence: [ka] (SEQ ID NO: 24) or a pharmaceutically acceptable salt thereof, RLN analog 2, which contains a VHH portion (underlined), (PGPA)8L1 (italicized), the A chain of RLN2, 10 residues of L2 (bold), and the B chain of RLN2 (desB1), has the following amino acid sequence: [ka] (SEQ ID NO: 25) or a pharmaceutically acceptable salt thereof, RLN analog 3, which contains a VHH portion (underlined), (G4Q)5L1 (italicized), the B chain of RLN2 (desB1), 10 residues of L2 (bold), and the A chain of RLN2, has the following amino acid sequence: [ka] (SEQ ID NO: 26) or a pharmaceutically acceptable salt thereof, RLN analog 4, which contains a VHH portion (underlined), (PGPQ)8L1 (italicized), the B chain of RLN2 (desB1), 10 residues of L2 (bold), and the A chain of RLN2, has the following amino acid sequence: [ka] (SEQ ID NO: 27) or a pharmaceutically acceptable salt thereof, RLN analog 5, which contains the B chain of RLN2 (desB1), the 10 residues of L2 (bold), the A chain of RLN2, (G4Q)5L1 (italic), and the VHH portion (underlined), has the following amino acid sequence: [ka] (SEQ ID NO: 28) or a pharmaceutically acceptable salt thereof, RLN analog 6, which contains the B chain of RLN2 (desB1), the 10 residues of L2 (bold), the A chain of RLN2, (PGPQ)8L1 (italic), and the VHH portion (underlined), has the following amino acid sequence: [ka] (SEQ ID NO: 29) or a pharmaceutically acceptable salt thereof, RLN analog 7, which contains a VHH portion (underlined), (G4Q)5L1 (italicized), the B chain of RLN2 (desB1), 10 residues of L2 (bold), and the A chain of RLN2, has the following amino acid sequence: [ka] (SEQ ID NO: 30) or a pharmaceutically acceptable salt thereof, RLN analog 8, which contains a VHH portion (underlined), (PGPQ)8-L1 (italicized), the B chain of RLN2 (desB1), 10 residues of L2 (bold), and the A chain of RLN2, has the following amino acid sequence: [ka] (SEQ ID NO: 31) or a pharmaceutically acceptable salt thereof, RLN analog 9, which contains the B chain of RLN2 (desB1), the 10 residues of L2 (bold), the A chain of RLN2, (G4Q)5L1 (italic), and the VHH portion (underlined), has the following amino acid sequence: [ka] (SEQ ID NO: 32) or a pharmaceutically acceptable salt thereof, RLN analog 10, which contains the B chain of RLN2 (desB1), the 10 residues of L2 (bold), the A chain of RLN2, (PGPQ)8L1 (italic), and the VHH portion (underlined), has the following amino acid sequence: [ka] (SEQ ID NO: 33) or a pharmaceutically acceptable salt thereof, RLN analog 11, which contains a VHH portion (underlined), (G4Q)5L1 (italicized), the B chain of RLN2 (desB1), the 13-residue L2 (bold), and the A chain of RLN2 (desA1-4), has the following amino acid sequence: [ka] (SEQ ID NO: 34) or a pharmaceutically acceptable salt thereof, RLN analog 12, which contains a VHH portion (underlined), (G4Q)5L1 (italicized), the B chain of RLN2 (desB1), the 13-residue L2 (bold), and the A chain of RLN2 (desA1-4), has the following amino acid sequence: [ka] (SEQ ID NO: 35) or a pharmaceutically acceptable salt thereof, RLN analog 13, which contains a VHH portion (underlined), (G4Q)5L1 (italicized), the A chain of RLN2 (desA1-4), 10 residues of L2 (bold), and the B chain of RLN2 (desB1), has the following amino acid sequence: [ka] (SEQ ID NO: 36) or a pharmaceutically acceptable salt thereof, RLN analog 14, which contains a VHH portion (underlined), (PGPQ)8L1 (italicized), the A chain of RLN2 (desA1-4), the 10 residues of L2 (bold), and the B chain of RLN2 (desB1), has the following amino acid sequence: [ka] (SEQ ID NO: 37) or a pharmaceutically acceptable salt thereof, RLN analog 15, which contains a VHH portion (underlined), (G4Q)5L1 (italicized), the B chain of RLN2 (native), 10 residues of L2 (bold), and the A chain of RLN2 (native), has the following amino acid sequence: [ka] (SEQ ID NO: 38) or a pharmaceutically acceptable salt thereof, RLN analog 16, which contains a VHH portion (underlined), (PGPA)8L1 (italicized), the B chain of RLN2 (native), 10 residues of L2 (bold), and the A chain of RLN2 (native), has the following amino acid sequence: [ka] (SEQ ID NO: 39) or a pharmaceutically acceptable salt thereof.

[0102] The half-life of the RLN analogs herein can be measured using methods known in the art, including, for example, the methods described in the Examples below. Similarly, the affinity of the present RLN analogs for albumin of different species can be measured using methods known in the art for measuring binding affinity, for example, the methods described in the Examples below, and generally corresponds to the equilibrium dissociation constant (K DAdditionally, the activity of the RLN analogs described herein at each of the RXFP receptors can be measured using methods known in the art, including, for example, the in vitro activity assays described below, and is generally expressed as an EC 50 It is expressed as a value.

[0103] As a result of the above modifications, the RLN analogs herein, when administered to mammals, particularly humans, have a t longer than that of native RLN, particularly native human RLN2 (SEQ ID NOS: 5 and 6). Because the VHH moieties herein target serum albumin as described above, the t of the RLN analogs herein can be expected to be similar to that of serum albumin of the species to which the RLN analogs are administered. In some cases, the RLN analogs may have a t of about 1 to about 31 days, about 5 to about 25 days, about 10 to about 20 days, or even about 15 days. In other cases, the RLN analogs may have a t of about 1 to about 5 days, about 6 to about 10 days, about 11 to about 15 days, about 16 to about 20 days, about 21 to about 25 days, or even about 26 to about 31 days. In other cases, the RLN analogs can have a t of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, or even about 31 days. In certain cases, the RLN analogs, when administered to a human, can have a t of about 20 days.

[0104] Similarly, the RLN analogs herein, when administered to humans, are, for example, within about 10- to about 100-fold more potent at the RXFP1 receptor than native human RLN2 (SEQ ID NOs: 5 and 6).

[0105] Pharmaceutical Compositions and Kits The RLN analogs described herein can be formulated as pharmaceutical compositions that can be administered parenterally (e.g., intravenously, intraperitoneally, intramuscularly, subcutaneously, or transdermally). Such pharmaceutical compositions and techniques for preparing them are well known in the art. See, for example, Remington, "The Science and Practice of Pharmacy" (D.B. Troy ed., 21 st See, e.g., Lippincott, Williams & Wilkins, Ed., 2006). In certain cases, the present RLN analogs are administered SQ or IV. Alternatively, however, the RLN analogs can be formulated in forms for other pharmaceutically acceptable routes, such as, for example, tablets or other solids for oral administration, sustained-release capsules, and any other form currently in use, including creams, lotions, inhalants, etc.

[0106] As mentioned above, to improve their in vivo compatibility and efficacy, the RLN analogs herein can be reacted with any number of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and general techniques for preparing them are well known in the art (e.g., Stahl et al., "Handbook of Pharmaceutical Salts: Properties, Selection and Use" (2002). nd Revised Ed. Wiley-VCH, 2011). Pharmaceutically acceptable salts for use herein include sodium salts, trifluoroacetate salts, hydrochloride salts, and / or acetate salts.

[0107] The RLN analogues herein can be administered by a physician or self-administered by injection.It is understood that the gauge size and injection volume can be easily determined by those skilled in the art.However, the injection volume can be about 2mL or less, or even about 1mL or less, and the needle gauge can be about 27G or more, or even about 29G or more.

[0108] The present disclosure also provides and thus encompasses novel intermediates and methods useful for synthesizing the RLN analogs described herein or pharmaceutically acceptable salts thereof. The intermediates and RLN analogs can be prepared by a variety of techniques well known in the art. For example, methods using recombinant synthesis are illustrated in the following examples. The specific steps for each of the described techniques can be combined in various methods for preparing the RLN analogs. The reagents and starting materials are readily available to those skilled in the art.

[0109] The RLN analogs herein are generally effective over a wide dosage range. Exemplary doses of the RLN analogs or pharmaceutical compositions containing them can be milligrams (mg), micrograms (μg), nanograms (ng), or picograms (pg) per kilogram (kg) of an individual. Thus, a daily dose can be from about 1 μg to about 100 mg.

[0110] Here, an effective amount of the RLN analog in the pharmaceutical composition may be a dose of about 0.25 mg to about 5.0 mg, although one skilled in the art will appreciate that in some cases, an effective amount (i.e., dose / dosage) below the lower end of the aforementioned range may be more than sufficient, while in other cases, an effective amount may be a higher dose and may be used with acceptable side effects.

[0111] In addition to the RLN analogs herein, the pharmaceutical compositions can also include at least one additional therapeutic agent, particularly a therapeutic agent typically used as a standard of care in cardiovascular, pulmonary, and renal conditions, diseases, and disorders.

[0112] Thus, the pharmaceutical compositions can comprise an effective amount of at least one RLN analog described herein, a pharmaceutically acceptable carrier, and optionally at least one additional therapeutic agent. For example, the pharmaceutical compositions can include an effective amount of an RLN analog of SEQ ID NO: 24 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 25 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 26 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 27 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 28 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 29 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 30 and a pharmaceutically acceptable carrier, or an effective amount of an RLN analog of SEQ ID NO: 31 and a pharmaceutically acceptable carrier. , an effective amount of an RLN analog of SEQ ID NO: 32 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 33 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 34 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 35 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 36 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 37 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 38 and a pharmaceutically acceptable carrier, or an effective amount of an RLN analog of SEQ ID NO: 39 and a pharmaceutically acceptable carrier.

[0113] Alternatively, the RLN analogs herein may be provided as part of a kit. In some cases, the kit includes a device for administering at least one RLN analog (and optionally at least one additional therapeutic agent) to an individual. In certain cases, the kit includes a syringe and needle for administering at least one RLN analog (and optionally at least one additional therapeutic agent). In certain cases, the RLN analog (and optionally at least one additional therapeutic agent) is pre-formulated in an aqueous solution in the syringe.

[0114] Methods for making and using RLN analogs The RLN analogs herein can be produced via any number of standard recombinant DNA methods known in the art or standard chemical peptide synthesis methods. With regard to recombinant DNA methods, standard recombinant techniques can be used to construct a polynucleotide having a nucleic acid sequence encoding the amino acid sequence of the RLN analog, incorporate the polynucleotide into a recombinant expression vector, and introduce the vector into host cells, such as bacteria, yeast, and mammalian cells, to produce the RLN analog. See, for example, Green & Sambrook, "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 4 th ed. 2012).

[0115] Regarding recombinant DNA methods, the compounds herein can be prepared by using recombinant DNA technology to produce protein or precursor protein molecules. DNA, including cDNA and synthetic DNA, can be double-stranded or single-stranded, and the coding sequence therein encoding the compounds herein can vary as a result of redundancy or degeneracy in the genetic code. Briefly, a DNA sequence encoding the compounds herein is introduced into a host cell to produce the compounds or their precursors. The host cell can be a bacterial cell, such as Escherichia coli K12 or B strain, a fungal cell, such as a yeast cell, or a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

[0116] To produce the compound of the present invention or its precursor, suitable host cells are transiently or stably transfected or transformed with expression systems such as expression vectors.Expression vectors are typically replicable in host organisms, either as episomes or as an integrated part of host chromosomal DNA.Generally, expression vectors contain selectable markers, such as tetracycline, neomycin, G418 and dihydrofolate reductase, to allow detection of cells transformed with desired DNA sequence.

[0117] The specific biosynthetic or synthetic steps for each of the steps described herein may be used, not used, or combined in different ways to prepare the compounds of the present invention.

[0118] For chemical peptide synthesis, standard manual or automated solid-phase synthesis procedures can be used. For example, automated peptide synthesizers are commercially available from, for example, Applied Biosystems (Foster City, CA) and Protein Technologies Inc. (Tucson, AZ). Reagents for solid-phase synthesis are readily available from commercial suppliers. Solid-phase synthesizers can be used according to the manufacturer's instructions for blocking interfering groups, protecting amino acids during the reaction, coupling, deprotecting, and capping unreacted amino acids. Additional details regarding the production of synthetic RLNs are described in U.S. Pat. Nos. 4,835,251 and 5,166,191.

[0119] One use of the RLN analogs herein is to treat cardiovascular conditions, diseases, and / or disorders. Exemplary cardiovascular conditions, diseases, and disorders include, but are not limited to, acute heart failure, chronic heart failure, atherosclerosis, coronary artery disease, diabetes, stroke, hypercholesterolemia, hypertension, ischemia, vasoconstriction, and ventricular hypertrophy.

[0120] Another use of the RLN analogs herein is for treating pulmonary conditions, diseases, and / or disorders. Exemplary pulmonary conditions, diseases, and disorders include, but are not limited to, pulmonary hypertension and chronic obstructive pulmonary disease (COPD).

[0121] Another use of the RLN analogs herein is for treating renal conditions, diseases, and / or disorders. Exemplary renal conditions, diseases, and disorders include, but are not limited to, chronic kidney disease and diabetic nephropathy.

[0122] The method may include the steps described herein, which may be, but are not necessarily, performed in the order described. However, other orders are contemplated. Furthermore, individual or multiple steps may be performed in parallel and / or overlapping time, and / or individually or in multiple repeated steps. Furthermore, the method may include additional, unspecified steps.

[0123] Therefore, such a method may include selecting an individual who has or is susceptible to a cardiovascular condition, disease, or disorder. Alternatively, the method may include selecting an individual who has or is susceptible to a pulmonary condition, disease, or disorder. Alternatively, the method may include selecting an individual who has or is susceptible to a renal condition, disease, or disorder. In certain cases, the method may include selecting an individual who has diabetes, hypertension with renal dysfunction, and / or obesity.

[0124] The methods may also include administering to the individual an effective amount of at least one RLN analog described herein, which may be in the form of a pharmaceutical composition, also as described herein. In some cases, the RLN analog / pharmaceutical composition includes additional therapeutic agents, such as anticoagulants, ACE inhibitors, ARBs, ARNIs, beta-blockers, diuretics, digitalis, digoxin, hydralazine nitrate / isorbide, MRAs or other aldosterone antagonists, SGLT2 inhibitors, statins, and / or antiglycemic agents, as well as other therapeutic agents for controlling co-morbidities, including, but not limited to, high cholesterol, high blood pressure, atrial fibrillation, and diabetes.

[0125] The concentrations / dosages / administration of the present RLN analogs and optional additional therapeutic agents are discussed elsewhere herein.

[0126] With regard to the route of administration, the RLN analog or pharmaceutical composition comprising same can be administered according to known methods, for example, orally, by injection (i.e., intra-arterially, intravenously, intraperitoneally, intracerebrally, intraventricularly, intramuscularly, intraocularly, intraportally, or intralesionally), by sustained release system, or by implanted device, etc. In certain cases, the RLN analog or pharmaceutical composition comprising same can be administered SQ by bolus injection or continuously.

[0127] With regard to dosing frequency, the RLN analog or pharmaceutical composition comprising it can be administered daily, every other day, three times a week, twice a week, once a week (i.e., every week), every other week (i.e., every other week), or monthly. In certain cases, the RLN analog or pharmaceutical composition comprising it is administered SQ every other day, SQ three times a week, SQ twice a week, SQ once a week, SQ every other week, or SQ monthly. In certain cases, the RLN analog or pharmaceutical composition comprising it is administered SQ once a week (QW).

[0128] When the RLN analog or pharmaceutical composition comprising the same is administered in combination with an effective amount of at least one additional therapeutic agent, the additional therapeutic agent may be administered simultaneously, separately, or sequentially with the RLN analog or pharmaceutical composition comprising the same.

[0129] Furthermore, the additional therapeutic agent may be administered at the same frequency as the RLN analog or pharmaceutical composition comprising same (i.e., every other day, twice a week, or even weekly). Alternatively, the additional therapeutic agent may be administered at a different frequency than the RLN analog or pharmaceutical composition comprising same. In other cases, the additional therapeutic agent may be administered SQ. In other cases, the additional therapeutic agent may be administered IV. In still other cases, the additional therapeutic agent may be administered orally.

[0130] It is further contemplated that these methods may be combined with diet and exercise, and / or may be combined with additional therapeutic agents other than those discussed above. [Example]

[0131] The following non-limiting examples are offered by way of illustration and not limitation.

[0132] Polypeptide Expression Example 1: Recombinant expression of RLN analog 1 Example 1 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLIT SKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQQLYSALANKCCHVGCTKRSLARFCGGGSGGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ ID NO: 24)

[0133] Here, the RLN analog of SEQ ID NO:24 is produced in a mammalian cell expression system using a CHOK1 cell derivative. The cDNA sequence encoding SEQ ID NO:24 is subcloned into an expression plasmid backbone (pEE12.4-based plasmid) containing GS. The cDNA sequence is fused in frame with the coding sequence for the signal peptide sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO:44) to enhance secretion of the RLN analog into tissue culture medium. Expression is driven by a viral CMV promoter.

[0134] To produce RLN analogs via transient transfection, CHOK1 cells are transfected with recombinant expression plasmids using a PEI-based method. Briefly, at a density of 4 x 10 6 An appropriate volume of CHOK1 suspension cells, in cells / mL, is transferred to a shake flask, and both PEI and recombinant plasmid DNA are added to the cells. The cells are incubated in suspension culture at 32°C for 6 days. At the end of the incubation period, the cells are removed by low-speed centrifugation, and the RLN analog protein is purified from the conditioned medium.

[0135] Alternatively, to generate RLN analogs via stable transfection, CHOK1 cells are stably transfected using electroporation and an appropriate amount of recombinant expression plasmid. Transfected cells are maintained in suspension culture at the appropriate cell density. Selection of transfected cells is achieved by growing them in serum-free medium containing 25 µM MSX and incubating at approximately 35°C-37°C and approximately 5-7% CO2.

[0136] RLN analogs are secreted from CHO cells into the culture medium and purified by Protein A affinity chromatography followed by ion exchange and hydrophobic interaction or size exclusion chromatography. Specifically, RLN analogs from harvested culture medium are captured on Mab Select Protein A resin (GE). The resin is then briefly washed with a running buffer, such as phosphate-buffered saline (PBS, pH 7.4) or a Tris-containing buffer, to remove nonspecifically bound material. The protein is eluted from the resin with a low pH solution, such as 10 mM citric acid, pH 3. Fractions containing RLN analogs are pooled and may be maintained at a low pH to inactivate latent virus. The pH may be neutralized by adding a base, such as 0.1 M Tris, pH 8.0. RLN analogs may be further purified by ion exchange chromatography using a resin, such as Poros 50 HS (ThermoFisher). The RLN analogues are eluted from the column using a NaCl gradient from 0 to 500 mM in 20 mM NaOAc at pH 5.0 over 15 column volumes.

[0137] RLN analogs may be further purified by hydrophobic interaction chromatography using a Capto Phenyl ImpRes HIC Column (GE Healthcare). Purification is achieved by adjusting the column load solution to approximately 0.5 M sodium sulfate and eluting using a 10 CV gradient from 0.5 M to 0 M sodium sulfate in 20 mM Tris at pH 8. After HIC, RLN analogs may be further purified by SEC by loading the concentrated Capto Phenyl ImpRes pool onto a Superdex200 (GE Healthcare) column using an isocratic elution in PBS at pH 7.4 or 20 mM histidine, 50 mM NaCl at pH 6.0.

[0138] The purified RLN analog is passed through a virus-retaining filter such as Planova 20N (Asahi Kasei Medical) and then concentrated / diafiltered into 20 mM histidine, 20 mM NaCl, pH 6 using tangential flow ultrafiltration on a regenerated cellulose membrane (Millipore).

[0139] Thus, RLN analogs are prepared in this manner, or in an analogous manner readily determined by one skilled in the art.

[0140] Example 2: Recombinant expression of RLN analogue 2 Example 2 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAQLYSALANKCCHVGCTKRSLARFCGGGSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ ID NO: 25)

[0141] Here, the RLN analog of SEQ ID NO:25 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO:25 is used in an expression plasmid.

[0142] Example 3: Recombinant expression of RLN analogue 3 Example 3 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLIT SKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 26)

[0143] Here, the RLN analog of SEQ ID NO:26 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO:26 is used in an expression plasmid.

[0144] Example 4: Recombinant expression of RLN analog 4 Example 4 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 27)

[0145] Here, the RLN analog of SEQ ID NO:27 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO:27 is used in an expression plasmid.

[0146] Example 5: Recombinant expression of RLN analog 5 Example 5 is an RLN analog having the following amino acid sequence: SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFCGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 28)

[0147] Here, the RLN analog of SEQ ID NO:28 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO:28 is used in an expression plasmid.

[0148] Example 6: Recombinant expression of RLN analog 6 Example 6 is an RLN analog having the following amino acid sequence: SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFCPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 29)

[0149] Here, the RLN analog of SEQ ID NO:29 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO:29 is used in an expression plasmid.

[0150] Example 7: Recombinant expression of RLN analog 7 Example 7 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLIT SKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 30)

[0151] Here, the RLN analog of SEQ ID NO: 30 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 30 is used in an expression plasmid.

[0152] Example 8: Recombinant expression of RLN analog 8 Example 8 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 31)

[0153] Here, the RLN analog of SEQ ID NO: 31 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 31 is used in an expression plasmid.

[0154] Example 9: Recombinant expression of RLN analog 9 Example 9 is an RLN analog having the following amino acid sequence: SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFCGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 32)

[0155] Here, the RLN analog of SEQ ID NO: 32 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 32 is used in an expression plasmid.

[0156] Example 10: Recombinant expression of RLN analog 10 Example 10 is an RLN analog having the following amino acid sequence: SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFCPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 33)

[0157] Here, the RLN analog of SEQ ID NO: 33 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 33 is used in an expression plasmid.

[0158] Example 11: Recombinant expression of RLN analog 11 Example 11 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLIT SKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGSGGGALANKCCHVGCTKRSLARFC (SEQ ID NO: 34)

[0159] Here, the RLN analog of SEQ ID NO: 34 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 34 is used in an expression plasmid.

[0160] Example 12: Recombinant expression of RLN analog 12 Example 12 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLIT SKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGSGGGALANKCCHVGCTKRSLARFC (SEQ ID NO: 35)

[0161] Here, the RLN analog of SEQ ID NO: 35 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 35 is used in an expression plasmid.

[0162] Example 13: Recombinant expression of RLN analog 13 Example 13 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQALANKCCHVGCTKRSLARFCGGGSGGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ ID NO: 36)

[0163] Here, the RLN analog of SEQ ID NO: 36 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 36 is used in an expression plasmid.

[0164] Example 14: Recombinant expression of RLN analog 14 Example 14 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSK VADLYPYWGQGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQALANKCCHVGCTKRSLARFCGGGSGGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ ID NO: 37)

[0165] Here, the RLN analog of SEQ ID NO: 37 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 37 is used in an expression plasmid.

[0166] Example 15: Recombinant expression of RLN analog 15 Example 15 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQDSWMEEVIKLCGRELVRAQIAICGMSTWSSGGGGSGGGGQLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 38)

[0167] Here, the RLN analog of SEQ ID NO: 38 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 38 is used in an expression plasmid.

[0168] Example 16: Recombinant expression of RLN analog 16 Example 16 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPADSWMEEVIKLCGRELVRAQIAICGMSTWSSGGGGSGGGGQLYSALANKCCHVGCTKRSLARFC (SEQ ID NO: 39)

[0169] Here, the RLN analog of SEQ ID NO: 39 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 39 is used in an expression plasmid.

[0170] In vitro function Example 17: RLN analogue albumin binding via SPR In vitro binding of RLN analogs to human, cynomolgus monkey, mouse, rat, pig, dog, bovine, and rabbit serum albumin is determined by SPR. Specifically, the affinities of the RLN analogs of Examples 1-14 for serum albumin of these species are summarized below in Tables 1-14.

[0171] The conjugation of the RLN analogs of Examples 1-14 to various serum albumins was performed on a Biacore 8K instrument. Immobilization of serum albumin onto the surface of a Series S Sensor Chip CM5 was performed according to the manufacturer's instructions (Amine Coupling Kit BR-1000-50). Briefly, the carboxyl groups on the sensor chip surface (flow cells 1 and 2) were activated by injecting 70 μL of a mixture containing 75 mg / mL EDC and 11.5 mg / mL NHS at 10 μL / min. Human, cynomolgus monkey, mouse, rat, porcine, canine, bovine, and rabbit serum albumin was diluted to 0.5, 0.5, 2.2, 0.6, 0.6, 0.8, 0.6, and 0.3 μg / mL in 10 mM sodium acetate (BR-1003-49) at pH 4.0 and then injected over the activated chip surface (flow cell 2, channels 1–8) at 10 μL / min for 180 s. (Human, mouse, rat, porcine, and bovine serum albumin was obtained from Sigma Aldrich (St. Louis, MO); cynomolgus monkey serum albumin was obtained from Holzel Diagnostika (Cologne, Germany); canine serum albumin was obtained from Molecular Innovations (Novi, MI); and rabbit serum albumin was obtained from Fitzgerald Industries.) Serum albumins were obtained from Biosciences Inc. (Acton, MA). Various serum albumins were covalently immobilized via free amines onto a carboxymethyldextran-coated sensor chip CM5, targeting a surface density of approximately 100 (62–145) RU. Excess reactive groups on the surface (flow cells 1 and 2) were deactivated by injecting 70 μL of 1 M ETA HCl-NaOH, pH 8.5.

[0172] The RLN analogs from Examples 1-14 were diluted in HBS-EP+ buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20) to concentrations of 1000, 333.3, 111.1, 37.04, 12.35, 4.12, 1.37, 0.457, 0.152, 0.051, and 0.017 nM. 150 μl of each sample was injected sequentially across the immobilized serum albumin on the chip surface and allowed to dissociate for 600 seconds at 25°C and a flow rate of 50 μL / min. The surface was regenerated by injecting 10 mM glycine-HCl (BR-1003-54), pH 1.5, at 50 μL / min for 100 seconds. The resulting sensorgrams are analyzed using a 1:1 binding kinetics model fitting in Biacore 8K Insight Evaluation Software (version 2.0.15.12933) to calculate the binding kinetic parameters association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD). [Table 1]

[0173] K D is determined for the RLN analog of Example 1 to be 0.1, 0.5, 3.3, 2.1, 7.1, 1.2, and 37 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 2]

[0174] K D is determined for the RLN analog of Example 2 to be 0.1, 0.45, 3.8, 3.0, 8.0, 1.4, and 50 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 3]

[0175] K Dis determined for the RLN analog of Example 3 to be 0.08, 0.36, 2.4, 1.6, 4.8, 0.7, and 26 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 4]

[0176] K D is determined for the RLN analog of Example 4 to be 0.09, 0.42, 3.0, 2.1, 6.2, 1.0, and 37 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 5]

[0177] K D is determined for the RLN analog of Example 5 to be 0.45, 1.8, 14, 10, 23, 4.7, and 120 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 6]

[0178] K D is determined for the RLN analog of Example 6 to be 0.51, 2.3, 12, 9.7, 24, 4.1, and 130 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 7]

[0179] K D is determined for the RLN analog of Example 7 to be 0.06, 0.29, 2.1, 1.4, 3.7, 0.69, and 25 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 8]

[0180] K D is determined for the RLN analog of Example 8 to be 0.08, 0.32, 1.8, 1.7, 4.1, 0.66, and 21 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 9]

[0181] K D is determined for the RLN analog of Example 9 to be 0.19, 0.86, 9.5, 8.2, 14, 3.8, and 84 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 10]

[0182] K D is determined for the RLN analog of Example 10 to be 0.37, 1.5, 9.7, 7.8, 20, 3.4, and 100 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 11]

[0183] K D is determined for the RLN analog of Example 11 to be 0.17, 0.48, 3.6, 1.3, 7.3, 1.3, and 27 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 12]

[0184] K D is determined for the RLN analog of Example 12 to be 0.14, 0.47, 2.6, 1.7, 4.2, 0.77, and 30 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 13]

[0185] K D is determined for the RLN analog of Example 13 to be 0.14, 0.49, 2.6, 2.0, 5.0, 0.92, and 29 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively. [Table 14]

[0186] K D is determined for the RLN analog of Example 14 to be 0.16, 0.61, 3.1, 2.2, 6.0, 1.1, and 34 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin binding, respectively.

[0187] Example 18: In vitro potency of RLN analogs at RXFP1 and RXFP2 receptors Generation of CHO-CRE-Luc cell line: CHO-K1 cells (ATCC) were cultured in DMEM-F12 3:1 containing 5% FBS with 20 mM HEPES, 40 μg / mL L-proline, and 1x antibiotics and split 1:5 every 2–3 days using TrypLE™ Express (Gibco). Cells were transfected with pGL4.29[luc2P / CRE / Hygro] (Promega) and Fugene HD (Promega) plasmid DNA according to the manufacturer's instructions. Transfected cells were selected with hygromycin B at 1 mg / mL for 3–4 weeks. Clonal lines were obtained by limiting dilution cloning into 96-well plates and confirmed by forskolin response in a luciferase assay using Bright-Glo Reagent (Promega). Clones were expanded, harvested, resuspended in freezing medium, aliquoted into cryovials, and stored in liquid nitrogen for long-term storage. The top responder, clonal line #2B6, was selected for subsequent transfection with human RXFP1 and RXFP2 receptors, demonstrating the best signal-to-background ratio for forskolin.

[0188] Generation of CHO human RXFP1 and human RXFP2-expressing cell lines: CHO-CRE-Luc line #2B6 cells were cultured in DMEM-F12 3:1 containing 5% FBS with 20 mM HEPES, 40 μg / mL L-proline, 1x antibiotics, and 1 mg / mL hygromycin B, and split 1:5 every 2–3 days using TrypLE™ Express (Gibco). Cells were transfected with plasmid DNA for the human RXFP1 receptor or human RXFP2 receptor and Fugene HD (Promega) according to the manufacturer's instructions. Transfected cells were selected for 3–4 weeks using hygromycin B (1 mg / mL) and puromycin (6 μg / mL). Clonal lines were obtained by limiting dilution cloning into 96-well plates. Clonal lines were confirmed by human RLN2 response. Clones were expanded, harvested, resuspended in freezing medium, aliquoted into cryovials, and kept frozen under liquid nitrogen for long-term storage. For assay validation, clonal lines with the best response (signal-to-background ratio) to human RLN2 were selected.

[0189] Human RXFP1 and RXFP2 receptor luciferase assay: CHO cell lines expressing human RXFP1 or human RXFP2 were cultured in selection medium (3:1 DMEM-F12 containing 5% FBS with 20 mM HEPES, 40 μg / mL L-proline, 1x antibiotics, 6 μg / mL puromycin, and 1 mg / mL hygromycin B). On day 1 (the day before the cAMP-CRE luciferase reporter assay), cells were washed once with PBS, lifted from the flask with cell dissociation solution (enzyme-free cell dissociation solution, GIBCO catalog number 13151-014: TrypLE™ Express = 30:1), and resuspended in plating medium (3:1 DMEM-F12 containing 20 mM HEPES, 1x antibiotics, and 0.5% FBS). Cells are plated at 20,000 cells / 0.1 mL / well in 96-well plates (Falcon catalog number 353219). Cells are cultured overnight at 37°C and 5% CO2. On day 1 (the day of the cAMP CRE luciferase reporter assay), the medium is removed and replaced with 90 μL of serum-free medium (DMEM-F12 3:1 with 20 mM HEPES and 1x antibiotics). The plate is incubated at 37°C for 2 hours, after which 10 μL of 10x ligand is added (RLN2, 1x final). The plate is incubated at 37°C for an additional 4 hours. After the incubation period is complete, the plate is allowed to come to room temperature for 15 minutes. 50 μL of Bright-Glo™ is then added to each well, and the plate is read on a Biotek Neo2 reader with Gen5 software.

[0190] Statistical analysis of data: Data are imported from the Biotek Neo2 reader into GraphPad Prism® software (GraphPad Software, LLC; La Jolla, CA; version 7). 50 Values ​​are generated by variable slope-four parameter dose-response curves. [Table 15]

[0191] In vitro function Example 19: Pharmacokinetics of RLN analogs in male Sprague Dawley rats Male Sprague Dawley rats were administered a single SQ dose of 200 nmol / kg of RLN analogs in His-NaCl buffer (pH 6.0) in a volume of 1.0 mL / kg. Blood samples were collected at 3, 6, 12, 24, 48, 72, 96, 120, 144, 168, and 240 hours post-dose for pharmacokinetic characterization.

[0192] Plasma concentrations of RLN analogs are determined by an LC / MS method certified by Eli Lilly and Company. Example compounds and analog internal standards are extracted from 100% rat plasma using a human RLN antibody, followed by detection of N-terminal tryptic peptides using a Q-Exactive™ Orbitrap® mass spectrometer.

[0193] Data for the RLN analogs of Examples 4 and 7 are provided in Table 16 below. [Table 16]

[0194] Note: Abbreviations: t = half-life, T max = time to maximum concentration, C max = maximum observed plasma concentration, AUC 0-inf = area under the curve from 0 hours to infinity, CL / F = clearance / bioavailability, N=3 animals / group / time point.

[0195] As shown in Table 16, the RLN analogs of Examples 4 and 7 demonstrate an extended pharmacokinetic profile in Sprague Dawley rats.

[0196] Example 20: In vivo effects of RLN analogs on renal blood flow after IV administration in male Sprague Dawley rats Male 5-week-old Sprague Dawley rats (Charles River Laboratories, Inc.) are housed in a vivarium with a normal light / dark cycle for one week prior to the start of the experiment. Rats are then randomized into treatment groups based on body weight, with vehicle (20 mM His / 20 mM NaCl pH 6.0 buffer) and the RLN analog of Example 7. The RLN analog is administered at a 2.44 μg / kg IV bolus followed by a 0.36 μg / kg / hour IV infusion, an 8.13 μg / kg IV bolus followed by a 1.2 μg / kg / hour IV infusion, a 24.4 μg / kg IV bolus followed by a 3.6 μg / kg / hour IV infusion, and an 81.3 μg / kg IV bolus followed by an 11.9 μg / kg / hour IV infusion.

[0197] To measure the effects of RLN analogs on renal blood flow, rats were anesthetized with urethane (1.2 g / kg, IP) and prepared for abdominal / renal ultrasound imaging and renal artery pulsed wave Doppler blood flow measurements (VisualSonics, Model Vevo 3100 Ultrasound System, Fujifilm). A chronic tail vein catheter was placed for IV bolus and infusion administration. After a 30-minute acclimation period, baseline and 3-hour post-dosing renal blood flow measurements were obtained. [Table 17]

[0198] Note: Results are expressed as mean ± SD. †Significantly different from baseline (p<0.001 ANOVA). * Significantly different from vehicle (p<0.001 ANOVA), N=5 per treatment group.

[0199] As can be seen in Table 17, the RLN analog of Example 7 significantly increases renal blood flow by 26.6, 48.8, and 60.4% in groups treated with an 8.13 μg / kg IV bolus followed by a 1.2 μg / kg / hour IV infusion, a 24.4 μg / kg IV bolus followed by a 3.6 μg / kg / hour IV infusion, and an 81.3 μg / kg IV bolus followed by an 11.9 μg / kg / hour IV infusion, at 3 hours post-exposure.

[0200] Example 21: In vivo effects of RLN analogs on renal blood flow after SQ administration in male Sprague Dawley rats Male 5-week-old Sprague Dawley rats (Charles River Laboratories, Inc.) are housed in a vivarium with a normal light-dark cycle for one week prior to the start of the experiment. Rats are then randomized into treatment groups based on body weight to receive either vehicle (20 mM His / 20 mM NaCl buffer, pH 6.0) or the RLN analog of Example 7. The RLN analog is administered SQ at 180 μg / kg.

[0201] To measure the effects of RLN analogs on renal blood flow, 48 hours after dosing, rats were anesthetized with urethane (1.2 g / kg, IP) and prepared for abdominal / renal ultrasound imaging and renal artery pulsed wave Doppler blood flow measurements (VisualSonics, Model Vevo 3100 Ultrasound System, Fujifilm). After a 30-minute acclimation period, renal blood flow measurements were obtained. [Table 18]

[0202] As can be seen in Table 18, the RLN analog of Example 7 significantly increases renal blood flow 48 hours after exposure to a 180 μg / kg SQ dose.

[0203] array The following nucleic acid and / or amino acid sequences are referred to in this disclosure and are provided below for reference. SEQ ID NO:1 - Human pro-RLN1 MPRLFLFHLLEFCLLNQFSRAVAAKWKDDVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDTETIIIMLEFIANLPPELKAALSERQPSLPELQQYVPALKDSNLSFEEFKKLIRNRQSEAADSNPSELKYLGLDTHSQKKRRPYVALFEKCCLIGCTKRSLAKYC SEQ ID NO:2 - Human RLN1 A chain PYVALFEKCCLIGCTKRSLAKYC SEQ ID NO:3 - Human RLN1 B chain VAAKWKDDVIKLCGRELVRAQIAICGMSTWS SEQ ID NO:4 - Human pro-RLN2 MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSPSELKYLGLDTHSRKKRQLYSALANKCCHVGCTKRSLARFC SEQ ID NO:5 - Human RLN2 A chain QLYSALANKCCHVGCTKRSLARFC SEQ ID NO:6 - Human RLN2 B chain DSWMEEVIKLCGRELVRAQIAICGMSTWS SEQ ID NO:7 - Human pro-RLN3 MARYMLLLLLAVWVLTGELWPGAEARAAPYGVRLCGREFIRAVIFTCGGSRWRRSDILAHEAMGDTFPDADADEDSLAGELDEAMGSSEWLALTKSPQAFYRGRPSWQGTPGVLRGSRDVLAGLSSSCCKWGCSKSEISSLC SEQ ID NO:8 - Human RLN3 A chain DVLAGLSSSCCKWGCSKSEISSLC SEQ ID NO:9 - Human RLN3 B chain RAAPYGVRLCGREFIRAVIFTCGGSRW SEQ ID NO:10 - VHH portion #1 (C22) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO:11 - VHH portion #2 (C22.43) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO:12 - VHH portion #3 (C80) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO: 13 - VHH portion #4 (C80.43) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 14-L1((GGGGQ) n (nucleotide sequence of GGGGQ SEQ ID NO: 15-L1((GGGQ) n (nucleotide sequence of GGGQ SEQ ID NO: 16-L1((GGGGS) n (nucleotide sequence of GGGGS SEQ ID NO: 17-L1((PGPQ) n (nucleotide sequence of PGPQ SEQ ID NO: 18-L1((PGPA) n (nucleotide sequence of PGPA SEQ ID NO: 19-L1#1((GGGGQ)5) GGGGQGGGGQGGGGQGGGGQGGGGQ SEQ ID NO: 20-L1#2((PGPQ)8) PGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQ SEQ ID NO: 21-L1#3(PGPA)8 PGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPA SEQ ID NO: 22-L2#1 GGGSGGSGGG SEQ ID NO: 23-L2#2 GGGSGGSGGSGGG SEQ ID NO:24 - RLN2 analog #1 (C22-(G4Q)5-A10B(desB1)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLIT SKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQQLYSALANKCCHVGCTKRSLARFCGGGSGGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS SEQ ID NO:25 - RLN2 analog #2 (C22-(PGPA)8-A10B(desB1)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVA DLYPYWGQGTLVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAQLYSALANKCCHVGCTKRSLARFCGGGSGGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS Array No. 26 - RLN2 analog #3 (C22-(G4Q)5-B10A(desB1)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC Array No. 27 - RLN2 analog #4 (C22-(PGPQ)8-B10A(desB1)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC Array No. 28 - RLN2 analog #5 (B10A(desB1)RLN-(G4Q)5-C22.43) SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFCGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP Array No. 29 - RLN2 analog #6 (B10A(desB1)RLN-(PGPQ)8-C22.43) SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFCPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 30 - RLN2 analog #7 (C80-(G4Q)5-B10A(desB1)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC SEQ ID NO: 31 - RLN2 analog #8 (C80-(PGPQ)8-B10A(desB1)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC SEQ ID NO: 32 - RLN2 analog #9 (B10A(desB1)RLN-(G4Q)5-C80.43) SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFCGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 33 - RLN2 analog #10 (B10A(desB1)RLN-(PGPQ)8-C80.43) SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFCPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 34 - RLN2 analog #11 (C22-(G4Q)5-B13A(desB1, desA1~4)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGSGGGALANKCCHVGCTKRSLARFC SEQ ID NO: 35 - RLN2 analog #12 (C80-(G4Q)5-B13A(desB1, desA1~4)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGSGGGALANKCCHVGCTKRSLARFC SEQ ID NO: 36 - RLN2 analog #13 (C80-(G4Q)5-A10B(desB1, desA1 - 4)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQALANKCCHVGCTKRSLARFCGGGSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS SEQ ID NO: 37 - RLN2 analog #14 (C80-(PGPQ)8-A10B(desB1, desA1 - 4)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQALANKCCHVGCTKRSLARFCGGGSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS SEQ ID NO: 38 - RLN2 analog #15 (C22-(G4Q)5-B10ARLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQDSWMEEVIKLCGRELVRAQIAICGMSTWSSGGGGSGGGGQLYSALANKCCHVGCTKRSLARFC SEQ ID NO:39 - RLN2 analog #16 (C22-(PGPA)8-B10ARLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVA DLYPYWGQGTLVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPADSWMEEVIKLCGRELVRAQIAICGMSTWSSGGGGSGGGGQLYSALANKCCHVGCTKRSLARFC SEQ ID NO: 40 - Human RXFP1 receptor MTSGSVFFYILIFGKYFSHGGGQDVKCSLGYFPCGNITKCLPQLLHCNGVDDCGNQADEDNCGDNNGWSLQFDKYFASYYKMTSQYPFEAETPECLVGSVPVQCLCQGLELDCDETNLRAVPSVSSNVTAMSLQWNLIRKLPPDCFKNYHDLQKLYLQNNKITSISIYAFRGLNSLTKLYLSHNRITFL KPGVFEDLHRLEWLIIEDNHLSRISPPTFYGLNSLILLVLMNNVLTRLPDKPLCQHMPRLHWLDLEGNHIHNLRNLTFISCSNLTVLVMRKNKINHLNENTFAPLQKLDELDLGSNKIENLPPLIFKDLKELSQLNLSYNPIQKIQANQFDYLVKLKSLSLEGIEISNIQQRMFRPLMNLSHIYFKKFQ YCGYAPHVRSCKPNTDGISSLENLLASIIQRVFVWVVSAVTCFGNIFVICMRPYIRSENKLYAMSIISLCCADCLMGIYLFVIGGFDLKFRGEYNKHAQLWMESTHCQLVGSLAILSTEVSVLLLTFLTLEKYICIVYPFRCVRPGKCRTITVLILIWITGFIVAFIPLSNKEFFKNYYGTNGVCFPLH SEDTESIGAQIYSVAIFLGINLAAFIIIVFSYGSMFYSVHQSAITATEIRNQVKKEMILAKRFFFIVFTDALCWIPIFVVKFLSLLQVEIPGTITSWVVIFILPINSALNPILYTLTTRPFKEMIHRFWYNYRQRKSMDSKGQKTYAPSFIWVEMWPLQEMPPELMKPDLFTYPCEMSLISQSTRLNSYS SEQ ID NO: 41 - Human RXFP2 receptor MIVFLVFKHLFSLRLITMFFLLHFIVLINVKDFALTQGSMITPSCQKGYFPCGNLTKCLPRAFHCDGKDDCGNGADEENCGDTSGWATIFGTVHGNANSVALTQECFLKQYPQCCDCKETELECVNGDLKSVPMISNNVTLLSLKKNKIHSLPDKVFIKYTKLKKIFLQHNCIRHISRKAFFGLCNLQ ILYLNHNCITTLRPGIFKDLHQLTWLILDDNPITRISQRLFTGLNSLFLSMVNNYLEALPKQMCAQMPQLNWVDLEGNRIKYLTNSTFLSCDSLTVLFLPRNQIGFVPEKTFSSLKNLGELDLSSNTITELSPHLFKDLKLLQKLNLSSNPLMYLHKNQFESLKQLQSLDLERIEIPNINTRMFQPMK NLSHIYFKNFRYCSYAPHVRICMPLTDGISSFEDLLANNILRIFVWVIAFITCFGNLFVIGMRSFIKAENTTHAMSIKILCCADCLMGVYLFFVGIFDIKYRGQYQKYALLWMESVQCRLMGFLAMLSTEVSVLLLTYLTLEKFLVIVFPFSNIRPGKRQTSVILICIWMAGFLIAVIPFWNKDYFGN FYGKNGVCFPLYYDQTEDIGSKGYSLGIFLGVNLLAFLIIVFSYITMFCSIQKTALQTTEVRNCFGREVAVANRFFFIVFSDAICWIPVFVVKILSLFRVEIPDTMTSWIVIFFLPVNSALNPILYTLTTNFFKDKLKQLLHKHQRKSIFKIKKKSLSTSIVWIEDSSSLKLGVLNKITLGDSIMKPVS SEQ ID NO: 42 - Human RXFP3 receptor MQMADAATIATMNKAAGGDKLAELFSLVPDLLEAANTSGNASLQLPDLWWELGLELPDGAPPGHPPGSGGAESADTEARVRILISVVYWVVCALGLAGNLLVLYLMKSMQGWRKSSI NLFVTNLALTDFQFVLTLPFWAVENALDFKWPFGKAMCKIVSMVTSMNMYASVFFLTAMSVTRYHSVASALKSHRTRGHGRGDCCGRSLGDSCCFSAKALCVWIWALAALASLPPSAI FSTTVKVMGEELCLVRFPDKLLGRDRQFWLGLYHSQKVLLGFVLPLGIIILCYLLLVRFIADRRAAGTKGGAAVAGGRPTGASARRLSKVTKSVTIVVLSFFLCWLPNQALTTWSIL IKFNAVPFSQEYFLCQVYAFPVSVCLAHSNSCLNPVLYCLVRREFRKALKSLLWRIASPSITSMRPFTATTKPEHEDQGLQAPAPPHAAAEPDLLYYPPGVVVYSGGRYDLLPSSSAY SEQ ID NO: 43 - Human RXFP4 receptor MPTLNTSASPPTFFWANASGGSVLSADDAPMPVKFLALRLMVALAYGLVGAIGLLGNLAVLWVLSNCARRAPGPPSDTFVFNLALADLGLALTLPFWAAESALDFHWPFGGALCKMVLTATVLNVYASIFLITALSVARYWVVAMAAGPGTHLSLFWARIATLAVWAAAALVTVPTAVFGVEGEVCG VRLCLLRFPSRYWLGAYQLQRVVLAFMVPLGVITTSYLLLLAFLQRRQRRRQDSRVVARSVRILVASFFLCWFPNHVVTLWGVLVKFDLVPWNSTFYTIQTYVFPVTTCLAHSNSCLNPVLYCLLRREPRQALAGTFRDLRLRLWPQGGGWVQQVALKQVGRRWVASNPRESRPSTLLTNLDRGTPG SEQ ID NO: 44 - Signal peptide METDTLLLWVLLLWVPGSTG SEQ ID NO: 45 - VHH portion #5 (MC6.1) EVQLLESGGGLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGTLVTVSS SEQ ID NO: 46-VHH part #6 (MC6.1C6) EVQLLESGGGLVQPGGSLRLSCAASGRYIDSTAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSRVANLYPYWGQGTLVTVSS SEQ ID NO: 47 - VHH portion #7 (C22-G26Y) EVQLLESGGGLVQPGGSLRLSCAASYRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO:48 - VHH portion #8 (C22-R27A) EVQLLESGGGLVQPGGSLRLSCAASGAYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO: 49 - VHH portion #9 (C22-I57E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDETYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO:50 - VHH portion #10 (C22-I57Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDQTYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO:51 - VHH portion #11 (C22-Y59A) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITAYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO:52 - VHH portion #12 (C22-Y59E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITEYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO:53 - VHH portion #13 (C22-Y59Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITQYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO:54 - VHH portion #14 (C22-Y59S) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO:55 - VHH portion #15 (C22-Y59T) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITTYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO:56 - VHH portion #16 (C22-R102K) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGKPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO:57 - VHH portion #17 (C22-R102Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGQPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO:58 - VHH portion #18 (C22-R102S) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGSPLITSKVADLYPYWGQGTLVTVSS SEQ ID NO:59 - VHH portion #19 (C22-P103E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRELITSKVADLYPYWGQGTLVTVSS SEQ ID NO:60 - VHH portion #20 (C22-P103Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRQLITSKVADLYPYWGQGTLVTVSS SEQ ID NO: 61 - VHH portion #21 (C22-P103S) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRSLITSKVADLYPYWGQGTLVTVSS SEQ ID NO: 62 - VHH portion #22 (C22-L104E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPEITSKVADLYPYWGQGTLVTVSS SEQ ID NO: 63 - VHH portion #23 (C22-L104G) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPGITSKVADLYPYWGQGTLVTVSS SEQ ID NO: 64 - VHH portion #24 (C22-L104Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPQITSKVADLYPYWGQGTLVTVSS SEQ ID NO: 65 - VHH portion #25 (C22-L104T) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPTITSKVADLYPYWGQGTLVTVSS Sequence number 66 - VHH part #26 (C22 - S107E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITEKVADLYPYWGQGTLVTVSS Sequence number 67 - L2 #3 SGGGGSGGGG

Claims

1. The following structure VHH-L １ -A-L ２ -B、 VHH-L １ -B-L ２ -A、 A-L ２ -BL １ -VHH, or B-L ２ -A-L １ -VHH, the VHH binds to serum albumin and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, and 13, or a sequence having at least 90% identity thereto; A is a relaxin A chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, and 8, or a sequence having at least 90% identity thereto; B is a relaxin B chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, and 9, or a sequence having at least 90% identity thereto; L １ is a first linker comprising an amino acid sequence selected from the group consisting of (GGGGQ)n, (PGPQ)n, and (PGPA)n, where n can be 1 to 10; L ２ is a second linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 67; or and pharmaceutically acceptable salts thereof.

2. 2. The compound of claim 1, wherein A is SEQ ID NO:

2.

3. 3. The compound of claim 1 or 2, wherein B is SEQ ID NO:

3.

4. 2. The compound of claim 1, wherein A is SEQ ID NO:

5.

5. 5. The compound of claim 1 or 4, wherein B is SEQ ID NO:

6.

6. The compound of claim 1, wherein A is SEQ ID NO: 5 and lacks the first four amino acids (desA1-4).

7. 10. The compound of claim 1, 4 or 6, wherein B is SEQ ID NO: 6 and lacks the first amino acid (desB1).

8. 2. The compound of claim 1, wherein A is SEQ ID NO: 5 lacking the first four amino acids (desA1-4), or A is SEQ ID NO: 5 and B is SEQ ID NO: 6 lacking the first amino acid (desB1).

9. 2. The compound of claim 1, wherein A is SEQ ID NO:

8.

10. 3. The compound of claim 1 or 2, wherein B is SEQ ID NO:

9.

11. L １ The compound according to any one of claims 1 to 10, wherein is SEQ ID NO:

19.

12. L １ The compound according to any one of claims 1 to 10, wherein: is SEQ ID NO:

20.

13. L １ is SEQ ID NO:

21.

14. A compound that binds to serum albumin, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 to 39, or a sequence having at least 90% identity thereto, or a pharmaceutically acceptable salt thereof.

15. A compound that binds to serum albumin, consisting solely of an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 to 39, or a sequence having at least 90% identity thereto, or a pharmaceutically acceptable salt thereof.

16. A compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable buffer.

17. 1. A pharmaceutical composition for treating a cardiac, pulmonary, and / or renal condition, disease, and / or disorder in an individual, comprising: A pharmaceutical composition comprising an effective amount for said individual of a compound according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16.

18. A medicament comprising a compound according to any one of claims 1 to 15 for use in therapy.

19. A medicament comprising a compound according to any one of claims 1 to 15 for use in the treatment of cardiac, pulmonary and / or renal conditions, diseases and / or disorders.

20. 20. Use of a compound according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment of cardiac, pulmonary and / or renal conditions, diseases and / or disorders.

21. A compound that binds to serum albumin, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-13 and 45-66, or a sequence having at least 90% identity thereto.

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