Relaxin analogs and methods of use thereof

Abstract

To provide compounds that function as an agonist of relaxin / insulin like family peptide receptor or pharmaceutically acceptable salt thereof.SOLUTION: In an aspect, disclosed is a compound or a pharmaceutically acceptable salt thereof comprising VHH-L1-A-L2-B, VHH-L1-B-L2-A, A-L2-B-L1-VHH, or B-L2-A-L1-VHH, where VHH comprises a specific amino acid sequence or a sequence having at least 90% sequence homology thereto, A is relaxin A chain comprising a specific amino acid sequence or a sequence having at least 90% homology thereto, B is relaxin B chain comprising a specific amino acid sequence or a sequence having at least 90% homology thereto, L1 is a first linker comprising an amino acid sequence selected from a specific group, L2 is a second linker comprising an amino acid sequence selected from a specific group.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates generally to biology and medicine, and more particularly to relaxation RLN analogs, especially RLN / insulin-like family receptors such as the RXFP1 receptor. It functions as an RXFP receptor agonist by binding to the RXFP receptor. The present disclosure further relates to compositions containing the same. and compositions for use in the treatment of cardiovascular, pulmonary, and / or renal conditions, diseases, or disorders. and their use. [Background technology]

[0002] Relaxin (RLN) is a member of the insulin superfamily and in humans The seven peptides with high structural similarity but low sequence homology were identified: RLN1 (H1RLX, RL XH1, or H1), RLN2 (H2RLX, RLXH2, or H2), RLN3 ( RXN3, ZINS4, or H3), insulin-like (INSL) peptide 3 (INSL 3), INSL4, INSL5, and INSL6. Of particular interest here is R LN2, which consists of 24 amino acids linked by two disulfide bonds. It is a heterodimer of two peptide chains (chain A and chain B) of 29 amino acids each. The A chain also contains one intramolecular disulfide bond (Schwabe & McDowell, nald (1977) Science 197:914-915). By cleaving the C-peptide from prorelaxin, the precursor hormone of is produced.

[0003] Physiologically, RLN2 was initially thought to be a pregnancy hormone, but it also has vasodilatory effects, Anti-fibrotic and angiogenic effects, including cardiovascular, hepatic, neurological, pancreatic, pulmonary, and renal indications RLN2 signaling regulates two different classes of GTPases. These are now called the RXFP1 receptor and the RXFP2 receptor, respectively. The leucine-rich repeat-containing GPCRs LGR7 and RXFP2 receptors are called Two other receptors in this family are RXFP3 receptors and LGR8. RLN2 has a short half-life (t1 / 2), and This presents a challenge when using RLN2 as a therapeutic agent. The in vivo t1 / 2 of RLN2 is several minutes. As a result, clinicians administer RLN2 continuously intravenously. Injections are administered, which often result in the individual receiving the RLN compound being unable to It causes inconvenience and is of short-term effectiveness.

[0004] There are several RLN2 analogs with improved t1 / 2. For example, International Patent Application Publication No. In No. 2018 / 148419, linkers, polymers, and Paraacetylphenylalanine, which can be coupled with a pharmacokinetic enhancer and / or Analogs containing unnatural amino acid residues such as methylamino acids have been described. In issue 8 / 138170, a linker of at least 15 amino acids and a t1 / 2 ratio were used to improve the An analogue has been described that is a fusion of the A and B chains with a half-life extending moiety of Patent application publication no. 2017 / 201340 describes variable light chain fragments for improving t1 / 2. An analogue has been described which is a fusion of an A chain and a B chain having the following structure: In 15 / 067791, a carrier-linked prodrug analogue was developed to improve t1 / 2. Carriers, particularly PEG-based carriers, have been described (see WO for additional PEG-linked analogs). (See also International Patent Application Publication No. 2012 / 024452). In International Patent Application Publication No. 2014 / 102179: Fusion of A and B chains with the Fc portion of IgG2 or IgG4 to improve t1 / 2 WO 2013 / 004607 describes analogs that are compounds of the formula: A linker of at least 5 amino acids but less than 15 amino acids to improve t1 / 2 Analogs that are fusions of A and B chains with Fc to improve t1 / 2 Analogs that are fusions of A and B chains with domains have been described.

[0005] Given the increasing understanding of the various physiological roles of the RLN, long-term treatment with improved t1 / 2 is needed. There remains a need for intermediate acting RLN analogs.

[0006] To address this need, the present disclosure first provides a method for identifying a compound that exhibits primary activity in the RXFP1 receptor. (i.e., act as an RXFP1 receptor agonist) Such RXFP1 receptor agonists include those that are fused to the amino terminus (N-terminus) of the RXFP1 receptor. Basic structure to the carboxyl terminal (C-terminal) 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, and A is RLN L1 is the A chain, B is the RLN B chain, L1 is the first linker, and L2 is the second linker. It's a car.

[0007] In some cases the VHH moiety is selected from SEQ ID NOs: 10, 11, 12, or 13, particularly SEQ ID NOs: In other cases, the VHH moiety may have the amino acid sequence of VHH moiety No. 10 or 12. is at least about 9 to any one of SEQ ID NOs: 10, 11, 12, or 13 to have an amino acid sequence with 0% to about 99% sequence identity (e.g., SEQ ID NO: 4 5-66), and may have one or more additions, deletions, insertions, or substitutions.

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

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

[0010] In some cases, L1 is (GGGGQ) n (SEQ ID NO: 14), (GGGQ) n (Array number No. 15), (GGGGS) n (SEQ ID NO: 16), (PGPQ) n (SEQ ID NO: 17), and (PGPA)n (SEQ ID NO: 18), where n is In other cases, L1 is selected from the group consisting of SEQ ID NOs: 19, 20, and 21, and in particular from about 5 to about 8. In still other cases, L1 may have the amino acid sequence of SEQ ID NO: 14 to 21. 21 sequences that share at least about 90% to about 99% sequence identity with any one of the 21 sequences As with the amino acid sequence, it may have one or more additions, deletions, insertions, or substitutions.

[0011] In some cases, L2 can 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 analogue comprises a VHH of SEQ ID NO: 10, 11, 12, or 13. , A chain of SEQ ID NO: 2, 5, or 8, B chain of SEQ ID NO: 3, 6, or 9, SEQ ID NO: 19, An amino acid sequence containing L1 of SEQ ID NO: 20 or 21 and L2 of SEQ ID NO: 22, 23, or 67 Alternatively, the RLN analog may have a VH of SEQ ID NO: 10, 11, 12, or 13. H, A chain of SEQ ID NO: 2, 5, or 8, B chain of SEQ ID NO: 3, 6, or 9, SEQ ID NO: 19 , 20, or 21, and L2 of SEQ ID NO: 22, 23, or 67. an amino acid sequence having at least about 90% to about 99% sequence identity to an amino acid sequence containing It may have an acid sequence.

[0013] In certain instances, the RLN analogs are selected from the group consisting of SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39, in particular SEQ ID NO: Alternatively, the R may have an amino acid sequence of 26, 27, 30, 31, 34, or 35. LN analogs are SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, or 39, in particular SEQ ID NOs: 26, 27, 30, 31 , 34, or 35. The amino acid sequence may be:

[0014] In some cases, the RLN analogs have a binding affinity at the RXFP1 receptor that is greater than that of natural human RLN. In other cases, the binding affinity of the RLNs is comparable to that of the RLN2 (SEQ ID NOs: 5 and 6). The analogs have binding affinities at the RXFP1 receptor similar to those of native human RLN2 (SEQ ID NO: 5 and and 6). In still other cases, the RLN analogs bind to RXFP1 The binding affinity at the receptor is the same as that of native human RLN2 (SEQ ID NOs: 5 and 6). is smaller than.

[0015] In some cases, the RLN analogs have a t1 / 2 greater than that of native human RLN2 (SEQ ID NO: 5 and and 6) longer than the t1 / 2 of α- and β-actin, which includes a maximum of about 20 to about 30 days in the case of administration to humans. It involves a long period.

[0016] The compositions may alternatively comprise nucleic acid sequences encoding the amino acid sequences described herein, such as and vectors and host cells containing nucleic acid sequences for expressing the RLN analogs herein. It could be.

[0017] Second, at least one RLN analogue or a pharmaceutically acceptable salt thereof (e.g., For example, trifluoroacetate, acetate, or hydrochloride salts) and a pharmaceutically acceptable carrier. In some cases, the pharmaceutically acceptable carrier is, for example, a physiologically acceptable carrier. Saline, phosphate-buffered saline, citrate-buffered saline, or histidine-buffered saline In certain cases, the buffer is histidine, a histidine buffer, or a salt solution. In other cases, the pharmaceutical composition comprises a carrier, It may further comprise a diluent and / or an excipient.

[0018] Additionally, the pharmaceutical compositions may be used to treat, for example, cardiovascular, pulmonary, and / or renal conditions, diseases, or at least one additional treatment, such as a drug used as standard of care in the disorder In some cases, the at least one additional therapeutic agent may be an anticoagulant, an angiotensin inhibitor, or a combination thereof. ACE inhibitors, angiotensin II receptor blockers (ARBs), RB neprilysin inhibitors (ARNI), beta-blockers, diuretics, digitalis, digoxin, Hydralazine nitrate / isorbide, mineralocorticoid receptor antagonist agonist (MRA, or aldosterone antagonist), sodium-glucose The therapeutic agent may be a SGLT2 inhibitor, a statin, and / or an anti-glycemic agent.

[0019] Third, methods for using the RLN analogs herein, particularly methods for using the RLN analogs, and methods for treating cardiovascular, pulmonary, and / or renal conditions, diseases, or disorders. The method includes administering to a subject an effective amount of at least one RLN analogue described herein. or a pharmaceutically acceptable salt thereof to an individual in need thereof. can be.

[0020] In some cases, the RLN analogs are administered, for example, intramuscularly, intravenously, parenterally, subcutaneously, or intravenously. The RLN analogs may be administered via any standard route of administration, such as transdermal. The body may be administered subcutaneously (SQ), intramuscularly (IM), or intravenously (IV). The RLN analogs may be administered to an individual SQ or IV.

[0021] Similarly, in some cases, the RLN analogs may be administered daily, every other day, three times a week, twice a week, once a week (or more). i.e., weekly), biweekly (i.e., every other week), monthly (i.e., every month), bimonthly ( i.e., every other month), or even every three months. This RLN analogue is administered SQ every other day, SQ three times a week, SQ twice a week, SQ once a week, or SQ In certain cases, the RLN analogs may be administered SQ every other week or SQ once a month. It is administered Q once weekly (QW).

[0022] Alternatively, the RLN analogs may be administered IV to an individual. As noted above, the RLN analogs is daily, every other day, three times a week, twice a week, once a week (i.e., every week), every other week (i.e., once a week) In certain cases, the RLN analogs may be administered IV every other day. , IV 3 times a week, IV 2 times a week, IV once a week, IV every other week, or IV once a month In certain instances, the RLN analog is administered IV once a week.

[0023] The method includes administering the RLN analog in combination with an effective amount of at least one additional therapeutic agent. In short, for many of the conditions / diseases / disorders described herein, Standard treatments include anticoagulants, ACE inhibitors, ARBs, ARNIs, beta-blockers, diuretics, and digital Lithium, digoxin, hydralazine nitrate / isorbide, MRA or other aldosterone antagonists), SGLT2 inhibitors, statins, and / or antiglycemic agents, and hyperglycemic agents Co-morbidities including but not limited to cholesterol, hypertension, atrial fibrillation, and diabetes In some cases, the additional therapeutic agent may be a RLN-like agent. The compounds may be administered simultaneously, separately or sequentially.

[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, every In other cases, the additional therapeutic agent may be administered as an 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 administered orally. Alternatively, the RLN analogs may be administered IV and the additional therapeutic agent may be administered IV. is administered SQ.

[0025] In some cases, the individual in need may have diabetic renal dysfunction and / or obesity. I have high blood pressure.

[0026] The method includes measuring or obtaining blood pressure and measuring or obtaining one or more such obtained values. Evaluate the effectiveness of treatment / therapy by comparing with baseline values ​​or previously obtained values ​​above Steps such as the following may also be included.

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

[0028] Fourth, uses are described that include at least one of the RLN analogs herein. For example, the RLN analogs herein can be used in therapy, particularly for treating cardiovascular, pulmonary, and / or renal conditions, The RLN analogs may be provided for use in the treatment of a disease or disorder. simultaneously, separately, or sequentially (i.e., in combination) with at least one additional therapeutic agent. Similarly, the use of RLN analogs herein may be used in cardiovascular, pulmonary, and and / or provided in manufacturing a medicine to treat a renal condition, disease, or disorder The drug may optionally further comprise one or more additional therapeutic agents as described above. good.

[0029] Fifth, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 10). In some cases, the compound has a lower affinity to SEQ ID NO: 10. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0030] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPP (SEQ ID NO: 11). In some cases, the compound has a sequence similar to that of SEQ ID NO: 11. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0031] Alternatively, a compound is provided that comprises the following amino acid sequence: EVQLLESGGGLV QPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIG GGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTA VYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 1 2) In some cases, the compound has a sequence identity that is at least about 90% to about 99% of SEQ ID NO: 12. The amino acid sequence may have a sequence homology of:

[0032] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPP (SEQ ID NO: 13). In some cases, the compound has a The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0033] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRTVSSTAVAWFRQA PGKEREFVAGIGGSVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGT LVTVSS (SEQ ID NO: 45). In some cases, the compound has a lower affinity to SEQ ID NO: 45. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0034] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDSTAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSRVANLYPYWGQGT LVTVSS (SEQ ID NO: 46). In some cases, the compound has a lower affinity to SEQ ID NO: 46. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0035] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASYRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 47). In some cases, the compound has a lower affinity to SEQ ID NO: 47. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0036] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGAYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 48). In some cases, the compound has a lower affinity to SEQ ID NO: 48. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0037] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDETYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 49). In some cases, the compound has a low affinity for SEQ ID NO: 49. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0038] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDQTYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 50). In some cases, the compound has a lower affinity to SEQ ID NO: 50. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0039] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITAYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 51). In some cases, the compound has a lower affinity to SEQ ID NO: 51. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0040] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITEYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 52). In some cases, the compound has a lower affinity to SEQ ID NO: 52. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0041] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITQYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 53). In some cases, the compound has a lower affinity to SEQ ID NO: 53. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0042] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITSYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 54). In some cases, the compound has a lower affinity to SEQ ID NO: 54. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0043] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITTYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 55). In some cases, the compound has a lower affinity to SEQ ID NO: 55. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0044] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGKPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 56). In some cases, the compound has a lower affinity to SEQ ID NO: 56. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0045] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGQPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 57). In some cases, the compound has a lower affinity to SEQ ID NO: 57. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0046] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGSPLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 58). In some cases, the compound has a lower affinity to SEQ ID NO: 58. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0047] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRELITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 59). In some cases, the compound has a low affinity for SEQ ID NO: 59. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0048] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRQLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 60). In some cases, the compound has a lower affinity to SEQ ID NO: 60. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0049] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRSLITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 61). In some cases, the compound has a lower affinity to SEQ ID NO: 61. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0050] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPEITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 62). In some cases, the compound has a lower affinity to SEQ ID NO: 62. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0051] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPGITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 63). In some cases, the compound has a lower affinity to SEQ ID NO: 63. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0052] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPQITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 64). In some cases, the compound has a lower affinity to SEQ ID NO: 64. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0053] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPTITSKVADLYPYWGQGT LVTVSS (SEQ ID NO: 65). In some cases, the compound has a lower affinity to SEQ ID NO: 65. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0054] Alternatively, there is provided a compound comprising the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITEKVADLYPYWGQGT LVTVSS (SEQ ID NO: 66). In some cases, the compound has a lower affinity to SEQ ID NO: 66. The amino acid sequence may have at least about 90% to about 99% sequence homology.

[0055] The advantage of the RLN analogs herein is that they are single-chain polypeptides (i.e., monomers). can be synthesized chemically or recombinantly as a biosynthetic compound, and therefore can be used in cells for biological activity. However, in some cases, VH The H moiety can be conjugated to single-chain RLN as well as double-chain RLN (e.g., native). In the VHH moiety, this is thought to be possible not only at the N-terminus and C-terminus but also at the VH It can also be conjugated to any surface-exposed amino acid of H (although such conjugation does not It doesn't disable binding completely.

[0056] The advantage of the RLN analogues herein is that the VHH moiety does not only contain the natural A and B chain sequences, but also Furthermore, the VHH moiety can be used in combination with other peptides. Enhanced expression of VHHs via peptide / protein fusions or small molecules attached to VHH moieties Or it may be further modified to have additional functions.

[0057] The advantage of the RLN analogues herein is that the VHH moiety is elongated in mammals such as humans. and can provide a prolonged duration of action and have a t1 / 2 of about 20 days to about 30 days, thereby , when compared to native human RLN, and in particular native human RLN2 (SEQ ID NOs: 5 and 6) , allowing for at least weekly or biweekly dosing, which may improve compliance That is the thing.

[0058] The advantages of the RLN analogs herein are compared to native human RLN2 (SEQ ID NOs: 5 and 6). similar or better selectivity for RXFP1 over the RXFP2 receptor, Alternatively stated, the present invention provides RLN analogs have sufficient activity at the RXFP1 receptor, and RXFP2 and RXFP3 , and RXFP4 receptors, resulting in reduced or insufficient activity at one or more of the receptors.

[0059] The advantage of the RLN analogues herein is that they do not alter the albumin affinity of the VHH moiety. The goal is to have tunable pharmacokinetics, which is achieved by adjusting the dosage.

[0060] The advantage of the RLN analogs herein is that they are similar to native human RLN2 (SEQ ID NOs: 5 and 6) or Improved stability in stored formulations compared to RLN analogs with Fc fusions To do this.

[0061] Furthermore, the advantage of VHH moieties is that they can be used not only with human serum albumin but also with dog, monkey, and other , binds equally well to mouse, pig, and rat serum albumin, making it suitable for pharmacodynamics, drug The kinetic and toxicological studies from these species can be more easily translated to humans.

[0062] The advantage of the VHH moieties is that they are similar to the native human RLN2 (SEQ ID NOs: 5 and 6). can be used to improve the t1 / 2 of the RLN analogs herein, as well as, e.g. For example, insulin, growth differentiation factor 15 (GDF-15), or glucose-dependent insulin Other biologically active peptides and proteins, such as phospholipid-stimulating peptide 1 (GLP-1), It can also be used to improve the t1 / 2 of protein.

[0063] Unless otherwise defined, all technical and scientific terms used herein are It has the same meaning as commonly understood by a person skilled in the art to which this disclosure pertains. Any methods and materials similar or equivalent to those described herein may be used to prepare RLN analogs, drugs, can be used in the practice or testing of pharmaceutical compositions and methods, but preferred methods and and materials are described herein.

[0064] Also, reference to an element by the indefinite article "a" or "an" means that the element is unique depending on the context. Unless specifically required, the existence of more than one element is not excluded. The indefinite article "a" or "an" usually means "at least one."

[0065] definition As used herein, "about" refers to, for example, a specified concentration, length, molecular weight, pH Statistically meaningful values ​​of sequence identity, time frame, temperature, volume, etc. Within a range means that such a value or range is typically 20% of the given value or range. Within a range of 10%, more typically within 5%, and even more typically within 10% of the original size. The allowed variation encompassed by "about" depends on the particular system under study. , which can be easily understood by those skilled in the art.

[0066] As used herein, the terms "activity," ... "Activate," "activating," and the like refer to the activity of the protein in the in vitro assay described below. The activity of compounds such as the RLN analogs herein can be measured using assays known in the art. By "antibody" is meant the ability of a compound to bind to its receptor and induce a response thereat.

[0067] As used herein, an "amino acid" is, from a chemical standpoint, an amino acid having one or more amine groups and and one or more carboxylic acid groups, and may contain other functional groups. As known in the art, the amino acids designated as standard amino acids and used by all living organisms are: The 20 amino acids that can be used as building blocks for the majority of peptides / proteins produced by The amino acid sequences of the present disclosure contain a set of standard amino acids for the 20 standard amino acids. It contains a standard one-letter or three-letter code.

[0068] As used herein, an "analog" is an analog that activates a target receptor and does not mimic the natural activity of that receptor. elicit at least one in vivo or in vitro effect induced by a gonist. , a synthetic peptide or polypeptide.

[0069] As used herein, a "conservative substitution" refers to a substitution that has one or more amino acid residues in its amino acid sequence relative to a reference molecule. a reference peptide or polypeptide that is identical except for having the above conservative amino acid substitutions Conservatively modified variants generally refer to variants of a reference amino acid sequence. At least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the target gene. More specifically, conservative substitutions are made based on characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone The resulting substituted peptides or polypeptides are similar in structure, rigidity, etc. Functional refers to the substitution of an amino acid with an amino acid that has minimal effect on the biological activity of the compound. Conservative substitution of amino acids similar to There's no need to explain.

[0070] As used herein, an "effective amount" refers to one or more of the RLN analogs herein, or The amount or dose of a pharmaceutically acceptable salt thereof administered to an individual in need thereof in a single or multiple doses. When administered multiple times, the compound has the desired effect (i.e., 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, decreased blood pressure, decreased inflammation ( and / or reducing (or preventing) cardiac, renal, hepatic, or pulmonary fibrosis. provide a clinically measurable difference in an individual's condition, such as prevention An effective amount can be determined by the use of known techniques and under similar circumstances. This can be readily determined by one skilled in the art by observing the results obtained in the previous section. In determining an effective amount for an individual, the species of mammal, its size, age, and general the specific health condition, the specific disease or disorder involved, the extent of the disease or disorder or whether it is involved the severity of the condition, the individual's response, the particular RLN analog administered, the mode of administration, and the formulation administered. The bioavailability characteristics of the product, the selected dosing regimen, the use of concomitant medications, and other relevant Many factors are taken into consideration, including but not limited to the circumstances under which the

[0071] As used herein, "long-lasting action" refers to the binding affinity of the RLN analogs herein. and activity is higher than that of native RLN, particularly native human RLN2 (SEQ ID NOs: 5 and 6). For a long period of time, it is recommended to use at least once a day or even three times a week, twice a week, if This means that it will be possible to administer the drug as frequently as once a week. The activity profile can be determined using known pharmacokinetic testing methods, such as those utilized in the Examples below. It may be measured as follows.

[0072] As used herein, "half-life" or "t1 / 2" refers to the half-life of a native RLN or the present invention. Half of the amount of compounds, such as the RLN analogs described in the specification, is released into the serum or blood of an individual through biological processes. or the time it takes for a substance to be removed from a fluid such as plasma or other physiological space. Alternatively, t1 / 2 is the time at which the amount of such a compound reaches its pharmacological, physiological, or radiological potential. It can also mean the time it takes to lose half of its biochemical activity.

[0073] As used herein, "median effective concentration" or "EC 50 " is a dose-response curve ( For example, cAMP, PI3K-Akt, NFκβ, VEGF, and / or nitric oxide ( 50% activation / stimulation of assay endpoints such as NO signaling pathways This refers to the concentration of the compound.

[0074] As used herein, "in combination with" refers to at least one of the RLN analogs herein. At least one of these drugs may be administered simultaneously, sequentially, or in combination with one or more additional therapeutic agents. This means administering it in a combined preparation.

[0075] As used herein, an "individual in need thereof" is defined as, for example, an individual who is a patient enumerated herein. a person with a condition, disease, disorder, or symptom requiring treatment or therapy, including In particular, the individual to be treated is a human.

[0076] As used herein, "long-acting" refers to the binding affinity of the RLN analogs herein. and activity lasted for a longer period than that of native human RLN2 (SEQ ID NOs: 5 and 6), Therefore, at least once a day, or even three times a week, twice a week, once a week, or once a month. This means that it is possible to administer the drug more frequently. The pharmacokinetic profiles of the drug are measured using known pharmacokinetic test methods, such as those described in the Examples below. This may also be done.

[0077] As used herein, a "non-standard amino acid" refers to an amino acid that may occur naturally in cells but is not readily available in peptides. Non-standard amino acids are amino acids that are not involved in peptide synthesis. This may be due to modifications of standard amino acids within the peptide (i.e., posttranslational modification). Non-standard amino acids have absolute opposite positions to the standard amino acids listed above. Chiral D-amino acids may be included.

[0078] As used herein, "pharmaceutically acceptable buffer" refers to a standard solution known to those skilled in the art. By "buffer" is meant any of the standard pharmaceutical buffers.

[0079] As used herein, "RLN" refers to a gene obtained from any species, including mammalian species, particularly humans. The term "relaxin" refers to relaxin produced or derived from relaxin, wherein the native form is composed of two peptide chains ( For example, the A and B chains are connected via two disulfide bonds, and the A chain is a single RLN is a heterodimeric peptide that further contains an intramolecular disulfide bond. Native RLN (i.e., full-length) and its variants (i.e., additions, deletions, insertions, In humans, RLN1, RLN2, and RLN3 are both nucleotides of the RLN1 and RLN2 subunits. There are three natural RLN isoforms of N3. RLN processing is initiated by preprorelaxin. This preprorelaxin then produces prorelaxin (containing A chain, B chain, and C peptide). Natural human proRLN1 is processed into the BCA structure. The sequence is shown in SEQ ID NO: 1 (UniProt / SwissProt database accession no. See also No. P04808), and the sequence of native human proRLN2 is shown in SEQ ID NO: 4 (Un See also iProt / SwissProt database accession number P04090), The sequence of proRLN3 is shown in SEQ ID NO: 7 (UniProt / SwissProt (See also database accession number Q8WXF3). Prorelaxin is involved in further processing. During this processing, the C-peptide is cleaved and reaches the RLN. The sequences of the A chains of RLN1, RLN2, and RLN3 are SEQ ID NOs: 2, 5, and 6, respectively. and 8. Similarly, the B chains of native human RLN1, RLN2, and RLN3 The sequences are shown in SEQ ID NOs: 3, 6, and 9, respectively.

[0080] Humans have four RLN receptors that act as GPCRs: RXFP1 (SEQ ID NO: 40; See also UniProt / SwissProt database accession number Q9HBX9), RX FP2 (SEQ ID NO: 41; UniProt / SwissProt database accession number Q8 See also WXD0), RXFP3 (SEQ ID NO: 42; UniProt / SwissProt data See also database accession number Q9NSD7), and RXFP4 (SEQ ID NO: 43; UniP See also rot / SwissProt database accession number Q8TDU9 (Hal ls et al.(2007)Br.J.Pharmacol.150:677-69 1). Of interest here are the RXFP1 and RXFP2 receptors, both of which are R It can bind to LN1 and RLN2. The RXFP1 receptor is involved in the brain, blood cells, bones, and heart. While the RXFP2 receptor is found in the liver, kidney, lung, liver, and vasculature, The RXFP1 receptor and RXFP2 receptor are restricted to the nucleus and are found in bone and the conductance zone. Upon receptor stimulation, adenylate cyclase, protein kinase A, and protein kinase enzyme C, phosphatidylinositol 3-kinase, and / or extracellular signal regulation A signaling network involving kinases (Erk1 / 2) is activated.

[0081] As used herein, "RLN analogs" and the like refer to compounds that bind to one or more RXFP receptors. elicit one or more of the effects of native RLN, but differ in amino acid sequence compared to native RLN. RLN means a compound, such as a peptide or polypeptide, that differs in some way from other compounds. Analogs can also include variants of these compounds, which variants functionally interact with RLN. Equivalent but fragmented, or complete sequence but with additions, deletions, insertions, and and / or substitutions. All references to amino acid positions in the selected RLN refer to the amino acid positions in the naturally occurring human RLN unless otherwise specified. The sequences are based on the corresponding positions in the A chain of SEQ ID NO: 5 or the B chain of SEQ ID NO: 6 of human RLN2. In some cases, the RLN analogs herein may have a higher or lower affinity for RXFP. RLN2 (SEQ ID NOs: 5 and 6) In this case, the compound may exhibit a longer t1 / 2 in vivo or in vitro. The RLN analogs described herein are synthetic compounds that act as RXFP receptor agonists.

[0082] As used herein, "sequence homology" refers to, for example, the overall length or ratio of two or more sequences. Matching of two or more nucleic acid or amino acid sequences of a biological compound, such as a match over a comparison window Sequence identity refers to a quantitative property of a sequence. Sequence identity can be expressed as (1) percent identity or (2) similarity. Percent identity can be measured by the percentage of identical sequences between two biological compounds. The percentage of residues divided by the length of the shortest sequence is measured, while the percent similarity measures identity and also includes sequence gaps and residue similarity in the assessment. Methods and algorithms for determining It is not necessary to go into detail. A specific percentage of identical nucleotide or amino acid positions The age is at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or Or even more.

[0083] As used herein, "single-chain relaxin," "scRLN," and the like, refers to A-L2- B or B-L2-A, where the A and B chains are mutually connected by a linker (i.e., L2). Furthermore, scRLN refers to the RLN polypeptides that are connected to each other. To maintain folding, few of the native inter- and / or intra-chain disulfide bonds are present. It may contain at least one.

[0084] As used herein, "two-chain relaxin," "tcRLN," and the like refer to naturally occurring RLN. In order to maintain the correct structural folding, the A and B chains must be attached to either linker. connected to each other by one or more inter- and / or intra-chain disulfide bonds rather than by The term "RLN polypeptide" refers to a RLN polypeptide that is

[0085] As used herein, "treating" or "to treat" "eat" means to suppress or reduce the progression or severity of a condition, disease, disorder, or symptom. Administration of RLN analogs is indicated to inhibit, reverse, slow, or stop Treatment means the management or care of an individual who has a condition, disease, disorder, or symptom. The treatment may include administering an RLN analogue as described herein or a composition comprising an RLN analogue as described herein. Administered to an individual to prevent the onset of symptoms or complications or to alleviate symptoms or complications. Treating includes treating or eliminating a condition, disease, disorder, or symptom. , an RLN analog or a composition comprising an RLN analog herein may be administered to an individual to, e.g., Increased angiogenesis, increased vascular compliance, increased cardiac blood flow, increased hepatic blood flow, and increased pulmonary blood flow Increased renal blood flow, increased glomerular filtration rate, decreased blood pressure, decreased (or prevented) inflammation, and and / or reduction (or prevention) of cardiac, renal, liver, or pulmonary fibrosis. The individual to be treated is a mammal, particularly a human.

[0086] As used herein, "individual," "patient," and "subject" are used interchangeably and It refers to mammals, particularly humans. In certain instances, individuals may be administered the RLN analogs described herein. Further, conditions, diseases, disorders, and / or symptoms that would benefit from the administration of It is characterized by:

[0087] As used herein, a "VHH" or "VHH portion" refers to a VHH in the form of a single domain antibody. In particular, the single heavy chain antibody (HcAb) is very small, approximately 15 kDa in size. It refers to an antibody fragment of a monomeric variable region. The VHH portion is used as a pharmacokinetic enhancer. This may extend the duration of action and / or improve the t1 / 2 of the RLN analogs herein. It has now been found that the VHH moieties herein bind to serum albumin, but this V The HH portion may be derived from IgG (containing the Fc domain), neonatal Fc receptor (FcRn), or other The VHHs herein can be used to bind to long-lasting serum proteins. The moiety is used to improve the t1 / 2 of RLN, but similarly, e.g., insulin of other biologically active peptides / proteins such as α-glucan, GDF-15, or GLP-1. It can be used to improve t1 / 2.

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

[0089] RLN analogue The RLN analogs herein are structurally similar to native human RLN, but possess many structural differences. For example, when compared to native human RLN2 (SEQ ID NOs: 5 and 6), LN analogs lack one or more of the amino acids present in native human RLN2 and contain the A chain and It contains a peptide linker between the B chain and the albumin-binding VHH portion. Depending on the body, activity at the RXFP1 receptor is sufficient to stimulate RXFP2, RXFP3, and R The activity at one or more of the XFP4 receptors is reduced or insufficient. LN analogs have the potential to improve solubility in aqueous solutions, chemical and physical formulation stability, and drug Extended kinetic profile (can be tailored based on VHH affinity to serum albumin) their potential as therapeutic treatments, including minimizing potential immunogenicity, and / or Possess beneficial attributes related to development potential.

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

[0091] With respect to the A chain, this refers to a naturally occurring RLN A chain, e.g., a naturally occurring human RLN1 A chain (sequence column number 2), native human RLN2 A chain (SEQ ID NO: 5), or native human RLN3 A Alternatively, the A chain may be a variant thereof. For example, The A-chain mutant has an amino acid sequence lacking residues 1-4 of SEQ ID NO:5 (i.e., des1-4hi The RLN2 A chain may be present (or desA1-4).

[0092] Similarly, and with respect to the B chain, this may be a native RLN B chain, e.g., a native human RLN 1 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 may be a variant thereof. For example, one B chain variant has the amino acid sequence of SEQ ID NO:6 lacking residue 1 (i.e., des 1 human RLN2 B chain or desB1).

[0093] In some cases, the A chain is the native human RLN1 A chain (SEQ ID NO: 2) and the B chain is the native human RLN1 A chain (SEQ ID NO: 3). The A chain can be the native human RLN1 B chain (SEQ ID NO: 3), or the A chain can be the native human RLN2 A chain (SEQ ID NO: 4). 5), the B chain can be the native human RLN2 B chain (SEQ ID NO: 6) or the A chain can be the native human RLN3 A chain (SEQ ID NO: 8), B chain is natural human RLN3 B chain (SEQ ID NO: 9) or the A chain is the natural human RLN1 A chain (SEQ ID NO: 2) and the B chain is the natural human RLN2 The A chain can be the natural human RLN1 A chain (SEQ ID NO: 2), B The B chain can be the native human RLN3 B chain (SEQ ID NO: 9) or the A chain can be the native human RLN2 The A chain (SEQ ID NO: 5), the B chain can be the natural human RLN1 B chain (SEQ ID NO: 3), The A chain is the natural human RLN2 A chain (SEQ ID NO: 5), and the B chain is the natural human RLN3 B chain (SEQ ID NO: 6). RLN3 A chain (SEQ ID NO: 8) and the B chain (SEQ ID NO: 9) or or the A chain can be the natural human RLN3 The A chain (SEQ ID NO: 8) and the B chain can be the native human RLN2 B chain (SEQ ID NO: 6).

[0094] In some cases, the A chain is an RLN2 A chain mutant lacking residues 1-4 (desA1-4). In other cases, the A chain may be any naturally occurring B chain. The B chain may be the native A chain, and the B chain may be an RLN2 B chain mutant lacking residue 1 (desB1). In still other cases, the A chain may be an RLN2 A chain variant lacking residues 1-4 ( desA1-4), and the B chain may be an RLN2 B chain mutant lacking residue 1 (des In certain cases, the A chain is a desA1-4 mutant. In this case, the B chain is a desB1 mutant.

[0095] Other A and B chains that can be used in the RLN analogs herein are described, for example, in the International Patent Publication No. Patent application publication numbers 2018 / 148419, 2018 / 138170, and 2017 / 201340, 2016 / 149501, 2015 / 157829, same No. 2015 / 067791, No. 2015 / 067113, No. 2014 / 1021 No. 79, No. 2013 / 177529, No. 2013 / 007563, No. 2013 / 004607, 2012 / 031326, and 2012 / 024452 No. 2011 / 0243942, as well as U.S. Patent Application Publication No. Cha. n et al. (2012) J. Biol. Chem. 287:41152-4116 4, Claasz et al. (2002) Eur.J.Biochem.269:6 287-6293, Hossain et al. (2015)Org.Biomol. Chem.13:10895-10890, Hossain et al. (2016) Chem.Sci.7:3805-3819, Park et al. (2008)J. Biol.Chem.283:32099-32109, and Wilkinson e See also t 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. L1 is about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, Alternatively, L1 can be about 45 or about 50 amino acids. 10 amino acids, about 10 to about 15 amino acids, about 15 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 to about 40 amino acids, about 40 to about 45 amino acids, or or about 45 to about 50 amino acids. In some cases, the A chain may be composed of a sequence of amino acids excluding L1. Alternatively, the B strand may be directly conjugated to the VHH moiety. In some cases, L1 may be (G GGGQ) n (SEQ ID NO: 14), where n is about 1 to It may be about 10, particularly 5 (i.e., (GGGGQ)5, SEQ ID NO: 19). L1 is (PGPQ) n (SEQ ID NO: 17), , n can be from about 1 to about 10, particularly 8 (i.e., (PGPQ)8, SEQ ID NO: 20). In still other cases, L1 is (PGPA) n Contains a repeat sequence of (SEQ ID NO: 18) wherein n is from about 1 to about 10, particularly 8 (i.e., (PGPA)8, SEQ ID NO: 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) I can't.

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

[0099] For VHHs, this may be due to their affinity to serum albumin or another serum protein with a long t1 / 2. A sequence of about 50 to about 200 amino acids, particularly about 125 to about 150 amino acids, that can be bound to the amino acid sequence. In some cases, the VHH may be a polypeptide of 150 amino acids. Alternatively, the VHH may be any one of SEQ ID NOs: 45 to 66. The structural features of these VHH moieties make it possible to distinguish natural RLNs, especially natural RLN analogs with longer t1 / 2 when compared to human RLN2 (SEQ ID NOs: 5 and 6) Given that the VHH moieties herein target serum albumin, For this reason, the t1 / 2 of RLN analogs is approximately the same as that of serum albumin of the species to which the RLN analog is administered. It can be expected to be similar to the t1 / 2 (taking into account target-mediated drug disposition).

[0100] In addition to the modifications described in this disclosure, the RLN analogs herein may also be used in combination with other analogs, such as RLN analogs containing .... one or more, provided that they are capable of binding to and activating the RXFP1 receptor; may contain additional amino acid modifications, particularly conservative substitutions.

[0101] In summary, exemplary RLN analogs are: VHH moiety (underlined), (G4Q)5L1 (italicized), A chain of RLN2, 10 residues of L2 (bold), and RLN analog 1, which contains the B chain of RLN2 (desB1), contains the following amino acids: array: [ka] (SEQ ID NO: 24) or a pharmaceutically acceptable salt thereof, VHH moiety (underlined), (PGPA)8L1 (italicized), A chain of RLN2, 10 residues of L 2 (bold), and RLN analog 2, which contains the B chain of RLN2 (desB1), Acid sequence: [ka] (SEQ ID NO: 25) or a pharmaceutically acceptable salt thereof, VHH portion (underlined), (G4Q)5L1 (italicized), B chain of RLN2 (desB1), RLN analogue 3, which contains the 10-residue L2 (bold) and the A chain of RLN2, contains the following amino acids: array: [ka] (SEQ ID NO: 26) or a pharmaceutically acceptable salt thereof, VHH moiety (underlined), (PGPQ)8L1 (italicized), B chain of RLN2 (desB1) RLN analog 4, which contains the 10-residue L2 (bold), and the A chain of RLN2, has the following amino acid sequence: Acid sequence: [ka] (SEQ ID NO: 27) or a pharmaceutically acceptable salt thereof, RLN2 B chain (desB1), 10 residues of L2 (bold), RLN2 A chain, (G4Q ) 5L1 (italicized), and RLN analog 5, which contains the VHH portion (underlined), has the following amino acids: array: [ka] (SEQ ID NO: 28) or a pharmaceutically acceptable salt thereof, RLN2 B chain (desB1), 10 residues of L2 (bold), RLN2 A chain, (PGP Q) RLN analog 6, which contains 8L1 (italicized) and a VHH moiety (underlined), has the following amino acid sequence: Acid sequence: [ka] (SEQ ID NO: 29) or a pharmaceutically acceptable salt thereof, VHH portion (underlined), (G4Q)5L1 (italicized), B chain of RLN2 (desB1), RLN analog 7, which contains the 10-residue L2 (bold) and the A chain of RLN2, contains the following amino acids: array: [ka] (SEQ ID NO: 30) or a pharmaceutically acceptable salt thereof, VHH moiety (underlined), (PGPQ)8-L1 (italicized), B chain of RLN2 (desB1) RLN analog 8, which contains the 10-residue L2 (bold), and the A chain of RLN2, has the following amino acid sequence: Acid sequence: [ka] (SEQ ID NO: 31) or a pharmaceutically acceptable salt thereof, RLN2 B chain (desB1), 10 residues of L2 (bold), RLN2 A chain, (G4Q RLN analog 9, which contains 5L1 (italicized), and a VHH portion (underlined), has the following amino acids: array: [ka] (SEQ ID NO: 32) or a pharmaceutically acceptable salt thereof, RLN2 B chain (desB1), 10 residues of L2 (bold), RLN2 A chain, (PGP Q) RLN analog 10, which contains 8L1 (italicized) and a VHH moiety (underlined), has the following amino acid sequence: Acid sequence: [ka] (SEQ ID NO: 33) or a pharmaceutically acceptable salt thereof, VHH portion (underlined), (G4Q)5L1 (italicized), B chain of RLN2 (desB1), RLN analogues containing the 13-residue L2 (bold) and the A chain of RLN2 (desA1–4) 11 has the following amino acid sequence: [ka] (SEQ ID NO: 34) or a pharmaceutically acceptable salt thereof, VHH portion (underlined), (G4Q)5L1 (italicized), B chain of RLN2 (desB1), RLN analogues containing the 13-residue L2 (bold) and the A chain of RLN2 (desA1–4) 12 has the following amino acid sequence: [ka] (SEQ ID NO: 35) or a pharmaceutically acceptable salt thereof, VHH portion (underlined), (G4Q)5L1 (italicized), A chain of RLN2 (desA1-4 ), a 10-residue L2 (bold), and an RLN analogue containing the B chain of RLN2 (desB1). 13 has the following amino acid sequence: [ka] (SEQ ID NO: 36) or a pharmaceutically acceptable salt thereof, VHH portion (underlined), (PGPQ)8L1 (italicized), A chain of RLN2 (desA1~ 4), an RLN-like protein containing the 10-residue L2 (bold), and the B chain of RLN2 (desB1). Enantiomer 14 has the following amino acid sequence: [ka] (SEQ ID NO: 37) or a pharmaceutically acceptable salt thereof, VHH moiety (underlined), (G4Q)5L1 (italicized), B chain of RLN2 (natural), 10 residues RLN analog 15, containing the group L2 (bold) and the A chain of RLN2 (native), was synthesized using the following amino acid sequence: Acid sequence: [ka] (SEQ ID NO: 38) or a pharmaceutically acceptable salt thereof, VHH moiety (underlined), (PGPA)8L1 (italicized), B chain of RLN2 (natural), 10 RLN analog 16, which contains residues L2 (bold) and the A chain of RLN2 (native), has the following structure: Amino acid sequence: [ka] (SEQ ID NO: 39) or a pharmaceutically acceptable salt thereof.

[0102] The half-lives of the RLN analogs herein can be determined by methods such as those described in the Examples below. This can be measured using methods known in the art. The affinity of the present RLN analogs for agonists can be determined using methods known in the art for measuring binding affinity. can be measured using various methods, such as those described in the Examples below, and generally Equilibrium dissociation constant (K D ) values. Furthermore, the values ​​described herein for each of the RXFP receptors are The activity of the RLN analogs described herein can be assayed, for example, using in vitro activity assays, including those described below. can be measured using methods known in the art and is generally 50 represented as a value will be done.

[0103] As a result of the above modifications, the RLN analogs herein can be administered to mammals, particularly humans, with In this case, the t1 / 2 is the same as that of the native RLN, particularly the native human RLN2 (SEQ ID NOs: 5 and 6). As mentioned above, the VHH moieties herein are intended to target serovars. Therefore, the t1 / 2 of the RLN analogs herein is determined based on the serum albumin levels of the species to which the RLN analog is administered. It can be expected that the t1 / 2 of RLN is similar to that of RLN. The body has a t1 / 2 of about 1 day to about 31 days, about 5 days to about 25 days, about 10 days to about 20 days, or In other cases, the RLN analogs have a t of about 1 day to about 15 days. Approximately 5 days, approximately 6 to 10 days, approximately 11 to 15 days, approximately 16 to 20 days, approximately 21 to 2 5 days, or even about 26 days to about 31 days. In other cases, the RLN analogs , t1 / 2 is 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 9th, about 10th, about 11th, about 12th, about 13th, about 14th, about 15th, about 16th, about 1 7th, about 18th, about 19th, about 20th, about 21st, about 22nd, about 23rd, about 24th, about 2 5 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, or even about 31 days In certain instances, the RLN analogs have a t of about 20 when administered to humans. It could be a day.

[0104] Similarly, the RLN analogs herein exhibit efficacy at the RXFP1 receptor when administered to humans. For example, the potency is about 10 to about 100 times that of natural human RLN2 (SEQ ID NOs: 5 and 6). is.

[0105] Pharmaceutical Compositions and Kits The RLN analogs described herein can be administered parenterally (e.g., intravenously, intraperitoneally, intramuscularly, or intradermally). The composition can be formulated as a pharmaceutical composition that can be administered intradermally (subcutaneously, intramuscularly, or transdermally). Such pharmaceutical compositions and techniques for their preparation are well known in the art. For example, Remington, "The Science and Pr actice of Pharmacy” (DBTroy ed.,21 st Ed ., Lippincott, Williams & Wilkins, 2006) In certain cases, the RLN analogs are administered SQ or IV. Alternatively, the RLN analogs may be administered, for example, in tablets or other solid forms for oral administration, sustained release capsules, or other solid forms for oral administration. capsules, and any other currently used formulations including creams, lotions, inhalants, etc. The pharmaceutical composition may be formulated in a form suitable for other pharmaceutically acceptable routes, such as in a pharmaceutical form.

[0106] As mentioned above, to improve their in vivo compatibility and efficacy, the RL The N analogs can be reacted with any number of inorganic and organic acids / bases to produce pharmaceutically acceptable acids / salts. Pharmaceutically acceptable salts and the general techniques for preparing them may be used. Methods are well known in the art (see, e.g., Stahl et al., "Handbo ok of Pharmaceutical Salts:Properties,Se lection and Use" (2 nd Revised Ed. Wiley-VC H, 2011). Pharmaceutically acceptable salts for use herein include sodium salts. Examples of suitable salts include sodium salts, trifluoroacetate salts, hydrochloride salts, and / or acetate salts.

[0107] The RLN analogs herein may be administered by a physician or self-administered using injections. It is understood that the gauge size and injection volume can be readily determined by one skilled in the art. However, the injection volume is about 2 mL or less, or even about 1 mL or less, and the needle The gauge can be about 27G or greater, or even about 29G or greater.

[0108] The present disclosure also provides a method for synthesizing the RLN analogs described herein or pharmaceutically acceptable salts thereof. The present invention provides and therefore encompasses novel intermediates and methods that are useful for 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 examples below. The specific steps for each method are described in various methods for preparing the present RLN analogs. The reagents and starting materials are readily available to one skilled in the art. It is Noh.

[0109] The RLN analogs herein are generally effective over a wide dosage range. An exemplary dose of an analog or a pharmaceutical composition containing same is: milligrams (mg), micrograms (μg), nanograms (ng), or picograms Thus, the daily dose may be from about 1 μg to about 100 mg. It could be.

[0110] Here, the effective amount of the RLN analog in the pharmaceutical composition is about 0.25 mg to about 5.0 mg. However, those skilled in the art will appreciate that in some cases, the effective amount (i.e., dose / administration) may be dose) may be more than sufficient even if it is below the lower limit of the aforementioned range, while in other cases The effective dose may be higher and may be used with tolerable side effects. Understand that.

[0111] In addition to the RLN analogs herein, the pharmaceutical compositions may contain at least one additional therapeutic agent. , especially as a standard of care in cardiovascular, pulmonary, and renal conditions, diseases, and disorders. Therapeutic agents may also be included that are used therapeutically.

[0112] Thus, the pharmaceutical composition comprises an effective amount of at least one RLN analogue described herein, and optionally at least one additional therapeutic agent. For example, the pharmaceutical composition may comprise an effective amount of an RLN analog of SEQ ID NO: 24 and a pharmaceutical a pharmaceutically acceptable carrier, an effective amount of the 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: 26; No. 27 RLN analogue and a pharmaceutically acceptable carrier, an effective amount of RLNs of SEQ ID NO: 28 an analog of SEQ ID NO: 29 and a pharmaceutically acceptable carrier; an effective amount of the RLN analog of SEQ ID NO: 29 and a pharmaceutically acceptable carrier; a pharmaceutically acceptable carrier, an effective amount of the RLN analog of SEQ ID NO: 30, and a pharmaceutically acceptable carrier. an effective amount of an RLN analog of SEQ ID NO: 31 and a pharmaceutically acceptable carrier; RLN analogs of SEQ ID NO: 32 and a pharmaceutically acceptable carrier, an effective amount of RLNs of SEQ ID NO: 33 an analog of SEQ ID NO: 34 and a pharmaceutically acceptable carrier; an effective amount of the RLN analog of SEQ ID NO: 34 and a pharmaceutically acceptable carrier; a pharmaceutically acceptable carrier, an effective amount of the 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; No. 37 RLN analogue and a pharmaceutically acceptable carrier, an effective amount of RLNs of SEQ ID NO: 38 an analog of SEQ ID NO: 39 and a pharmaceutically acceptable carrier, or an effective amount of an RLN analog of SEQ ID NO: 39 and A pharmaceutically acceptable carrier may be included.

[0113] Alternatively, the RLN analogs herein may be provided as part of a kit. The kit includes at least one RLN analog (and optionally at least one In certain instances, the present invention may include a device for administering a therapeutic agent (e.g., a steroid or other therapeutic agent) to an individual. The kit contains at least one RLN analogue (and optionally at least one additional therapeutic agent). In certain cases, the syringes and needles used to administer RLN analogs (and and optionally at least one additional therapeutic agent) are pre-formulated in an aqueous solution in a syringe. can be.

[0114] Methods for making and using RLN analogs The RLN analogs herein can be synthesized using any number of standard recombinant DNA methods known in the art. Alternatively, it can be produced via standard chemical peptide synthesis methods. For example, nucleic acids encoding the amino acid sequences of RLN analogs can be synthesized using standard recombinant techniques. constructing a polynucleotide having the sequence and inserting the polynucleotide into a recombinant expression vector; Integrative vectors can be introduced into host cells, such as bacteria, yeast, and mammalian cells, to produce R LN analogs can be produced. See, e.g., Green & Sambrook, "Mo "lecular Cloning: A Laboratory Manual" (Col. d Spring Harbor Laboratory Press,4 th ed. 2012).

[0115] With respect to recombinant DNA methods, the compounds herein are those derived from proteins produced using recombinant DNA technology. It can be prepared by producing a protein or precursor protein molecule. and DNA, including synthetic DNA, may be double-stranded or single-stranded and may be any of the DNAs described herein. The coding sequences therein that encode compounds may vary as a result of redundancy or degeneracy in the genetic code. Briefly, a DNA sequence encoding the compounds of the present invention may be introduced into a host cell. The host cell is Escherichia coli, which produces the compound or its precursor. bacterial cells, such as strain K12 or strain B of B. li, fungal cells, such as yeast cells, or Chinese The cell may be a mammalian cell such as a Chinese hamster ovary (CHO) cell.

[0116] To produce the compounds of the invention or their precursors, suitable host cells can be cultured with an expression vector The vector is transiently or stably transfected or transformed with an expression system such as an expression vector. are typically expressed either as episomes or as an integrated part of the host chromosomal DNA. Generally, expression vectors are used to transform a desired DNA sequence into a vector capable of replicating in the host organism. To allow detection of transformed cells, a selectable marker, e.g., tetracycline, necrosis factor, Contains omycin, G418, and dihydrofolate reductase.

[0117] Specific procedures for each of the steps described herein for preparing the compounds of the present invention biosynthetic or synthetic steps may or may not be used, and may be combined in different ways.

[0118] For chemical peptide synthesis, standard manual or automated solid-phase synthesis procedures can be used. For example, an automated peptide synthesizer can be manufactured by, for example, Applied Biosystems. tems (Foster City, CA) and Protein Technology Reagents for solid phase synthesis are commercially available from ies Inc. (Tucson, AZ). These are readily available from commercial suppliers. Solid phase synthesis equipment for coupling, deprotection, and capping of unreacted amino acids The manufacturer's instructions can be followed. Additional details regarding the preparation of synthetic RLNs can be found in , and are described in U.S. Patent 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 acute heart failure, , chronic heart failure, atherosclerosis, coronary artery disease, diabetes, stroke, high cholesterol These include, but are not limited to, vasoconstriction, hypertension, ischemia, vasoconstriction, and ventricular hypertrophy. .

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

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

[0122] The method may include, but is not necessarily limited to, the steps described herein. The steps may be performed in the order listed; however, other orders are also contemplated. or multiple steps may be performed in parallel and / or overlapping time, and / or may be performed individually or in multiple repeated steps. , additional unspecified steps may be included.

[0123] Thus, such methods include administering to a patient having or suffering from a cardiovascular condition, disease, or disorder. Alternatively, the method may include selecting an individual who is or is susceptible to pulmonary Selecting individuals who have or are susceptible to a condition, disease, or disorder Alternatively, the method may include administering to a patient having a renal condition, disease, or disorder, or In certain instances, the method may include selecting individuals who are susceptible to the disease. This includes selecting individuals with diabetes, hypertension with renal dysfunction, and / or obesity. obtain.

[0124] The method also includes administering to the individual an effective amount of at least one RLN analogue described herein. The analogs may also be used in pharmaceutical compositions as described herein. In some cases, the RLN analog / pharmaceutical composition may contain an additional therapeutic agent. , e.g., anticoagulants, ACE inhibitors, ARBs, ARNIs, beta-blockers, diuretics, digitalis , digoxin, hydralazine nitrate / isorbide, MRA or other aldosterone antagonists agonists), SGLT2 inhibitors, statins, and / or antiglycemic agents, and hypercholesterolemia Control comorbidities including, but not limited to, sterols, hypertension, atrial fibrillation, and diabetes Other therapeutic agents to control

[0125] The concentrations / dosages / administration of the present RLN analogs and optional additional therapeutic agents are described elsewhere herein. It is being considered in the place.

[0126] Regarding the route of administration, RLN analogs or pharmaceutical compositions containing them can be administered, for example, orally, by injection, or injection (i.e., intra-arterial, intravenous, intraperitoneal, intracerebral, intraventricular, intramuscular, intraocular, intraportal, or The therapeutic agent may be administered in accordance with known methods, such as by intralesional administration, sustained release systems, or implanted devices. In certain cases, the RLN analog or a pharmaceutical composition comprising the same is administered as a bolus injection. It can be administered SQ by intravenous or continuously.

[0127] Regarding the frequency of administration, the RLN analog or a pharmaceutical composition containing the same may be administered daily, every other day, or 3 times a week. once 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 a pharmaceutical composition comprising the same may be administered in a manner similar to that described above. Q 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, RLN analogs or pharmaceutical compositions containing same The substance is administered SQ weekly (QW).

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

[0129] Additionally, the additional therapeutic agent may be administered as frequently as the RLN analog or a pharmaceutical composition containing same (e.g., i.e., every other day, twice a week, or even weekly). Alternatively, the additional therapeutic agent The RLN analogue or pharmaceutical composition comprising the same may be administered at a different frequency. In some 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] These methods may be combined with diet and exercise and / or include the methods discussed above. It is further contemplated that the compounds may be combined with additional therapeutic agents other than those mentioned 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: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQQLYSALANK CCHVGCTKRSLARFCGGGSGGSGGGGSWMEEVIKLCGRELV RAQIAICGMSTWS (SEQ ID NO: 24)

[0133] Here, the RLN analog of SEQ ID NO: 24 was synthesized in mammalian cells using CHOK1 cell derivatives. The cDNA sequence encoding SEQ ID NO: 24 is produced in a cell expression system. Subcloning into the current plasmid backbone (pEE12.4-based plasmid). The NA sequence was amplified by the signal peptide sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: RLN analogs were fused in-frame to the coding sequence of RLN (No. 44) to deliver the analogs to tissue culture media. Expression is driven by the viral CMV promoter.

[0134] PEI-based transfection to generate RLN analogues Using the method, CHOK1 cells are transfected with the recombinant expression plasmid. has a density of 4 x 10 6 Add the CHOK1 suspension cells to a shake flask in an appropriate volume of cells / mL. The cells are then transferred to a suspension culture medium and both PEI and recombinant plasmid DNA are added to the cells. Incubate the cells at 32°C for 6 days. At the end of the incubation period, The RLN analogue protein is purified from the conditioned medium by centrifugation.

[0135] Alternatively, to generate RLN analogs via stable transfection, CHOK1 cells were transfected with electroporation and an appropriate amount of recombinant expression plasmid. The transfected cells were stably transfected using PBS and then suspended at the appropriate cell density. The transfected cells were maintained in culture. Selection of the cells was performed in serum-free medium containing 25 μM MSX. This is achieved by growing the cells in a medium at approximately 35°C to 37°C and approximately 5 to 7% CO2. Incubate.

[0136] The RLN analogs were secreted from CHO cells into the culture medium and purified by protein A affinity chromatography. followed by ion exchange and hydrophobic interaction chromatography or Specifically, RLNs were purified from the collected culture medium by size exclusion chromatography. The analogs are captured on Mab Select Protein A resin (GE). Wash the resin with a running buffer such as saline (PBS, pH 7.4) or a Tris-containing buffer. A brief wash removes non-specifically bound material. Proteins are then resuspended in 10 mM citric acid. The resin is eluted with a low pH solution, such as pH 3. Fractions containing the RLN analogs are pooled and the latent fractions are analyzed. A low pH may be used to inactivate resident viruses. Any base may be added to neutralize the pH. Ion exchange chromatography using resins such as HS (ThermoFisher) It may be further purified by 15 column volumes of 20 mM NaO at pH 5.0. A gradient of 0 to 500 mM NaCl in Ac was used to remove the RLN analogs from the column. Elute from the

[0137] RLN analogs were purified using a Capto Phenyl ImpRes HIC Column ( GE Healthcare) for hydrophobic interaction chromatography. Further purification may be performed by adjusting the column packing solution to about 0.5 M sodium sulfate. and a 10 CV gradient from 0.5 M to 0 M sodium sulfate in 20 mM Tris solution at pH 8. After HIC, the RLN analogs were eluted using PBMC at pH 7.4. Isocratic incubation in S or in 20 mM histidine, 50 mM NaCl at pH 6.0 Using the backflow elution, the concentrated Capto Phenyl ImpRes pool was purified by Sup Loaded onto Erdex200 (GE Healthcare) and analyzed by SEC. It may be further purified.

[0138] The purified RLN analogue was purified using Planova 20N (Asahi Kasei Meteorological Agency). After passing it through a virus-retaining filter such as a regenerated cellulose membrane (Milli Using tangential flow ultrafiltration on a 200-well pore system, the lysate was filtered with 20 mM histidine, 20 mM N-acetylglucosamine, and 100 mM PEG / PPG solution at pH 6. Concentrate / diafilter to aCl.

[0139] Thus, RLN analogs can be synthesized in this manner or by other methods readily determined by one skilled in the art. It is prepared in a similar manner.

[0140] Example 2: Recombinant expression of RLN analogue 2 Example 2 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAQL YSALANKCCHVGCTKRSLARFCGGGSGGSGGGGSWMEEVIK LCGRELVRAQIAICGMSTWS (SEQ ID NO: 25)

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

[0142] Example 3: Recombinant expression of RLN analogue 3 Example 3 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKL CGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCC HVGCTKRSLARFC (SEQ ID NO: 26)

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

[0144] Example 4: Recombinant expression of RLN analogue 4 Example 4 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSW MEEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYS ALANKCCHVGCTKRSLARFC (SEQ ID NO: 27)

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

[0146] Example 5: Recombinant expression of RLN analog 5 Example 5 is an RLN analog having the following amino acid sequence: SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQL YSALANKCCHVGCTKRSLARFCGGGGQGGGGQGGGGQGGG GQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETA VAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRD NSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLY PYWGQGTLVTVSSPP (SEQ ID NO: 28)

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

[0148] Example 6: Recombinant expression of RLN analog 6 Example 6 is an RLN analog having the following amino acid sequence: SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQL YSALANKCCHVGCTKRSLARFCPGPQPGPQPGPQPGPQPG PQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLSCAASG RYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKG RFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLIT SKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 29)

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

[0150] Example 7: Recombinant expression of RLN analog 7 Example 7 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKL CGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCC HVGCTKRSLARFC (SEQ ID NO: 30)

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

[0152] Example 8: Recombinant expression of RLN analog 8 Example 8 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSW MEEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYS ALANKCCHVGCTKRSLARFC (SEQ ID NO: 31)

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

[0154] Example 9: Recombinant expression of RLN analog 9 Example 9 is an RLN analog having the following amino acid sequence: SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQL YSALANKCCHVGCTKRSLARFCGGGGQGGGGQGGGGQGGG GQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETA VAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRD NSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLY PYWGQGTLVTVSSPP (SEQ ID NO: 32)

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

[0156] Example 10: Recombinant expression of RLN analog 10 Example 10 is an RLN analog having the following amino acid sequence: SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQL YSALANKCCHVGCTKRSLARFCPGPQPGPQPGPQPGPQPG PQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLSCAASG RYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKG RFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLIT SKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 33)

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

[0158] Example 11: Recombinant expression of RLN analog 11 Example 11 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKL CGRELVRAQIAICGMSTWSGGGSGGSGGSGGGALANKCCH VGCTKRSLARFC (SEQ ID NO: 34)

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

[0160] Example 12: Recombinant expression of RLN analog 12 Example 12 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKL CGRELVRAQIAICGMSTWSGGGSGGSGGSGGGALANKCCH VGCTKRSLARFC (SEQ ID NO: 35)

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

[0162] Example 13: Recombinant expression of RLN analog 13 Example 13 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQALANKCCHV GCTKRSLARFCGGGSGGSGGGGSWMEEVIKLCGRELVRAQI AICGMSTWS (SEQ ID NO: 36)

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

[0164] Example 14: Recombinant expression of RLN analog 14 Example 14 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQAL ANKCCHVGCTKRSLARFCGGGSGGSGGGGSWMEEVIKLCGR ELVRAQIAICGMSTWS (SEQ ID NO: 37)

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

[0166] Example 15: Recombinant expression of RLN analog 15 Example 15 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQDSWMEEVIK LCGRELVRAQIAICGMSTWSSGGGGSGGGGQLYSALANKC CHVGCTKRSLARFC (SEQ ID NO: 38)

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

[0168] Example 16: Recombinant expression of RLN analog 16 Example 16 is an RLN analog having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPADS WMEEVIKLCGRELVRAQIAICGMSTWSSGGGGSGGGGQLY SALANKCCHVGCTKRSLARFC (SEQ ID NO: 39)

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

[0170] In vitro function Example 17: RLN analogue albumin binding via SPR RLN analogs in humans, cynomolgus monkeys, mice, rats, pigs, dogs, cattle, and rabbits The in vitro binding of these species to serum albumin is determined by SPR. The affinities of the RLN analogs of Examples 1 to 14 for serum albumin are summarized below in Tables 1 to 14. To promise.

[0171] The binding of the RLN analogs of Examples 1-14 to various serum albumins was measured using Biacore Performed with 8K equipment. Serum alginate on the surface of Series S Sensor Chip CM5 Immobilization of amine was performed according to the manufacturer's instructions (Amine Coupling Kit BR-10 00-50) is performed. Briefly, the sensor chip surface (flow cells 1 and 2) of carboxyl groups in a solution containing 75 mg / mL EDC and 11.5 mg / mL NHS Activation was performed by injecting 70 μL of the mixture at 10 μL / min. Serum albumins from mice, rats, pigs, dogs, cattle, and rabbits were diluted to 100% ethanol at pH 4.0. 0 mM sodium acetate (BR-1003-49), 0.5, 0.5, 2.2, 0.6, Dilutions of 0.6, 0.8, 0.6, and 0.3 μg / mL were then applied to the activated chip surface. (Flow cell 2, channels 1-8) were injected at 10 μL / min for 180 seconds (human, mouse) , rat, porcine, and bovine serum albumins were purchased from Sigma Aldrich (St. Serum albumin from cynomolgus monkeys was obtained from Holzel Diagrams, Inc. Dog serum albumin was obtained from Gnostika (Cologne, Germany). The lysine was obtained from Molecular Innovations (Novi, MI) and rabbit Guy serum albumin is from Fitzgerald Industries Intl. (Ac) Various serum albumins were obtained from the National Institute of Allergy and Immunology (NIA) at approximately 100 (62-145) RU. The sensor chip was coated with carboxymethyl dextran, aiming for a surface density of Covalently immobilized via free amines on the CM5 surface (flow cells 1 and 2). ) by injecting 70 μL of 1 M ETA HCl-NaOH at pH 8.5. It is inactivated by

[0172] The RLN analogs of Examples 1-14 were dissolved in HBS-EP+ buffer (10 mM HEPES p H7.6, 150mM NaCl, 3mM EDTA, 0.05% polysorbate 20) , 1000, 333.3, 111.1, 37.04, 12.35, 4.12, 1.37 Dilute to concentrations of 0.457, 0.152, 0.051, and 0.017 nM. 0 μl of sample was injected individually and sequentially across the immobilized serum albumin on the surface of the chip and incubated at 25°C. Dissociate for 600 seconds at a flow rate of 50 μL / min in 10 mM glycine-HCl, pH 1.5. The surface is regenerated by injecting (BR-1003-54) at 50 μL / min for 100 seconds. The resulting sensorgrams were analyzed using Biacore 8K Insight Evaluation 1:1 binding kinetic model in the software (version 2.0.15.12933) The binding rate parameters, association rate (kA), dissociation rate (kA), and Calculate the equilibrium dissociation constant (KD), and the equilibrium dissociation constant (KD). [Table 1]

[0173] K DThe RLN analogue of Example 1 was used in humans, cynomolgus monkeys, mice, rats, pigs, and Nu and bovine serum albumin binding was 0.1, 0.5, 3.3, and 2.0, respectively. The concentrations were determined to be 0.1, 7.1, 1.2, and 37 nM. [Table 2]

[0174] K D The RLN analogue of Example 2 was used in humans, cynomolgus monkeys, mice, rats, pigs, and Nu and bovine serum albumin binding were 0.1, 0.45, and 3.8, respectively. Determined to be 3.0, 8.0, 1.4, and 50 nM. [Table 3]

[0175] K D The RLN analogue of Example 3 was used in humans, cynomolgus monkeys, mice, rats, pigs, and The binding of 0.08, 0.36, and 2.4g to human serum albumin and bovine serum albumin, respectively. , 1.6, 4.8, 0.7, and 26 nM. [Table 4]

[0176] K D The RLN analogue of Example 4 was used in humans, cynomolgus monkeys, mice, rats, pigs, and The binding of 0.09, 0.42, and 3.0g of bovine serum albumin was observed, respectively. , 2.1, 6.2, 1.0, and 37 nM. [Table 5]

[0177] K DThe RLN analogue of Example 5 was used in humans, cynomolgus monkeys, mice, rats, pigs, and rabbits. The binding of guinea pig serum and bovine serum albumin was 0.45, 1.8, 1.4, and 1.0, respectively. Determined to be 0, 23, 4.7, and 120 nM. [Table 6]

[0178] K D The RLN analogue of Example 6 was used in humans, cynomolgus monkeys, mice, rats, pigs, and rabbits. Nu and bovine serum albumin binding was 0.51, 2.3, 12, and 9, respectively. The concentrations are determined to be 0.7, 24, 4.1, and 130 nM. [Table 7]

[0179] K D The RLN analogue of Example 7 was used in humans, cynomolgus monkeys, mice, rats, pigs, and The binding of 0.06, 0.29, and 2.1g to bovine serum albumin was observed, respectively. , 1.4, 3.7, 0.69, and 25 nM. [Table 8]

[0180] K D The RLN analogue of Example 8 was used in humans, cynomolgus monkeys, mice, rats, pigs, and Nu and bovine serum albumin binding was 0.08, 0.32, and 1.8, respectively. , 1.7, 4.1, 0.66, and 21 nM. [Table 9]

[0181] K DThe RLN analogue of Example 9 was used in humans, cynomolgus monkeys, mice, rats, pigs, and rabbits. Nu and bovine serum albumin binding was 0.19, 0.86, and 9.5, respectively. , 8.2, 14, 3.8, and 84 nM. [Table 10]

[0182] K D is the RLN analog of Example 10, and is effective in humans, cynomolgus monkeys, mice, rats, pigs, Binding to dog and bovine serum albumin was 0.37, 1.5, and 9.7, respectively. , 7.8, 20, 3.4, and 100 nM. [Table 11]

[0183] K D is the RLN analog of Example 11, and is effective in humans, cynomolgus monkeys, mice, rats, pigs, For dog and bovine serum albumin binding, 0.17, 0.48, and 3. The concentrations are determined to be 6, 1.3, 7.3, 1.3, and 27 nM. [Table 12]

[0184] K D is the RLN analog of Example 12, and is effective in humans, cynomolgus monkeys, mice, rats, pigs, For dog and bovine serum albumin binding, 0.14, 0.47, and 2. The concentrations are determined to be 6, 1.7, 4.2, 0.77, and 30 nM. [Table 13]

[0185] KD is the RLN analog of Example 13, and is effective in humans, cynomolgus monkeys, mice, rats, pigs, For dog and bovine serum albumin binding, 0.14, 0.49, and 2. The concentrations are determined to be 6, 2.0, 5.0, 0.92, and 29 nM. [Table 14]

[0186] K D is the RLN analog of Example 14, and is effective in humans, cynomolgus monkeys, mice, rats, pigs, For dog and bovine serum albumin binding, 0.16, 0.61, and 3. The concentrations are determined to be 1, 2.2, 6.0, 1.1, and 34 nM.

[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 20 mM DM containing 5% FBS with HEPES, 40 μg / mL L-proline, and 1x antibiotics The cells were cultured in EM-F12 3:1 and lysed in TrypLE™ Express (Gibco). Split cells 1:5 every 2-3 days at RT. Dissociate cells into pGL4.29 cells according to the manufacturer's instructions. [luc2P / CRE / Hygro] (Promega) and Fugene HD (P Transfect the cells with the plasmid DNA of romega. The clones were selected using 1 mg / mL hygromycin B for 3-4 weeks. Obtained by limiting dilution cloning into 96-well plates and labeled with Bright-Glo Re Forskolin was detected by luciferase assay using a 5'-hydroxybenzoate (Promega) The clones are expanded, harvested, resuspended in freezing medium, and cryovialed. The resulting mixture was aliquoted into 500 ml aliquots and kept in liquid nitrogen for long-term storage. For subsequent transfection with the inhibitor, the forskolin response is best ( The top responder (signal to background ratio) clonal line #2B6 is selected.

[0188] Generation of CHO human RXFP1 and human RXFP2 expressing cell lines: CHO-CRE-Lu c Strain #2B6 cells were cultured in 20 mM HEPES, 40 μg / mL L-proline, 1x antibiotic Substances: DMEM-F12 with 5% FBS containing 1 mg / mL hygromycin B 3: 1 and rehydrated every 2-3 days with TrypLE™ Express (Gibco). Split at 1:5. Cells were incubated with either human RXFP1 receptor or human R according to the manufacturer's instructions. Transfected with plasmid DNA of the XFP2 receptor and Fugene HD (Promega). The transfected cells were incubated for 3-4 weeks in the presence of hygromycin B (1 Select using 500 mg / mL of ATP and puromycin (6 μg / mL). The clonal lines were obtained by limiting dilution cloning into 96-well plates. The clones are expanded, harvested, resuspended in freezing medium, and cryovialized. The assay is then aliquoted and kept frozen under liquid nitrogen for long-term storage. Therefore, the clone with the best response (signal to background ratio) for human RLN2 was selected. Select a stock.

[0189] Human RXFP1 and RXFP2 receptor luciferase assay: Human RXFP1 or A CHO cell line expressing human RXFP2 was cultured in selective medium (20 mM HEPES, 40 μL) g / mL L-proline, 1x antibiotics, 6 μg / mL puromycin, 1 mg / mL Culture in DMEM-F12 (3:1) containing 5% FBS and hygromycin B. On day 1 (the day before the cAMP CRE luciferase reporter assay), cells were incubated with PBS. Wash once and add cell dissociation solution (enzyme-free cell dissociation solution, GIBCO catalog number 13151 -014:TrypLE™ Express = 30:1) to float it from the flask and Incubation medium (DME containing 20 mM HEPES, 1x antibiotics, 0.5% FBS) The cells were resuspended in M-F12 3:1. The cells were plated in a 96-well plate (Falcon Catalog). Plate 20,000 cells / 0.1 mL / well onto a 100% PBS-containing plate (product number 353219). The cells are cultured overnight at 37°C, 5% CO2. Day 1 (cAMP CRE luciferase On the day of the reporter assay, remove the medium and replace with 90 µL of serum-free medium (20 mM HEP Replace the plate with DMEM-F12 (3:1) containing ES and 1x antibiotics. Keep the plate at 37°C. After 2 hours of incubation, add 10 μL of 10x ligand (RLN2, final 1x ) Incubate the plate at 37°C for an additional 4 hours. After the incubation is complete, allow the plate to come to room temperature for 15 minutes. Then, add 50 μL of Bright-Glo (TM) was added to each well and the plate was incubated in a Biotek HPLC system equipped with Gen5 software. Read with Neo2 reader.

[0190] Statistical analysis of data: Data were transferred from the Biotek Neo2 reader to GraphPad Prism® software (GraphPad Software, LLC) Import into EC (La Jolla, CA; version 7). 50 Values, variable gradient Generated by a four-parameter dose-response curve. [Table 15]

[0191] In vitro function Example 19: Pharmacokinetics of RLN analogs in male Sprague Dawley rats Male Sprague Dawley rats were given His-NaCl buffer (pH 6.0) A single SQ dose of 200 nmol / kg of RLN analogue in a volume of 1.0 mL / kg To evaluate the pharmacokinetic properties, the following doses were administered: 3, 6, 12, 24, 48, 72, 96, and 120 days after administration. Blood samples are taken at 144, 168, and 240 hours.

[0192] Plasma concentrations of RLN analogs were measured using a clinically approved The internal standard for the example compounds and analogs is determined by an LC / MS method using human RL70. Extraction from 100% rat plasma using N antibody followed by Q-Exactive™ Detecting N-terminal tryptic peptides using an Orbitrap® mass spectrometer do.

[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 observation Plasma concentration, AUC 0-inf= area under the curve from time 0 to infinity, CL / F = clearance Efficiency / bioavailability, N=3 animals / group / time point.

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

[0196] Example 20: IV administration of RLN analogs in male Sprague Dawley rats In vivo effects on subsequent renal blood flow Male 5-week-old Sprague Dawley rats (Charles River L The mice (aborators, Inc.) were kept under a normal light-dark cycle for one week before the start of the experiment. The rats were then placed in a rearing room. (pH 6.0 buffer) and randomized to treatment with the RLN analog of Example 7 based on body weight. The RLN analogue was administered as an IV bolus of 2.44 μg / kg followed by 0.36 μg / kg g / hour IV infusion, followed by an 8.13 μg / kg IV bolus and 1.2 μg / kg / hour IV infusion, 24.4 μg / kg IV bolus followed by 3.6 μg / kg / hour IV infusion, and an IV bolus of 81.3 μg / kg followed by 11.9 μg / kg / hour I Administer medication via V injection.

[0197] To measure the effect of RLN analogs on renal blood flow, rats were treated with urethane (1.2 g / The animals were anesthetized with 100 mg / kg IP and underwent abdominal / renal ultrasound imaging and renal artery pulsed wave Doppler blood flow measurement. VisualSonics, Model Vevo 3100 ultrasound system, Fu A chronic tail vein catheter is prepared for IV bolus and infusion administration. After a 30-minute acclimation period, baseline and 3-hour post-dose renal function tests were performed. Blood flow measurements are obtained. [Table 17]

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

[0199] As seen in Table 17, the RLN analog of Example 7 resulted in an IV bolus followed by 1.2 μg / kg / hour IV infusion, 24.4 μg / kg IV bolus followed by an IV infusion of 3.6 μg / kg / hour and an IV bolus of 81.3 μg / kg. Renal blood flow in the group treated with 11.9 μg / kg / hour IV infusion was significantly higher during the 3 hours of exposure. Later, there was a significant increase of 26.6, 48.8, and 60.4%.

[0200] Example 21: RLN analogues administered SQ in male Sprague Dawley rats In vivo effects on subsequent renal blood flow Male 5-week-old Sprague Dawley rats (Charles River L The mice (aborators, Inc.) were kept under a normal light-dark cycle for one week before the start of the experiment. The rats were then placed in a rearing room. (pH 6.0 buffer) and randomized to treatment with the RLN analog of Example 7 based on body weight. The RLN analogue is administered at 180 μg / kg SQ.

[0201] To measure the effect of RLN analogs on renal blood flow, rats were treated 48 hours after administration. The subjects were anesthetized with urethane (1.2 g / kg, IP), and abdominal / renal ultrasound imaging and renal artery pulsation were performed. Wave Doppler blood flow measurement (VisualSonics, Model Vevo 3100 After a 30-minute acclimation period, the renal blood flow was measured. A volume measurement is obtained. [Table 18]

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

[0203] array The following nucleic acid and / or amino acid sequences are referred to in this disclosure and are provided for reference: Provided below. SEQ ID NO:1 - Human pro-RLN1 MPRLFLFHLLEFCLLLNQFSRAVAAKWKDDVIKLCGRELV RAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKD TETIIIMLEFIANLPPELKAALSERQPSLPELQQYVPALK DSNLSFEEFKKLIRNRQSEAADSNPSELKYLGLDTHSQKK RRPYVALFEKCCLIGCTKRSLAKYC 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 MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELV RAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKD TETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLK DSSLLFEEFKKLIRNRQSEAADSPSELKYLGLDTHSRKK RQLYSALANKCCHVGCTKRSLARFC 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 MARYMLLLLLAVWVLTGELWPGAEARAAPYGVRLCGREFI RAVIFTCGGSRWRRSDILAHEAMGDTFPDADADEDSLAGE LDEAMGSSEWLALTKSPQAFYRGRPSWQGTPGVLRGSRDV LAGLSSSCCKWGCSKSEISSLC 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) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO:11 - VHH portion #2 (C22.43) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPP SEQ ID NO:12 - VHH portion #3 (C80) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO: 13 - VHH portion #4 (C80.43) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPP 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) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQQLYSALANK CCHVGCTKRSLARFCGGGSGGSGGGGSWMEEVIKLCGRELV RAQIAICGMSTWS SEQ ID NO:25 -RLN2 analog #2 (C22-(PGPA)8-A10B(desB1 )RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAQL YSALANKCCHVGCTKRSLARFCGGGSGGSGGGGSWMEEVIK LCGRELVRAQIAICGMSTWS Array No. 26 - Relaxin-2 analog #3 (C22-(G4Q)5-B10A(desB1) RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKL CGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCC HVGCTKRSLARFC Array No. 27 - Relaxin-2 analog #4 (C22-(PGPQ)8-B10A(desB1 )RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY [[ID=2,4]]LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSW MEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYS ALANKCCHVGCTKRSLARFC Array No. 28 - Relaxin-2 analog #5 (B10A(desB1)RLN-(G4Q)5- C22.43) SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQL YSALANKCCHVGCTKRSLARFCGGGGQGGGGQGGGGQGGG GQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETA VAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRD NSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLY PYWGQGTLVTVSSPP SEQ ID NO: 29 - RLN2 analog #6 (B10A(desB1)RLN-(PGPQ)8 -C22.43) SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQL YSALANKCCHVGCTKRSLARFCPGPQPGPQPGPQPGPQPG PQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLSCAASG RYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKG RFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLIT SKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 30 - RLN2 analog #7 (C80-(G4Q)5-B10A(desB1) RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA<00> PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKL CGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCC HVGCTKRSLARFC SEQ ID NO: 31 - RLN2 analog #8 (C80-(PGPQ)8-B10A(desB1<000> )RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSW MEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYS ALANKCCHVGCTKRSLARFC SEQ ID NO: 32 - RLN2 analog #9 (B10A(desB1)RLN-(G4Q)5 - C80.43) SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQL[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKL CGRELVRAQIAICGMSTWSGGGSGGSGGSGGGALANKCCH VGCTKRSLARFC SEQ ID NO: 35 - RLN2 analog #12 (C80-(G4Q)5-B13A(desB1 、desA1~4)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKL CGRELVRAQIAICGMSTWSGGGSGGSGGSGGGALANKCCH VGCTKRSLARFC SEQ ID NO: 36 - RLN2 analog #13 (C80-(G4Q)5-A10B(desB1 [[ID=3l]]、desA1~4)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQALANKCCHV GCTKRSLARFCGGGSGGSGGGSWMEEVIKLCGRELVRAQI AICGMSTWS SEQ ID NO:37 - RLN2 analog #14 (C80-(PGPQ)8-A10B(desB 1, desA1-4)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQAL ANKCCHVGCTKRSLARFCGGGSGGSGGGGSWMEEVIKLCGR ELVRAQIAICGMSTWS SEQ ID NO:38 - RLN2 analog #15 (C22-(G4Q)5-B10ARLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQDSWMEEVIK LCGRELVRAQIAICGMSTWSSGGGGSGGGGQLYSALANKC CHVGCTKRSLARFC SEQ ID NO:39 - RLN2 analog #16 (C22-(PGPA)8-B10ARLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPADS WMEEVIKLCGRELVRAQIAICGMSTWSSGGGGSGGGGQLY SALANKCCHVGCTKRSLARFC SEQ ID NO: 40 - Human RXFP1 receptor MTSGSVFFYILIFGKYFSHGGGQDVKCSLGYFPCGNITKC LPQLLHCNGVDDCGNQADEDNCGDNNGWSLQFDKYFASYY KMTSQYPFEAETPECLVGSVPVQCLCQGLELDCDETNLRA VPSVSSNVTAMSLQWNLIRKLPPDCFKNYHDLQKLYLQNN KITSISIYAFRGLNSLTKLYLSHNRITFLKPGVFEDLHRL EWLIIEDNHLSRISPPTFYGLNSLILLVLMNNVLTRLPDK PLCQHMPRLHWLDLEGNHIHNLRNLTFISCSNLTVLVMRK NKINHLNENTFAPLQKLDELDGSNKIENLPPLIFKDLKE LSQLNLSYNPIQKIQANQFDYLVKLKSLSLEGIEISNIQQ RMFRPLMNLSHIYFKKFQYCGYAPHVRSCKPNTDGISSLE NLLASIIQRVFVWVVSAVTCFGGNIFVICMRPYIRSENKLY AMSIISLCCADCLMGIYLFVIGGFDLKFRGEYNKHAQLWM ESTHCQLVGSLAILSTEVSVLLLTFLTLEKYICIVYPFRC VRPGKCRTITVLILIWITGFIVAFIPLSNKEFFKNYYGTN GVCFPLHSEDTESIGAQIYSVAIFLGINLAAFIIIVFSYG SMFYSVHQSAITATEIRNQVKKEMILAKRFFFIVFTDALC WIPIFVVKFLSLLQVEIPGTITSWVVIFILPINSALNPIL YTLTTRPFKEMIHRFWYNYRQRKSMDSKGQKTYAPSFIWV EMWPLQEMPPELMKPDLFTYPCEMSLISQSTRLNSYS SEQ ID NO: 41 - Human RXFP2 receptor MIVFLVFKHLFSLRLITMFFLLHFIVLINVKDFALTQGSM ITPSCQKGYFPCGNLTKCLPRAFHCDGKDDCGNGADEENC GDTSGWATIFGTVHGNANSVALTQECFLKQYPQCCDCKET ELECVNGDLKSVPMISNNVTLLSLKKNKIHSLPDKVFIKY TKLKKIFLQHNCIRHISRKAFFGLCNLQILYLNHNCITTL RPGIFKDLHQLTWLILDDNPITRISQRLFTGLNSLFFLSM VNNYLEALPKQMCAQMPQLNWVDLEGNRIKYLTNSTFLSC DSLTVLFLPRNQIGFVPEKTFSSLKNLGELDLSSNTITEL SPHLFKDLKLLQKLNLSSNPLMYLHKNQFESLKQLQSLDL ERIEIPNINTRMFQPMKNLSHIYFKNFRYCSYAPHVRICM PLTDGISSFEDLLANNILRIFVWVIAFITCFGNLFVIGMR SFIKAENTTHAMSIKILCCADCLMGVYLFFVGIFDIKYRG QYQKYALLWMESVQCRLMGFLAMLSTEVSVLLLTYLTLEK FLVIVFPFSNIRPGKRQTSVILICIWMAGFLIAVIPFWNK DYFGNFYGKNGVCFPLYYDQTEDIGSKGYSLGIFLGVNLL AFLIIVFSYITMFCSIQKTALQTTEVRNCFGREVAVANRF FFIVFSDAICWIPVFVVKILSLFRVEIPDTMTSWIVIFFL PVNSALNPILYTLTTNFFKDKLKQLLHKHQRKSIFKIKKK SLSTSIVWIEDSSSLKLGVLNKITLGDSIMKPVS SEQ ID NO: 42 - Human RXFP3 receptor MQMADAATIATMNKAAGGDKLAELFSLVPDLLEAANTSGN ASLQLPDLWWELGLELPDGAPPGHPPGSGGAESADTEARV RILISVVYWVVCALGLAGNLLVLYLMKSMQGWRKSSINLF VTNLALTDFQFVLTLPFWAVENALDFKWPFGKAMCKIVSM VTSMNMYASVFFLTAMSVTRYHSVASALKSHRTRGHGRGD CCGRSLGDSCCFSAKALCVWIWALAALASLPSAIFSTTVK VMGEELCLVRFPDKLLGRDRQFWLGLYHSQKVLLGFVLPL GIIILCYLLLVRFIADRRAAGTKGGAAVAGGRPTGASARR LSKVTKSVTIVVLSFFLCWLPNQALTTWSILIKFNAVPFS QEYFLCQVYAFPVSVCLAHSNSCLNPVLYCLVRREFRKAL KSLLWRIASPSITSMRPFTATTKPEHEDQGLQAPAPPHAA AEPDLLYYPPGVVVYSGGRYDLLPSSSAY SEQ ID NO: 43 - Human RXFP4 receptor MPTLNTSASPPTFFWANASGGSVLSADDAPMPVKFLALRL MVALAYGLVGAIGLLGNLAVLWVLSNCARRAPGPPSDTFV FNLALADLGLALTLPFWAAESALDFHWPFGGALCKMVLTA TVLNVYASIFLITALSVARYWVVAMAAGPGTHLSLFWARI ATLAVWAAAALVTVPTAVFGVEGEVCGVRLCLLRFPSRYW LGAYQLQRVVLAFMVPLGVITTSYLLLLAFLQRRQRRRQD SRVVARSVRILVASFFLCWFPNHVVTLWGVLVKFDLVPWN STFYTIQTYVFPVTTCLAHSNSCLNPVLYCLLRREPRQAL AGTFRDLRLRLWPQGGGWVQQVALKQVGRRWVASNPRESR PSTLLTNLDRGTPG SEQ ID NO: 44 - Signal peptide METDTLLLWVLLLWVPGSTG SEQ ID NO: 45 - VHH portion #5 (MC6.1) EVQLLESGGGLVQPGGSLRLSCAASGRTVSSTAVAWFRQA PGKEREFVAGIGGSVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGT LVTVSS SEQ ID NO: 46-VHH part #6 (MC6.1C6) EVQLLESGGGLVQPGGSLRLSCAASGRYIDSTAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSRVANLYPYWGQGT LVTVSS SEQ ID NO: 47 - VHH portion #7 (C22-G26Y) EVQLLESGGGLVQPGGSLRLSCAASYRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO:48 - VHH portion #8 (C22-R27A) EVQLLESGGGLVQPGGSLRLSCAASGAYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO: 49 - VHH portion #9 (C22-I57E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDETYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO:50 - VHH portion #10 (C22-I57Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDQTYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO:51 - VHH portion #11 (C22-Y59A) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITAYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO:52 - VHH portion #12 (C22-Y59E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITEYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO:53 - VHH portion #13 (C22-Y59Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITQYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO:54 - VHH portion #14 (C22-Y59S) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITSYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO:55 - VHH portion #15 (C22-Y59T) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITTYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO:56 - VHH portion #16 (C22-R102K) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGKPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO:57 - VHH portion #17 (C22-R102Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGQPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO:58 - VHH portion #18 (C22-R102S) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGSPLITSKVADLYPYWGQGT LVTVSS SEQ ID NO:59 - VHH portion #19 (C22-P103E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRELITSKVADLYPYWGQGT LVTVSS SEQ ID NO:60 - VHH portion #20 (C22-P103Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRQLITSKVADLYPYWGQGT LVTVSS SEQ ID NO: 61 - VHH portion #21 (C22-P103S) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRSLITSKVADLYPYWGQGT LVTVSS SEQ ID NO: 62 - VHH portion #22 (C22-L104E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPEITSKVADLYPYWGQGT LVTVSS SEQ ID NO: 63 - VHH portion #23 (C22-L104G) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPGITSKVADLYPYWGQGT LVTVSS SEQ ID NO: 64 - VHH portion #24 (C22-L104Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPQITSKVADLYPYWGQGT LVTVSS SEQ ID NO: 65 - VHH portion #25 (C22-L104T) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPTITSKVADLYPYWGQGT LVTVSS SEQ ID NO: 66 - VHH portion #26 (C22-S107E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQA PGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITEKVADLYPYWGQGT LVTVSS SEQ ID NO: 67-L2#3 SGGGGSGGGG

Claims

1. The following structure VHH-L 1 -BL 2 -A, A compound comprising the VHH comprises the amino acid sequence of SEQ ID NO: 12; A is SEQ ID NO: 5, B is SEQ ID NO: 6 and lacks the first amino acid (desB1), L 1 is SEQ ID NO: 19, and L 2 is SEQ ID NO: 22; The compound, or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable buffer.

3. A pharmaceutical composition for treating a cardiac, pulmonary, and / or renal condition, disease, and / or disorder in an individual, comprising an effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof.

4. A pharmaceutical comprising a compound or a pharmaceutically acceptable salt of claim 1 for use in therapy.

5. A pharmaceutical comprising the compound or pharmaceutically acceptable salt of claim 1 for use in treating cardiac, pulmonary, and / or renal conditions, diseases, and / or disorders.

6. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a pharmaceutical for the treatment of cardiac, pulmonary, and / or renal conditions, diseases, and / or disorders.

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