EA046279B1 - ANALOGUES OF RELAXIN AND METHODS OF THEIR USE - Google Patents

Description

The disclosure relates generally to biology and medicine and, in particular, to relaxin analogs (RLNrelaxin), especially long-acting single-chain RLN analogs that can bind to the RLN/insulin-like family peptide receptor (RXFP-RLN/insulin-like family peptide). , such as the RXFP1 receptor, thereby acting as RXFP receptor agonists. In addition, the disclosure relates to compositions containing these analogs and their use for the treatment of pathological conditions, diseases or disorders of the cardiovascular system, lungs and/or kidneys.

Relaxins (RLNs) are part of the insulin superfamily and in humans include seven peptides with high structural similarity but low sequence similarity - RLN1 (H1RLX, RLXH1 or H1), RLN2 (H2RLX, RLXH2 or H2), RLN3 (RXN3, ZINS4 or H3) , insulin-like (INSL-insulin-like) peptide 3 (INSL3), INSL4, INSL5 and INSL6. Of particular interest here is RLN2, which is a heterodimer of two peptide chains of twenty-four and twenty-nine amino acids (A-chain and B-chain), respectively, linked by two disulfide bonds, with the A-chain additionally having one intramolecular disulfide connection (see Schwabe & McDonald (1977) Science 197:914-915). RLN2 is formed from its prohormone, prorelaxin, by cleavage of its C-peptide.

Physiologically, RLN2 has a diverse set of functions that regulate adaptations in the cardiovascular system, liver, nervous system, pancreas, lung and kidney, such as vasodilatory, antifibrotic and angiogenic effects, although it was originally described as a pregnancy hormone. RLN2 signaling occurs through two different classes of G protein-coupled receptors (GPCRs), namely the leucine-rich repeat GPCRs, LGR7 and LGR8, now called RXFP1 and RXFP2 receptors, respectively. Two other receptors in this family include the RXFP3 and RXFP4 receptors. RLN2 has a short half-life (t ¹ /2), which makes it difficult to use as a therapeutic agent. In fact, the t ¹ /2 of native RLN2 in vivo is minutes. Consequently, practitioners administer RLN2 by continuous intravenous infusion, which often results in inconvenience for people receiving the RLN compound and short-term effectiveness.

There are a number of RLN2 analogues with improved t ¹ /2. For example, International Patent Application Publication No. WO 2018/148419 describes analogs that include a non-native amino acid residue, such as para-acetyl-phenylalanine, to which linkers, polymers and/or pharmacokinetic enhancers can be attached to improve t ¹ /2. International Patent Application Publication No. WO 2018/138170 describes analogues that are A-chain and B-chain fusions containing a linker of at least fifteen amino acids and a half-life extending moiety to improve t1/ ₂ . International Patent Application Publication No. WO 2017/201340 describes analogues that are A-chain-B-chain fusions having a light chain variable region fragment to improve t1/ ₂ . International Patent Application Publication No. WO 2015/067791 describes analogs that are prodrugs linked to a carrier, particularly PEG-based carriers, to improve t1/ ₂ (see also WO 2012/024452 for additional PEG-linked analogs) . International Patent Application Publication No. WO 2014/102179 describes analogues that are A-chain-B-chain fusions containing an IgG2 or IgG4 Fc fragment to improve t1/ ₂ . International Patent Application Publication No. WO 2013/004607 describes analogues which are A-chain and B-chain fusions containing a linker of at least five amino acids but less than fifteen amino acids to improve t1/ ₂ , or which are A-chain and B-chain fusions containing the Fc domain of antibodies to improve t1/ ₂ .

With increasing understanding of the various physiological functions of RLN, there remains a need for long-acting RLN analogues having improved t1/ ₂ .

To address this issue, the disclosure first describes single-chain RLN analogues having primary activity at the RXFP1 receptor (ie, acting as RXFP1 receptor agonists). Such RXFP1 receptor agonists contain a basic structure from the amino terminus (N-terminus) to the carboxy terminus (C-terminus):

VHH-L1-A-L2-B;

VHH-L1-B-L2-A;

A-L2-B-L1-VHH; or

B-L2-A-L1-VHH;

where VHH is a moiety that can act as a pharmacokinetic enhancer, A is an RLN A chain, B is an RLN B chain, L1 is a first linker, and L2 is a second linker.

In some cases, the VHH fragment may have the amino acid sequence of SEQ ID NO: 10, 11, 12 or 13, especially SEQ ID NO: 10 or 12. In other cases, the VHH fragment may contain one or more additions, deletions, insertions or substitutions, such as that the VHH fragment has an amino acid sequence having at least about 90% to about 99% sequence similarity to any of SEQ ID NO: 10, 11, 12 or 13 (see, for example, SEQ ID NO: 10, 11, 12 or 13

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NO: 45-66).

In some cases, the A chain may have an amino acid sequence of SEQ ID NO: 2, 5 or 8, particularly the sequence of SEQ ID NO: 5. In other cases, the A chain may contain one or more additions, deletions, insertions or substitutions, such that The A chain has an amino acid sequence having at least about 90% to about 99% sequence similarity to any of SEQ ID NOs: 2, 5, or 8. For example, the A chain may be the sequence des1-4 of SEQ ID NO: 5.

In some cases, the B chain may have the amino acid sequence of SEQ ID NO: 3, 6 or 9, especially SEQ ID NO: 6. In other cases, the B chain may contain one or more additions, deletions, insertions or substitutions, such that B The β-chain has an amino acid sequence having at least about 90% to about 99% sequence similarity to any of SEQ ID NO: 3, 6, or 9. For example, the B-chain may be the sequence desl SEQ ID NO: 6.

In some cases, L1 may have the amino acid sequence (GGGGQ) _n (SEQ ID NO: 14), (GGGQ) _n (SEQ ID NO: 15), (GGGGS) _n (SEQ ID NO: 16), (PGPQ) _n (SEQ ID NO: 17) or (PGPA) _n (SEQ ID NO: 18), where n can be from 1 to 10, especially from about 5 to about 8. In other cases, L1 may have the amino acid sequence of SEQ ID NO: 19, 20 or 21. In other cases, L1 may contain one or more additions, deletions, insertions or substitutions such that _L1 has an amino acid sequence having at least about 90% to about 99% sequence similarity to any of the sequences of SEQ ID NO: 14-21.

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

In some cases, RLN analogues may have an amino acid sequence that contains VHH with the sequence SEQ ID NO: 10, 11, 12 or 13; A chain having the sequence SEQ ID NO: 2, 5 or 8; B chain having the sequence SEQ ID NO: 3, 6 or 9; L1 with the sequence SEQ ID NO: 19, 20 or 21; and L2 with the sequence SEQ ID NO: 22, 23, or 67. Alternatively, the RLN analogues may have an amino acid sequence having at least about 90% to about 99% sequence similarity to an amino acid sequence that contains an amino acid sequence containing VHH with the sequence SEQ ID NO: 10, 11, 12 or 13; A chain having the sequence SEQ ID NO: 2, 5 or 8; B chain having the sequence SEQ ID NO: 3, 6 or 9; L1 with the sequence SEQ ID NO: 19, 20 or 21; and L2 with SEQ ID NO: 22, 23 or 67.

In specific cases, RLN analogues may have the amino acid sequence SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39, especially SEQ ID NO : 26, 27, 30, 31, 34 or 35. Alternatively, the RLN analogues may have an amino acid sequence having at least about 90% to about 99% sequence similarity to the amino acid sequence of SEQ ID NO: 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39, especially SEQ ID NO: 26, 27, 30, 31, 34 or 35.

In some cases, the RLN analogues have binding affinities for the RXFP1 receptor comparable to those of native human RLN2 (SEQ ID NOs: 5 and 6). In other cases, RLN analogues have binding affinities for the RXFP1 receptor that are higher than those of native human RLN2 (SEQ ID NOs: 5 and 6). In other cases, RLN analogues have binding affinities for the RXFP1 receptor that are less than those of native human RLN2 (SEQ ID NOs: 5 and 6). In some cases, the RLN analogues have a ^t1 / ₂ that is longer than that of native human RLN2 (SEQ ID NOs: 5 and 6), including about 20 days to about 30 days longer when administered to humans.

The above compositions may alternatively be nucleic acid sequences encoding the amino acid sequences described herein, as well as vectors and host cells that contain such sequences, for the expression of the RLN analogues described herein.

Secondly, pharmaceutical compositions are described that contain at least one RLN analogue described herein, or a pharmaceutically acceptable salt thereof (eg, trifluoroacetate salts, acetate salts or hydrochloride salts) and a pharmaceutically acceptable carrier. In some cases, the pharmaceutically acceptable carrier is a buffer, such as, for example, saline, phosphate buffered saline, citrate buffered saline, or histidine buffered saline. In some cases, the buffer is histidine, histidine buffer, or histidine-buffered saline. In other cases, the pharmaceutical compositions may further include carriers, diluents and/or excipients.

Moreover, the pharmaceutical compositions may include at least one additional therapeutic agent, such as, for example, an agent used as a standard for treating a cardiovascular, pulmonary, and/or renal condition, disease, or disorder. In some cases, at least one additional therapeutic agent may

- 2 046279 is an anticoagulant, angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB-angiotensin II receptor blocker), ARB/neprilysin inhibitor (ARNI-angiotensin receptor II blocker-neprilysin inhibitor), β-blocker, diuretic, digitalis, digoxin, hydralazine/isorbide dinitrate, mineralocorticoid receptor antagonist (MCRA; or aldosterone antagonist), sodium glucose transporter type 2 (SGLT2) inhibitor, statin and/or antiglycemic agent.

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

In some cases, the RLN analogue can be administered by any standard route of administration, such as, for example, intramuscularly, intravenously, parenterally, subcutaneously or transdermally. In some cases, the RLN analogue is administered subcutaneously (SC), intramuscularly (IM), or intravenously (IV). In specific cases, the RLN analogue can be administered to the individual SC or IV.

Likewise, and in some cases, the RLN analogue can be administered daily, every other day, three times a week, twice a week, once a week (i.e., weekly), once every two weeks (i.e., every two weeks), once a month (i.e. monthly), once every two months (i.e. every two months) or even every three months. In some cases, the RLN analog may be administered SC every other day, SC three times a week, SC twice a week, SC once a week, SC every two weeks, or SC once every month. In specific cases, the RLN analogue is administered subcutaneously once a week (once a week, QW).

Alternatively, the RLN analogue can be administered to the individual intravenously. As stated above, the RLN analogue can be administered daily, every other day, three times a week, twice a week, once a week (i.e., weekly), biweekly (i.e., every two weeks), or monthly . In some cases, the RLN analog may be given IV every other day, IV three times a week, IV twice a week, IV once a week, IV every two weeks, or IV once every month. In specific cases, an RNL analogue is administered intravenously once a week. The methods may also include the step of administering an RLN analog in combination with an effective amount of at least one additional therapeutic agent. In brief, the standard of care for many of the conditions/diseases/disorders described herein includes an anticoagulant, ACE inhibitor, ARB, ARNI, β-blocker, diuretic, digitalis, digoxin, hydralazine/isosorbitol dinitrate, AMKR or other aldosterone antagonist, SGLT2 inhibitor , a statin and/or antiglycemic agent, as well as other therapeutic agents to control comorbidities including, but not limited to, high cholesterol, high blood pressure, atrial fibrillation and diabetes. In some cases, the additional therapeutic agent may be administered simultaneously, separately, or sequentially with the RLN analogue.

For example, the additional therapeutic agent can be administered at the same frequency as the RLN analogue (ie, every other day, twice a week, weekly, or even monthly). In other cases, the additional therapeutic agent may be administered at a different frequency than the RLN analogue. In some cases, an additional therapeutic agent can be administered subcutaneously or intravenously. In other cases, the RLN analogue is administered SC, and the additional therapeutic agent can be administered orally or IV. Alternatively, the RLN analogue is administered IV and the additional therapeutic agent is administered SC.

In some cases, the individual in need is diabetic, hypertensive with impaired renal function, and/or obese. The methods may also include the steps of measuring or obtaining blood pressure data and comparing such obtained values with one or more baseline values or previously obtained values to evaluate the effectiveness of the treatment/therapy.

The methods may also be combined with diet and exercise and/or may be combined with additional therapeutic agents other than those discussed above.

Fourth, this document describes applications that include at least one of the RLN analogues described herein. For example, the RLN analogues described herein may be provided for use in therapy, particularly in the treatment of cardiovascular, pulmonary and/or renal conditions, diseases or disorders. RLN analogs may optionally be administered simultaneously, separately, or sequentially (ie, in combination) with at least one additional therapeutic agent.

Likewise, the use of the RLN analogues described herein is proposed in the manufacture of a medicament for the treatment of pathological conditions, diseases or disorders of the cardiovascular system, lungs and/or kidneys, wherein the medicament may optionally further include one or more additional therapeutic agents , as mentioned above.

Fifth, this document provides a compound that contains an amino acid after

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EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO: 10)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 10. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSSPP (SEQ Ш NO: И)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 11. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS (SEQ ID NO: 12)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 12. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSSPP (SEQ Ш NO: 13)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 13. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWG

QGTLVTVSS (SEQ ID NO:45)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 45. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDSTAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSRVANLYPYWG

QGTLVTVSS (SEQ ID NO:46)

In some cases, a compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 46. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASYRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:47)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 47. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGAYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:48)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 48. Alternatively, there is provided a compound that contains the amino acid sequence:

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EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDET

YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYW GQGTLVTVSS (SEQ Ш NO:49)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 49. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDQT

YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYW

GQGTLVTVSS (SEQ Ш NO:50)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 50. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITA

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:51)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 51. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITE

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:52)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 52. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITQ

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:53)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 53. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITS

YAD S VKGRFTISRDNSKNTL YLQMNSLRPEDT AVYYC AARPGRPLIT SK VADLYP YWG

QGTLVTVSS (SEQ ID NO:54)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 54. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITT

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:55)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 55. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGKPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:56)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 56. Alternatively, there is provided a compound that contains the amino acid sequence:

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EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGQPLITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:57)

In some cases, a compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 57. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGSPLITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:58)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 58. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRELITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:59)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 59. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRQLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:60)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 60. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YAD S VKGRFTISRDNSKNTL YLQMNSLRPEDT AVYYC AARPGRSLIT SK VADLYP YWG

QGTLVTVSS (SEQ ID NO:61)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 61. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPEITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:62)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 62. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPGITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:63)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 63. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPQITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:64)

In some cases, a compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 64. Alternatively, there is provided a compound that contains the amino acid sequence:

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EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPTITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:65)

In some cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 65. Alternatively, there is provided a compound that contains the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITEKVADLYPYWG

QGTLVTVSS (SEQ ID NO:66)

In some cases, a compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 66. An advantage of the RLN analogs described herein is that they can be chemically or recombinantly synthesized as a single chain polypeptide (i.e., monomeric) and thus do not require endoproteolytic processing to provide biological activity. However, it is believed that in some cases the VHH fragment may be conjugated not only to single-stranded RNLs, but also to double-stranded RLNs (eg, native ones). On the VHH fragment, the polypeptide can be conjugated not only to the N- and C-terminus, but also to any exposed VHH amino acid (provided that such conjugation does not completely abolish albumin binding).

The advantage of the RLN analogues described herein is that VHH fragments can be used not only with native A-chain and B-chain sequences, but also with modified sequences thereof. Moreover, VHH fragments can be further modified to obtain enhanced or additional functionality by fusing other peptides/proteins or small molecules attached to the VHH fragments. The advantage of the RLN analogues described herein is that the VHH fragments provide an extended duration of action in mammals such as humans and can have a t1/ ₂ of about 20 days to about 30 days, allowing them to be administered at least , once weekly or once every two weeks compared to native human RLN, particularly native human RLN2 (SEQ ID NOs: 5 and 6), which may improve compliance.

The advantage of the RLN analogues described herein is that they have similar or better selectivity, affinity and/or potency for RXFP1 receptors than RXFP2 receptors compared to native human RLN2 (SEQ ID NO: 5 and 6) . Alternatively, the RLN analogs described herein result in sufficient activity of the RXFP1 receptor and reduced or insufficient activity of one or more of the RXFP2, RXFP3 and RXFP4 receptors. The advantage of the RLN analogues described herein is that they have tunable pharmacokinetics achieved by altering the albumin affinity of the VHH moieties.

An advantage of the RLN analogs described herein is that they have improved stability in canned preparation compared to native human RLN2 (SEQ ID NOs: 5 and 6) or Fc fusion RLN analogs.

Moreover, the advantage of VHH fragments is that they have equal binding not only to human serum albumin, but also to canine, monkey, mouse, pig and rat serum albumin, allowing pharmacodynamic, pharmacokinetic and toxicological studies to be carried out for easier transition from these species on people.

The advantage of VHH fragments is that not only can they be used to improve t1/ ₂ of the RLN analogues described herein compared to native human RLN2 (SEQ ID NO: 5 and 6), but they can also be used to improve t1 / ₂ other biologically active peptides and proteins, such as, for example, insulin, growth and differentiation factor 15 (GDF15) or glucose-dependent insulinotropic peptide 1 (GLP-1).

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice or research of RLN analogues, pharmaceutical compositions and methods, preferred methods and materials are described herein.

In addition, mentioning an element in the singular does not exclude the possibility of the presence of more than one of the elements unless the context clearly suggests the presence of one and only one of the elements. Accordingly, singular terms usually mean at least one.

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Definitions

As used herein, the term "about" means within a statistically significant range of value or values, such as, for example, stated concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc. Such value or range may be within the order of, typically within 20%, more typically within 10%, and even more typically within 5% of the given value or range. The tolerance covered by the term about will depend on the particular system under study and can be readily estimated by one skilled in the art.

As used herein and with respect to one or more RXFP receptors, the term activity, activate, activating, and the like means the activity of a compound, e.g., the RLN analogues described herein, in binding to and inducing a response at their receptor(s), as measured by using assays known in the art, such as the in vitro assays described below.

As used herein, the term amino acid means a molecule that is chemically characterized by the presence of one or more amino groups and one or more carboxylic acid groups and may contain other functional groups. As is known in the art, there are a set of twenty amino acids that are designated as standard amino acids and can be used as building blocks for most peptides/proteins produced by any living thing. The amino acid sequences of this disclosure contain standard one-letter or three-letter codes for twenty standard amino acids.

As used herein, the term analog means a compound, such as a synthetic peptide or polypeptide, that activates a target receptor and causes at least one in vivo or in vitro effect caused by a native agonist of that receptor.

As used herein, the term conservative substitution means a variant of a reference peptide or polypeptide that is identical to the reference molecule except for the presence of one or more conservative amino acid substitutions in its amino acid sequence. In general, the variant obtained by conservative modification includes an amino acid sequence that is at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the reference amino acid sequence. More specifically, a conservative substitution refers to the replacement of an amino acid with an amino acid that has similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) and has minimal effect on the biological activity of the resulting peptide or polypeptide. Conservative substitutions of functionally similar amino acids are well known in the art and therefore need not be described in detail herein. As used herein, the term effective amount means an amount or dose of one or more RLN analogues described herein, or a pharmaceutically acceptable salt thereof, which, when administered single or multiple doses to an individual in need thereof, provides the desired effect in such individual under treatment. on diagnosis or treatment (i.e., may cause clinically measurable differences in the individual's condition, such as, for example, increased angiogenesis, increased vascular elasticity, increased cardiac blood flow, increased hepatic blood flow, increased pulmonary blood flow, increased renal blood flow, increased glomerular filtration rate , lowering blood pressure, reducing (or preventing) inflammation and/or reducing (or preventing) fibrosis of the heart, kidneys, liver or lungs). The effective amount can be readily determined by one skilled in the art using known methods and by observing the results obtained under similar circumstances. In determining the effective amount for an individual, a number of factors are considered, including, but not limited to, the species of mammal, its size, age and general health, the specific disease or disorder, the extent or involvement or severity of the disease or disorder, the response of the individual, the specific analogue administered RLN, route of administration, bioavailability characteristics of the formulation administered, dosage schedule chosen, use of concomitant medications, and other relevant circumstances.

As used herein, the term extended duration of action means that the binding affinity and activity for the RLN analogue described herein is maintained for a period of time greater than that of native RLN, particularly native human RLN2 (SEQ ID NOs: 5 and 6 ), allowing the dose to be administered less frequently, such as once a day, three times a week, twice a week, or once a week. The temporal action profile of the RLN analogue can be measured using known pharmacokinetic assay methods, such as those used in the examples below.

As used herein, the term half-life or t ¹ / ₂ means the time required for half the amount of a compound, such as native RLN or an RLN analogue described herein, to be removed from a fluid or other physiological substance.

- 8 046279 space, such as the serum or plasma of an individual, through biological processes. Alternatively, t1/ ₂ may also mean the time at which a quantity of such compound loses half of its pharmacological, physiological or radiological activity. As used herein, the term half-maximal effective concentration or EC50 means the concentration of a compound that results in 50% activation/stimulation of an assay endpoint such as a dose-response curve (e.g., cAMP, PI3K-Akt, ΚΕκβ, VEGF, and /or nitric oxide (NO).

As used herein, the term in combination with means administering at least one of the RLN analogues described herein simultaneously, sequentially, or as a single combination formulation with one or more additional therapeutic agents.

As used herein, the term "individual in need" means a mammal, such as a human, having a condition, disease, disorder or symptom requiring treatment or therapy, including, for example, those listed herein. In particular, the subject to be treated is a human.

As used herein, the term extended-release means that the binding affinity and activity of the RLN analogue described herein is maintained for a period of time greater than that of native human RLN2 (SEQ ID NOs: 5 and 6), allowing administer the dose less frequently, such as once a day or even three times a week, twice a week or once a week or monthly. The temporal action profile of RLN analogues can be measured using known pharmacokinetic assay methods, such as those described in the examples below.

As used herein, the term non-standard amino acid means an amino acid that may occur naturally in cells but is not involved in peptide synthesis. Non-standard amino acids can be constituents of a peptide and are often formed by modifying standard amino acids in the peptide (ie, post-translational modification). Non-standard amino acids may include D-amino acids that have an absolute chirality opposite to the standard amino acids listed above.

As used herein, the term pharmaceutically acceptable buffer means any of the standard pharmaceutical buffers known to one skilled in the art.

As used herein, the term RLN means relaxin derived from or based on any species, such as mammalian species, especially humans, wherein the native form is a heterodimeric peptide having two peptide chains (e.g., an A chain and B chain) connected by two disulfide bonds, while the A chain additionally has one intramolecular disulfide bond. The RLN includes both the native RLN (i.e., full-length) and its variants (i.e., additions, deletions, insertions, and/or substitutions of the native RLN). In humans, there are three native isoforms of RLN - RLN1, RLN2 and RLN3. Processing of RLN begins with preprorelaxin, which is processed to prorelaxin (contains an A chain, a B chain and a C peptide; native RLN has a B-C-A structure), with the sequence of native human npoRLN1 presented in SEQ ID NO: 1 (see . also, UniProt/SwissProt database accession number P04808), the sequence of native human npoRLN2 is presented in SEQ ID NO: 4 (see also, UniProt/SwissProt database accession number P04090), and the sequence of native human npoRLN3 is given in SEQ ID NO: 7 (see also UniProt/SwissProt accession number Q8WXF3). Prorelaxin undergoes further processing, during which the C-peptide is cleaved to reach the RLN. The A chain sequences of native human RLN1, RLN2 and RLN3 are shown in SEQ ID NO: 2, 5 and 8, respectively. Likewise, the B chain sequences of native human RLN1, RLN2 and RLN3 are shown in SEQ ID NO: 3, 6 and 9, respectively.

Humans have four RLN receptors - RXFP1 (SEQ ID NO: 40; see also, accession number in the UniProt/SwissProt database No. Q9HBX9), RXFP2 (SEQ ID NO: 41; see also, accession number in the UniProt database/ SwissProt No. Q8WXD0), RXFP3 (SEQ ID NO: 42; see also UniProt/SwissProt accession no. Q9NSD7) and RXFP4 (SEQ ID NO: 43; see also UniProt/SwissProt accession no. Q8TDU9) - which act as GPCRs (see Halls et al. (2007) Br. J. Pharmacol. 150:677-691). The receptors of interest here are RXFP1 and RXFP2, both of which can bind RLN1 and RLN2. The RXFP1 receptor has been found in the brain, blood cells, bones, heart, kidneys, lungs, liver and vasculature, while the RXFP2 receptor has a much more limited occurrence and has been found in the bone and testicular ligament guide. Stimulation of RXFP1 and RXFP2 receptors activates signaling networks including adenylate cyclase, protein kinase A, protein kinase C, phosphatidylinositol 3-kinase and/or extracellular signal-regulated kinases (Erk1/2). As used in the context of this document, the term RLN analogue, etc. means a compound, such as a peptide or polypeptide, that has one or more native RLN effects on one or more RXFP receptors, but varies in some way with respect to the amino acid sequence

- 9 046279 value compared to native RLN. An RLN analog may also include variants of these compounds that are functionally equivalent to RLN but have sequences that are fragments of the sequence or are the complete sequence but with additions, deletions, insertions and/or substitutions. All references to amino acid positions in unmodified or modified RLNs described herein are based on the corresponding position in the A chain of SEQ ID NO: 5 or the B chain of SEQ ID NO: 6 of native human RLN2 unless otherwise noted. other. In some cases, the RLN analogues described herein may bind to RXFP with higher or lower affinity but exhibit a longer t1/ ₂ in vivo or in vitro compared to native RLN, particularly native human RLN2 (SEQ ID NO: 5 and 6). Thus, the RLN analogues described herein are synthetic compounds that act as RXFP receptor agonists.

As used herein, the term sequence similarity means a quantitative property of two or more nucleic acid sequences or amino acid sequences of biological compounds, such as, for example, full-length correspondence or windows of comparison between two or more sequences. Sequence similarity can be measured by (1) percent identity or (2) percent similarity. Percent identity measures the percentage of residues identical between two biological compounds divided by the length of the shortest sequence; whereas percent similarity measures identity and further includes sequence gaps and residue similarities in the score. Methods and algorithms for determining sequence similarity are well known in the art and therefore need not be described in detail herein. The determined percentage of identical nucleotide or amino acid positions is at least about 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or higher .

As used herein, the terms single chain relaxin, ohRLN, and the like. means an RLN polypeptide in which chains A and B are connected to each other by a linker (ie L2), as in A-L2-B or B-L2-A. Moreover, ohRLN may contain at least one of native interchain and/or intrachain disulfide bonds to maintain proper structural folding. The terms double-chain relaxin, dsIbN, etc. used in the context of this document. means an RLN polypeptide in which chains A and B are linked to each other by one or more interchain and/or intrachain disulfide bonds, but are not linked by any linkers, to maintain proper structural folding such as that of native RLN.

As used herein, the term treat or treat means the treatment and care of a person having a condition, disease, disorder or symptom for which administration of an RLN analogue is indicated for the purpose of attenuating, controlling, regressing, slowing or stopping the progression or reducing the severity of the condition. disease, disorder and/or symptom. Treatment involves administering an RLN analogue described herein, or a composition containing an RLN analogue described herein, to an individual to prevent the onset of symptoms or complications, alleviate symptoms or complications, or eliminate a pathological condition, disease, disorder or symptom. Treatment involves administering to the individual an RLN analog or a composition containing an RLN analog described herein, resulting in, for example, increased angiogenesis, increased vascular elasticity, increased cardiac blood flow, increased hepatic blood flow, increased pulmonary blood flow, increased renal blood flow, increased glomerular blood flow. filtration, lowering blood pressure, reducing (or preventing) inflammation and/or reducing (or preventing) fibrosis of the heart, kidneys, liver or lungs). The individual being treated is a mammal, in particular a human.

As used herein, the terms individual, patient and subject are used interchangeably to refer to a mammal, particularly a human. In some cases, the individual is further characterized by having a condition, disease, disorder and/or symptom that may be ameliorated by administration of the RLN analogue described herein. As used herein, the term VHH or VHH fragment means a single-domain antibody form, particularly a single monomeric heavy chain-only antibody variable region (HcAb) antibody fragment that is very small in size, about 15 kDa. This document concludes that VHH fragments can be used as a pharmacokinetic enhancer to increase the duration of action and/or improve the t1/ ₂ of the RLN analogues described herein. The VHH fragments described herein bind serum albumin; however, VHH fragments can be used to bind IgG (including the Fc domain), neonatal Fc receptor (FcRn), or other long-acting serum proteins. Although the VHH fragments described herein are used to increase the t1/ ₂ of RLN, they can also be used to improve the t1/ ₂ of other biologically active peptides/proteins, such as, for example, insulin, FDF-15 or GIP-1.

Some abbreviations are defined as follows: ACR refers to the Albu ratio

- 10 046279 min in urine/creatinine in urine; A. units m refers to the atomic mass unit; AUC refers to the area under the curve; Boc refers to tert-butoxycarbonyl; cAMP refers to cyclic adenosine monophosphate; CMV belongs to the cytomegalovirus; CV refers to the volume of the column; DNA refers to deoxyribonucleic acid; DMF refers to dimethylformamide; DMSO refers to dimethyl sulfoxide; EDC refers to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; EDTA refers to ethylenediaminetetraacetic acid; ELISA/RIA refers to enzyme immunoassay/radioimmunoassay; ETA refers to ethanolamine; GS refers to glutamine synthetase; CHB refers to hydrophobic interaction chromatography; h refers to an hour or hours; HRGF refers to time-resolved homogeneous fluorescence; IV refers to intravenous administration; IP refers to intraperitoneal administration; kDa refers to kilodaltons; LC/MS refers to liquid chromatography with mass spectrometry; min refers to a minute or minutes; MS refers to mass spectrometry; MSO refers to methionine sulfoximine; NaOAc refers to sodium acetate; NHS refers to N-hydroxysuccinimide; OtBu refers to O-tert-butyl; Pbf refers to NG-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PEA refers to polyethylenimine; RP-HPLC refers to reversed-phase high-performance liquid chromatography; RU refers to resonant units; sec refers to a second or seconds; SPR refers to surface plasmon resonance; s/c refers to subcutaneous administration; SEC refers to size exclusion chromatography; SEM refers to the standard error of the mean; TFA refers to trifluoroacetic acid; and Trt refers to trityl.

Analogues of RLN.

The RLN analogues described herein are structurally similar to native human RLN but also have many structural differences. For example, compared to native human RLN2 (SEQ ID NOs: 5 and 6), the RLN analogues lack one or more amino acids present in native human RLN2, the analogues include a peptide linker between the A chain and the B chain, and include albumin -VHH binding fragment. RLN analogues result in sufficient activity of the RXFP1 receptor and reduced or insufficient activity of one or more of the RXFP2, RXFP3 and RXFP4 receptors. Likewise, RLN analogues have beneficial properties relevant to their potential for use as therapeutic treatments, including improved solubility in aqueous solutions, improved chemical and physical stability of formulation, enhanced pharmacokinetic profile (which can be adjusted based on the affinity of VHH for serum albumin) and/or minimal potential for immunogenicity.

Briefly, the RLN analogs described herein contain an amino acid sequence from N-terminus to C-terminus having one of the following structures:

VHH-L ₁ -AL ₂ -B;

VHH-L ₁ -BL ₂ -A;

A-L2-B-L1-VHH; or

B-L2-A-L1-VHH;

wherein VHH is a moiety that acts as a pharmacokinetic enhancer, A is the A chain of RLN, B is the B chain of RLN, L ₁ is a first peptide linker and L ₂ is a second peptide linker, wherein L ₁ and L ₂ differ from each other (i.e., each of them does not have the same amino acid sequence).

As for the A chain, it may be a native RLN1 A chain, such as a native human RLN1 A chain (SEQ ID NO: 2); Native human RLN2 A chain (SEQ ID NO: 5); or native human RLN3 A chain (SEQ ID NO: 8). Alternatively, the A chain may be a variant thereof. For example, one A chain variant may have an amino acid sequence that lacks residues 1 to 4 of SEQ ID NO: 5 (ie, human RLN2 des1-4 A chain or desA14).

Likewise, as for the B chain, it may be a native RLN B chain, such as the human native RLN1 B chain (SEQ ID NO: 3); Native human RLN2 B chain (SEQ ID NO: 6); or native human RLN3 B chain (SEQ ID NO: 9). Alternatively, the B chain may be a variant thereof. For example, one B chain variant may have an amino acid sequence that lacks residue 1 of SEQ ID NO: 6 (ie, human des1 RLN2 or desB1 B chain).

In some cases, the A chain may be a native human RLN1 A chain (SEQ ID NO: 2) and the B chain may be a native human RLN1 B chain (SEQ ID NO: 3); The A chain may be a native human RLN2 A chain (SEQ ID NO: 5), and the B chain may be a native human RLN2 B chain (SEQ ID NO: 6); The A chain may be a native human RLN3 A chain (SEQ ID NO: 8), and the B chain may be a native human RLN3 B chain (SEQ ID NO: 9); The A chain may be a native human RLN1 A chain (SEQ ID NO: 2), and the B chain may be a native human RLN2 B chain (SEQ ID NO: 6); The A chain may be a native human RLN1 A chain (SEQ ID NO: 2), and the B chain may be a native human RLN3 B chain (SEQ ID NO: 9); The A-chain may be an A-chain

- 11 046279 native human RLN2 (SEQ ID NO: 5), and the B chain may be a native human RLN1 B chain (SEQ ID NO: 3); The A chain may be a native human RLN2 A chain (SEQ ID NO: 5), and the B chain may be a native human RLN3 B chain (SEQ ID NO: 9); The A chain may be a native human RLN3 A chain (SEQ ID NO: 8), and the B chain may be a native human RLN1 B chain (SEQ ID NO: 3); or the A chain may be a native human RLN3 A chain (SEQ ID NO: 8) and the B chain may be a native human RLN2 B chain (SEQ ID NO: 6).

In some cases, the A chain may be a variant of the RLN2 A chain lacking residues 1 to 4 (desA1-4), and the B chain may be any native B chain. In other cases, the A chain may be any native A chain, and the B chain may be a variant of the RLN2 B chain lacking residue 1 (desB1). In other cases, the A chain may be a variant of the RLN2 A chain lacking residues 1 to 4 (desA1-4), and the B chain may be a variant of the RLN2 B chain lacking residue 1 (desB1) . In some cases, the A chain is a desA1-4 variant. In some cases, the B chain is a desB1 variant.

Other A and B chains that can be used in the RLN analogues described herein are described, for example, in international patent application publication WO 2018/148419, WO 2018/138170, WO 2017/201340, WO 2016/149501, WO 2015 /157829, WO 2015/067791, WO 2015/067113, WO 2014/102179, WO 2013/177529, WO 2013/007563, WO 2013/004607, WO 2012/031326 and WO 2012/024 452; and US Patent Application Publication No. US2011/0243942. See also Chan et al. (2012) J. Biol. Chem. 287:41152-41164; Claasz et al. (2002) Em. J. Biochem. 269:6287-6293; 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 et al. (2005) BMC Evol. Biol. 5:14.

As for L1, it can be a peptide containing from about 1 amino acid to about 50 amino acids. Alternatively, L1 may consist of about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 amino acids. Alternatively, L1 may consist of from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids , from about 30 amino acids to about 35 amino acids, from about 35 amino acids to about 40 amino acids, from about 40 amino acids to about 45 amino acids, or from about 45 amino acids to about 50 amino acids. In some cases, L1 may be omitted so that the A chain or B chain is directly conjugated to the VHH moiety. In some cases, L1 may contain a repeat sequence (GGGGQ)n (SEQ ID NO: 14), where n can be from about 1 to about 10, especially 5 (i.e., (GGGGQ) ₅ ; SEQ ID NO: 19) . In other cases, L1 may contain a repeat sequence (PGPQ) _n (SEQ ID NO: 17), where n can be from about 1 to about 10, especially 8 (i.e. (PGPQ) ₈ ; SEQ ID NO: 20) . In other cases, L1 may contain a repeat sequence (PGPA) _n (SEQ ID NO: 18), where n can be from about 1 to about 10, especially 8 (i.e. (PGPA) ₈ ; SEQ ID nO: 21) .

Other linkers that can be used in RLN analogues as L1 include, but are not limited to, (GGGQ) _n (SEQ ID NO: 15) or (GGGGS) _n (SEQ ID NO: 16).

As for L ₂ , it can be a peptide containing from about 1 amino acid to about 15 amino acids. Alternatively, L2 may consist of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 amino acids. Alternatively, L2 may consist of from about 1 amino acid to about 5 amino acids, from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, especially from about 10 amino acids to about 15 amino acids. In some cases, L2 may contain a mixture of Ala/A, Gln/Q, Gly/G, Pro/P and Ser/S residues. In other cases, L2 may be SEQ ID NO: 22, 23, or 67.

As for VHH, it can be a polypeptide containing from about 50 to about 200 amino acids, in particular from about 125 to about 150 amino acids, which can bind serum albumin or other serum protein having a long t ¹ / ₂ . In some cases, VHH may be any of the sequences of SEQ ID NO: 10-13. Alternatively, VHH may be any of the sequences of SEQ ID NO: 45-66. The structural features of these VHH fragments result in the RLN analogs having a longer t1/ ₂ compared to native RLN, particularly compared to native human RLN2 (SEQ ID NOs: 5 and 6). Given that the VHH fragments described herein target serum albumin, the t1/ ₂ of the RLN analog would be expected to be similar to that of the serum albumin of the species to which the RLN analog is administered (taking into account any target-mediated distribution of the drug).

In addition to the changes described in the disclosure, the RLN analogs described herein may include one or more additional amino acid modifications, especially conservative substitutions, provided, however, that the RLN analogs remain capable of binding to the receptor

- 12 046279 torus RXFP1 and activate it. In general, illustrative RLN analogues are:

RLN Analog 1, which contains the VHH fragment (underlined), L1 (G4Q)5 (italics), the RLN2 achain, ten-residue _L2 (in bold), and the B chain (desB1) of RLN2, and has the following amino acid sequence :

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

OGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQOLYSALANKCCHVGCTKRSLAR

FCGGGSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ GO NO:24) or a pharmaceutically acceptable salt thereof;

RLN Analog 2, which contains the VHH fragment (underlined), L ₁ (PGPA)8 (italicized), the ten-residue RLN2 achain, L2 (in bold), and the B chain (desB1) of RLN2, and has the following amino acid sequence :

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAQLYSALANKCCHVGCTKR

SLARFCGGGSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ GO NO:25) or a pharmaceutically acceptable salt thereof;

RLN Analogue 3, which contains the VHH fragment (underlined), L1 (G4Q)5 (italics), RLN2 chain (desB1), ten-residue L2 (in bold), and the RLN2 A chain, and has the following amino acid sequence:

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

OGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAOIAICG

MSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC (SEQ GO NO:26) or a pharmaceutically acceptable salt thereof;

RLN Analog 4, which contains the VHH fragment (underlined), L1 (PGPQ)8 (italics), RLN2 chain (desB1), ten-residue _L2 (in bold), and the A chain of RLN2, and has the following amino acid sequence :

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

OGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSWMEEVIKLCGRELVRA

QIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC (SEQ GO NO:27) or a pharmaceutically acceptable salt thereof;

RLN Analog 5, which contains the ten-residue B chain (desB1) of RLN2, L2 (in bold), the A chain of RLN2, L ₁ (G4Q) ₅ (in italics), and the VHH fragment (underlined), and has the following amino acid sequence:

SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRS

LARFCGGGGQGGGGQGGGGQGGGGQGGGGQEVOLLESGGGLVOPGGSLRLSCAASG

RYIDETAVAWFROAPGKEREFVAGIGGVDITYYADSVKGRFTISRDNSKNTLYLOMNS

LRPEDTAVYYCAARPGRPLITSKVADLYPYWGOGTLVTVSSPP (SEQ GO NO:28) or a pharmaceutically acceptable salt thereof;

RLN Analog 6, which contains the ten-residue B chain (desB1) RLN2, L2 (in bold), the A chain RLN2, L ₁ (PGPQ) ₈ (in italics), and the VHH fragment (underlined), and has the following amino acid sequence:

SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRS

LARFCPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVOLLESGGGLVOPGGSLRLSC

AASGRYIDETAVAWFROAPGKEREFVAGIGGVDITYYADSVKGRFTISRDNSKNTLYL

OMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGOGTLVTVSSPP (SEQ GO NO:29) or a pharmaceutically acceptable salt thereof;

RLN Analogue 7, which contains the VHH fragment (underlined), L1(G4Q)5 (italics), RLN2 chain (desB1), _ten -residue L2 (in bold), and the RLN2 A chain, and has the following amino acid sequence :

- 13 046279

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKGREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG OGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAOIAICG MSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLAR FC (SEQ GO NO:30) or a pharmaceutically acceptable salt thereof;

RLN Analogue 8, which contains the VHH fragment (underlined), L1 (PGPQ) ₈ (italics), RLN2 chain (desB1), ten-residue L ₂ (in bold), and the A chain of RLN2, and has the following amino acid sequence :

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSWMEEVIKLCGRELLVRA

QIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC (SEQ Ш NO:31) or a pharmaceutically acceptable salt thereof;

RLN Analog 9, which contains the ten-residue B chain (desB1) of RLN2, L2 (in bold), the A chain of RLN2, L1 (G4Q)5 (in italics), and the VHH fragment (underlined), and has the following amino acid subsequence:

SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRS LARFCGGGGQGGGGQGGGGQGGGGQGGGGQEVOLLESGGGLVOPGGSLRLSCAASG RYIDETAVAWFROAPGKGREFVAGIGGVDITYYADSVKGRFTISRDNSKNTLYLQMNS LRPEDTAVYYCAARPGRPLITSKVADLYPYWGOGTLV TVSSPP (SEQ III NO:32) or a pharmaceutically acceptable salt thereof;

RLN Analogue 10, which contains the ten-residue B chain (desB1) RLN2, L2 (in bold), the A chain RLN2, L1 (PGPQ) ₈ (in italics) and the VHH fragment (underlined), and has the following amino acid subsequence:

SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRS LARFCPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVOLLESGGGLVQPGGSLRLSC AASGRYIDETAVAWFROAPGKGREFVAGIGGVDITYYADSVKGRFTISRDNSKNTLYL OMNSLRPEDTAVYYCAARPGRPLITSKVAD LYPYWGOGTLVTVSSPP (SEQ GO NO:33) or a pharmaceutically acceptable salt thereof;

RLN Analog 11, which contains the VHH fragment (underlined), L1 (G4Q)5 (italics), the RLN2 chain (desB1), the thirteen-residue L2 (bold), and the A chain (desA1-4) of RLN2, and has the following amino acid sequence:

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG OGTLVTVSSGGGGOGGGGOGGGGOGGGGOGGGGOSWMEEVIKLCGRELVRAOIAI CGMSTWSGGGSGGSGGSGGGALANKCCHVGCTKRSLARFC (SEQ GO NO:34) or a pharmaceutically acceptable salt thereof;

RLN Analog-12, which contains the VHH fragment (underlined), L ₁ (G4Q) ₅ (italics), Chain (desB1) RLN2, thirteen residue L ₂ (in bold), and A chain (desA1-4) RLN2 , and has the following amino acid sequence:

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAOIAICG

MSTWSGGGSGGSGGSGGGALANKCCHVGCTKRSLARFC (SEQ GO NO:35) or a pharmaceutically acceptable salt thereof;

RLN Analog-13, which contains the VHH fragment (underlined), L ₁ (G4Q)5 (in italics), A chain (desA1-4) RLN2, ten-residue L ₂ (in bold), and B chain (desB1) RLN2 , and has the following amino acid sequence:

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

OGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQALANKCCHVGCTKRSLARFCGG

GSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ GO NO:36) or a pharmaceutically acceptable salt thereof;

RLN-Analogue 14, which contains fragment VHH (underlined), L1 (PGPQ) ₈ (italicized),

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The A chain (desA1-4) of RLN2, L ₂ of ten residues (in bold) and the B chain (desB1) of RLN2, and has the following amino acid sequence:

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

OGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQALANKCCHVGCTKRSLA

RFCGGGSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ Ш NO:37) or a pharmaceutically acceptable salt thereof;

RLN Analog 15, which contains the VHH fragment (underlined), L ₁ (G4Q)5 (italics), RLN2 Chain (native), ten-residue L ₂ (bold), and A-chain (native) RLN2, and has the following amino acid sequence:

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQDSWMEEVIKLCGRELVRAOIAIC GMSTWSSGGGGSGGGGQLYSALANKCCHVGCTKRSLARFC (SEQ Ш NO:38) or a pharmaceutically acceptable salt thereof; And

RLN Analog 16, which contains the VHH fragment (underlined), L1 (PGPQ) ₈ (italics), RLN2 B chain (native), ten-residue L ₂ (bold), and RLN2 A chain (native), and has the following amino acid sequence:

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKEREFVAGIGGVDITY

or a pharmaceutically acceptable salt thereof.

The half-life of the RLN analogs described herein can be measured using methods known in the art, including, for example, the methods described in the examples below. Likewise, the affinity of RLN analogs for albumins of various species can be measured using methods known in the art for measuring binding affinity, such as those described in the examples below, and typically the affinity is expressed as the value of the equilibrium dissociation constant (KD ). Moreover, the activity of the RLN analogues for each of the RXFP receptors can be measured using methods known in the art, including, for example, the in vitro activity assays described below, and is typically expressed as an EC50 value.

As a result of the modifications described above, the RLN analogues described herein have a t1/ ₂ that is longer than that of native RLN, particularly native human RLN2 (SEQ ID NOs: 5 and 6) when administered to a mammal, particularly a human. As noted above, the VHH fragments described herein target the whey album; therefore, the t1/ ₂ of the RLN analogues described herein can be expected to be similar to that of the serum albumin of the species to which the RLN analogue is administered. In some cases, RLN analogues may have a t1/ ₂ of about 1 day to about 31 days, from about 5 days to about 25 days, from about 10 days to about 20 days, or from about 15 days. In other cases, RLN analogs may have t1/ ₂ from about 1 day to about 5 days, from about 6 days to about 10 days, from about 11 days to about 15 days, from about 16 days to about 20 days, from about 21 days to about 25 days or even from about 26 to about 31 days. In other cases, RLN analogues may have a ^t1 / ₂ of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days , about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days or even about 31 days. In specific cases, RLN analogues may have a t1/ ₂ of approximately 20 days when administered to humans. Likewise, the RLN analogues described herein have about 10- to about 100-fold greater activity at the RXFP1 receptor compared to, for example, native human RLN2 (SEQ ID NOs: 5 and 6) when administered to humans.

Pharmaceutical compositions and kits.

The RLN analogs described herein can be formulated into pharmaceutical compositions that can be administered by parenteral routes (eg, intravenous, intraperitoneal, intramuscular, subcutaneous, or transdermal). Such pharmaceutical compositions and methods for their preparation are well known in the art. See, for example, Remington, The Science and Practice of Pharmacy (DB Troy ed., ^21st Ed., Lippincott, Williams & Wilkins, 2006). In specific cases, RLN analogues are administered subcutaneously or intravenously. Alternatively, however, RLN analogues can be prepared in forms for other pharmaceutically acceptable routes, such as, for example, tablets or

- 15 046279 other solid dosage forms for oral administration; delayed release capsules; and any other forms currently in use, including creams, lotions, inhalants, etc.

As noted previously, and to improve their tolerability and effectiveness in vivo, the RLN analogs described herein can react with any of a number of inorganic and organic acid/bases to form pharmaceutically acceptable acid/base addition salts. Pharmaceutically acceptable salts and conventional methods for preparing them are well known in the art (see, for example, Stahl et al, Handbook of Pharmaceutical Salts: Properties, Selection and Use ( ^2nd Revised Ed. Wiley-VCH, 2011)). Pharmaceutically acceptable salts for use described herein include sodium, trifluoroacetate, hydrochloride and acetate salts.

The RLN analogues described herein may be administered by a physician or self-administered by injection. It will be understood that the needle gauge number and the injection volume can be easily determined by one skilled in the art. However, the injection volume may be < about 2 ml or even < about 1 ml, and the needle gauge may be > about 27 G or even > about 29 G.

The disclosure also provides and therefore covers new intermediates and methods useful for the synthesis of the RLN analogues described herein or pharmaceutically acceptable salts thereof. RLN intermediates and analogues can be prepared by various methods that are well known in the art. For example, a method using recombinant synthesis is illustrated in the examples below. The specific steps for each of the described methods can be combined in different ways to produce RLN analogues. Reagents and starting materials are readily available to one skilled in the art.

The RLN analogs described herein are generally effective over a wide dosage range. Exemplary dosages of the RLN analogues described herein, or pharmaceutical compositions comprising them, may be in milligrams (mg) or micrograms (mcg), nanograms (ng) or picograms (pg) per kilogram (kg) of the individual's body weight. Thus, the daily dose may range from about 1 mcg to about 100 mg.

In this case, the effective amount of the RLN analog in the pharmaceutical composition may be from about 0.25 mg to about 5.0 mg. However, one skilled in the art will appreciate that in some cases the effective amount (i.e. dose/dose) may be below the lower end of the above range and be more than sufficient, while in other cases the effective amount may be large dose and can be used with acceptable side effects.

In addition to the RLN analogue described herein, the pharmaceutical composition may also contain at least one additional therapeutic agent, particularly a therapeutic agent commonly used as a standard of care for the treatment of cardiovascular, pulmonary and renal conditions, diseases and disorders.

Thus, the pharmaceutical composition may contain an effective amount of at least one RLN analog described herein, a pharmaceutically acceptable carrier, and optionally at least one additional therapeutic agent. For example, a pharmaceutical composition may comprise an effective amount of an RLN analogue of SEQ ID NO: 24 and a pharmaceutically acceptable carrier, an effective amount of an RLN analogue of SEQ ID NO: 25 and a pharmaceutically acceptable carrier, an effective amount of an RLN analogue of SEQ ID NO: 26 and a pharmaceutically acceptable carrier, an effective amount of an RLN analogue of SEQ ID NO: 27 and a pharmaceutically acceptable carrier, an effective amount of an RLN analogue of SEQ ID NO: 28 and a pharmaceutically acceptable carrier, an effective amount of an RLN analogue of SEQ ID NO: 29 and a pharmaceutically acceptable carrier a carrier, an effective amount of an RLN analogue of SEQ ID NO: 30 and a pharmaceutically acceptable carrier, an effective amount of an RLN analogue of SEQ ID NO: 31 and a pharmaceutically acceptable carrier, an effective amount of an RLN analogue of SEQ ID NO: 32 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 33 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 34 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog of SEQ ID NO: 35 and a pharmaceutically acceptable carrier, an effective amount an RLN analog having the sequence SEQ ID NO: 36 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog having the sequence SEQ ID NO: 37 and a pharmaceutically acceptable carrier, an effective amount of an RLN analog having the sequence SEQ ID NO: 38 and a pharmaceutically acceptable carrier or an effective amount of an RLN analog with the sequence SEQ ID NO: 39 and a pharmaceutically acceptable carrier. Alternatively, the RLN analogs described herein may be offered as part of a kit. In some cases, the kit contains a device for administering to an individual at least one RLN analogue (and optionally at least one additional

- 16 046279 therapeutic agent). In some cases, the kit contains a syringe and a needle for administering at least one RLN analogue (and, optionally, at least one additional therapeutic agent). In specific cases, the RLN analogue (and optionally at least one additional therapeutic agent) is pre-formulated in an aqueous solution within a syringe.

Methods for obtaining and using RLN analogues.

The RLN analogues described herein can be prepared using any number of standard recombinant DNA methods or standard chemical peptide synthesis methods known in the art. With respect to recombinant DNA methods, standard recombinant technologies can be used to construct a polynucleotide having a nucleic acid sequence that encodes the amino acid sequence for an RLN analogue, incorporate the polynucleotide into recombinant expression vectors, and introduce the vectors into host cells such as bacterial cells, yeast cells and mammalian cells to produce an RLN analogue. See, for example, Green & Sambrook, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, ^4th ed. 2012).

With respect to recombinant DNA methods, the compounds described herein can be produced by producing a protein or precursor protein molecule using recombinant DNA technologies. DNA, including cDNA and synthetic DNA, may be double-stranded or single-stranded, and its coding sequences that encode a compound described herein may vary as a result of redundancy or degeneracy of the genetic code. Briefly, DNA sequences encoding the compounds described herein are introduced into a host cell to produce the compound or its precursor. Host cells may be bacterial cells such as K12 or B strains of Escherichia coli, fungal cells such as yeast cells, or mammalian cells such as Chinese hamster ovary (CHO) cells. A suitable host cell is transiently or stably transfected or transformed with an expression system, such as expression vectors, to produce a compound described herein or a precursor thereof. Expression vectors typically replicate in hosts either as episomes or as an integral part of the host's chromosomal DNA. Typically, expression vectors will contain selection markers, for example, tetracycline, neomycin, G418 and dihydrofolate reductase, to allow selection of those cells that are transformed with the desired DNA sequences.

Specific biosynthetic or synthetic steps for each described step may be used, not used, or combined in various combinations to produce the compounds described herein.

As for methods of chemical synthesis of peptides, standard solid-phase synthesis procedures can be used in manual or automated mode. For example, automated peptide synthesizers are commercially available from, for example, Applied Biosystems (Foster City, Calif.) and Protein Technologies Inc. Tucson, Arizona). Reagents for solid phase synthesis are readily available from commercial sources. Solid phase synthesizers can be used according to the manufacturer's instructions to block interfering groups, protect amino acids during reaction, bind, deprotect, and cap unreacted amino acids. Additional information on the creation of synthetic RLNs can be found in US Patents No. 4835251 and No. 5166191.

One of the uses of the RLN analogues described herein is the treatment of pathological conditions, diseases and/or disorders of the cardiovascular system. Illustrative cardiovascular conditions, diseases and disorders include, but are not limited to, acute heart failure, chronic heart failure, atherosclerosis, coronary artery disease, diabetes, stroke, hypercholesterolemia, hypertension, ischemia, vasoconstriction and ventricular hypertrophy.

Another use for the RLN analogues described herein is in the treatment of pulmonary conditions, diseases and/or disorders. Exemplary pulmonary conditions, diseases and disorders include, but are not limited to, pulmonary hypertension and chronic obstructive pulmonary disease (COPD).

Another use for the RLN analogues described herein is in the treatment of renal conditions, diseases and/or disorders. Exemplary renal conditions, diseases and disorders include, but are not limited to, chronic kidney disease and diabetic nephropathy. The methods may include the steps described herein, and they may not necessarily be performed in the sequence described. However, other execution sequences are also possible. Moreover, individual or multiple steps can be performed either in parallel and/or overlapping in time, and/or independently or in repeated steps. In addition, the methods may include additional unspecified steps.

Thus, such methods may involve selecting an individual who has, or is predisposed to, a cardiovascular condition, disease, or disorder. Alternatively, the methods may include selecting an individual having a pathological condition for

- 17 046279 disease or disorder of the lungs, or those predisposed to it. Alternatively, the methods may include selecting an individual having or predisposed to a renal condition, disease or disorder. In some cases, the methods may include selecting an individual who has diabetes, hypertension with renal impairment, and/or obesity.

The methods may also include administering to an individual an effective amount of at least one RLN analog described herein, which may be in the form of a pharmaceutical composition, as also described herein. In some cases, the RLN analogue/pharmaceutical composition may contain additional therapeutic agents such as an anticoagulant, ACE inhibitor, ARB, ARNI, β-blocker, diuretic, digitalis, digoxin, hydralazine/isosorbitol dinitrate, AMKR or other aldosterone antagonist, SGLT2 inhibitor, statin and/or an antiglycemic agent, as well as other therapeutic agents to control concomitant diseases, including, but not limited to, high cholesterol, high blood pressure, atrial fibrillation and diabetes.

The concentration/dose/dosage of the RLN analog and optional additional therapeutic agent are discussed elsewhere in this document. Regarding the route of administration, the RLN analogue or a pharmaceutical composition containing it can be administered by known methods, such as, for example, orally; by injection (ie, intraarterial, intravenous, intraperitoneal, intracerebral, intracerebroventricular, intramuscular, intraocular, intraportal, or intralesional); via sustained release systems or implantable devices. In some cases, the RLN analog or a pharmaceutical composition containing it can be administered subcutaneously by bolus injection or continuously.

With regard to dosing frequency, the RLN analog or a pharmaceutical composition containing it can be administered daily, every other day, three times a week, twice a week, once a week (i.e. weekly), biweekly (i.e. e. every two weeks) or monthly. In some cases, the RLN analog or a pharmaceutical composition containing it is administered subcutaneously every other day, subcutaneously three times a week, subcutaneously twice a week, subcutaneously once a week, subcutaneously every two weeks, or s/c monthly. In specific cases, an RLN analog or a pharmaceutical composition containing it is administered subcutaneously once a week (1 time/week).

That in some cases, an RLN analog or a pharmaceutical composition containing it is administered in combination with an effective amount of at least one additional therapeutic agent. The additional therapeutic agent may be administered simultaneously, separately, or sequentially with the RLN analog or a pharmaceutical composition containing the same.

Moreover, the additional therapeutic agent can be administered at the same frequency as the RLN analogue or pharmaceutical composition containing it (ie, every other day, twice a week, or even weekly). Alternatively, the additional therapeutic agent can be administered at a frequency different from the frequency of administration of the RLN analog or a pharmaceutical composition containing it. In other cases, an additional therapeutic agent may be administered subcutaneously. In other cases, an additional therapeutic agent may be administered IV. In other cases, an additional therapeutic agent may be administered orally. It is also contemplated that the methods may be combined with diet and exercise and/or may be combined with additional therapeutic agents other than those discussed above.

Examples

The following non-limiting examples are offered for purposes of illustration and not limitation. Expression of polypeptides.

Example 1. Recombinant expression of RLN-Analog 1.

Example 1 is an RLN analog having the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQQLYSALANKCCHVGCTKRSLAR FCGGGSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ Ш NO:24).

This document discloses that the RLN analogue of SEQ ID NO: 24 is produced in a mammalian cell expression system using CHOK1 cell derivatives. The cDNA sequence encoding the sequence of SEQ ID NO: 24 is subcloned into the GS-containing backbone of expression plasmids (pEE12.4-based plasmids). The cDNA sequence is fused in frame to the coding sequence of the signal peptide sequence, METDTLLLWVLLLWVPGSTG (SEQ ID NO: 44), to enhance secretion of the RLN analog into tissue culture medium. Expression is regulated by the CMV viral promoter.

To produce the RLN analogue by transient transfection, CHOK1 cells are transfected with a recombinant expression plasmid using a PEI-based method.

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Briefly, an appropriate volume of CHOK1 cell suspension at a density of 4 x 10 ⁶ cells/ml is transferred into shake flasks and both PEI and recombinant plasmid DNA are added to the cells. Cells are incubated in suspension culture at 32°C for 6 days. At the end of the incubation period, the cells are removed by low speed centrifugation and the RLN analogue protein is purified from the conditioned medium.

Alternatively, and to generate an RLN analogue through stable transfections, CHOK1 cells are stably transfected using electroporation and an appropriate amount of recombinant expression plasmid, and the transfected cells are maintained in suspension culture at an appropriate cell density. Selection of transfected cells is carried out by growing in serum-free medium containing 25 μM MSCs and incubating at about 35-37°C and about 5-7% CO ₂ .

The RLN analogue is secreted into the medium from CHO cells, which are purified by protein A affinity chromatography followed by ion exchange and hydrophobic interaction chromatography or size exclusion chromatography. Typically, the RLN analogue from the collected medium is captured on Mab Select Protein A (GE) resin. The resin is then quickly washed with a running buffer such as phosphate-buffered saline (PBS; pH 7.4) or Tris-containing buffer to remove nonspecifically bound material. The protein is eluted from the resin with a low pH solution such as 10 mM citric acid pH 3. Fractions containing the RLN analog are pooled and can be maintained at low pH to inactivate potential viruses. The pH may be neutralized by adding a base such as 0.1M Tris pH 8.0. The RLN analog can be further purified by ion exchange chromatography using resins such as Poros 50 HS (ThermoFisher).

The RLN analogue is eluted from the column using a 0 to 500 mM NaCl gradient in 20 mM NaOAc, pH 5.0, through 15 column volumes. The RLN analog can be further purified by hydrophobic interaction chromatography using a Capto Phenyl ImpRes hydrophobic interaction chromatography (HIC) column (GE Healthcare). Purification is performed by bringing the charged column solution to about 0.5 M sodium sulfate and performing elution using a 10 column volume (CV) gradient going from 0.5 M to 0 M sodium sulfate in 20 mM Tris pH 8. After CHB analogue RLN can be further purified by SEC by loading a concentrated pool of Capto Phenyl ImpRes onto a Superdex200 (GE Healthcare) with isocratic elution in PBS pH 7.4 or 20 mM histidine, 50 mM NaCl pH 6.0.

The purified RLN analogue can be passed through a virus retention filter such as Planova 20N (Asahi Kasei Medical), followed by concentration/diafiltration in 20 mM histidine, 20 mM NaCl pH 6, using tangential flow ultrafiltration on a regenerated cellulose membrane (Millipore) .

The RLN analogs provided herein are prepared by this or a similar method, which can be readily determined by one skilled in the art.

Example 2. Recombinant expression of RLN analogue 2.

Example 2 is an RLN analog having the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAQLYSALANKCCHVGCTKR SLARFCGGGSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ GO NO:25)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 25 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 25.

Example 3. Recombinant expression of RLN-Analogue 3.

Example 3 is an RLN analog having the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICG MSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC (SEQ GO NO:26)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 26 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 26.

Example 4. Recombinant expression of RLN-Analogue 4.

Example 4 is an RLN analog having the amino acid sequence:

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EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSWMEEVIKLCGRELVRAQI AICGMSTWSGGGSGGSGGGQLY SALANKCCHVGCTKRSLARFC (SEQ ID NO:27)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 27 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 27.

Example 5. Recombinant expression of RLN-Analog 5.

Example 5 is an RLN analog having the amino acid sequence: SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSL

ARFCGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGR YIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSL RPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ Ш NO:28)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 28 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 28.

Example 6. Recombinant expression of RLN-Analog 6.

Example 6 is an RLN analog having the amino acid sequence: SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSL

ARFCPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGKEREFVAGIGGVDITYYADSVKGRFTISRDNSKNTLYLQM NSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ Ш NO:29)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 29 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 29.

Example 7. Recombinant expression of RLN-Analog 7.

Example 7 is an RLN analog having the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICG MSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC (SEQ DI NO :30)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 30 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 30.

Example 8. Recombinant expression of RLN-Analog 8.

Example 8 is an RLN analog having the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSWMEEVIKLCGRELVRAQI AICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSLARFC (SEQ ID NO 3 I)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 31 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 31.

Example 9. Recombinant expression of RLN-Analog 9.

Example 9 is an RLN analog having the amino acid sequence: SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSL

ARFCGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGR YIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSL RPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ Ш NO:32)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 32 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 32.

Example 10. Recombinant expression of RLN-Analog 10.

Example 10 is an RLN analog having the amino acid sequence:

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SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSL ARFCPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGI<GREFVAGIGGVDITYYADSVI<GRFTISRDNSI<NTLYLQM NSLRPEDTAVYYCAARPG RPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO:33)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 33 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 33.

Example 11. Recombinant expression of RLN-Analog 11.

Example 11 is an RLN analog having the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICG MSTWSGGGSGGSGGSGGGALANKCCHVGCTKRSLARFC (SEQ Ш NO:34)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 34 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 34.

Example 12. Recombinant expression of RLN-Analog 12.

Example 12 is an RLN analog having the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICG MSTWSGGGSGGSGGSGGGALANKCCHVGCTKRSLARFC (SEQ Ш NO:35)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 35 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 35.

Example 13. Recombinant expression of RLN-Analogue 13.

Example 13 is an RLN analog having the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQALANKCCHVGCTKRSLARFCGG

GSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ Ш NO:36)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 36 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 36.

Example 14. Recombinant expression of RLN-Analogue 14.

Example 14 is an RLN analog having the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQALANKCCHVGCTKRSLAR FCGGGSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ Ш NO:3 7)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 37 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 37.

Example 15. Recombinant expression of RLN-Analogue 15.

Example 15 is an RLN analog having the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQDSWMEEVIKLCGRELVRAQIAIC GMSTWSSGGGGSGGGGQLYSALANKCCHVGCTKRSLARFC (SEQ ID NO:38)

This document describes that an RLN analogue with the sequence SEQ ID NO: 38 is created by

- 21 046279 essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence of SEQ ID NO: 38.

Example 16. Recombinant expression of RLN-Analog 16.

Example 16 is an RLN analog having the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPADSWMEEVIKLCGRELVRA QIAICGMSTWSSGGGGSGGGGQLYSALANKCCHVGCTKRSLARFC (SEQ Ш NO:39)

Disclosed herein is that an RLN analog having the sequence SEQ ID NO: 39 is created essentially as described in Example 1, except that the expression plasmid uses a cDNA sequence that encodes the sequence SEQ ID NO: 39.

In vitro activity.

Example 17 Albumin binding assay of RLN analogue using SPR.

In vitro binding of the RLN analogue to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin was determined by SPR. In particular, the affinities of the RLN analogues of Examples 1-14 for serum albumin of these species are summarized below in Table. 114.

The binding of the RLN analogues of Examples 1-14 to various serum albumins was examined on a Biacore 8K instrument. Immobilization of serum albumin onto the surface of the S Series CM5 sensor chip is performed according to the manufacturer's instructions (Amine Coupling Kit BR-100050). Briefly, carboxyl groups on the surfaces of the sensor chip (flow cell 1 and 2) are activated by injecting 70 μL of a mixture containing 75 mg/mL EDC and 11.5 mg/mL NHS at 10 μL/min. Serum albumin from human, cynomolgus, mouse, rat, pig, dog, cow and rabbit is diluted in 10 mM sodium acetate, pH 4.0 (BR-1003-49) at 0.5, 0.5, 2.2, 0 .6, 0.6, 0.8, 0.6 and 0.3 μg/ml, and then injected through the activated chip surfaces (flow cell 2, channels 1 to 8) at a rate of 10 μl/min for 180 s (human, mouse, rat, pig, and bovine serum albumin were obtained from Sigma Aldrich (St. Louis, MO); cynomolgus serum albumin was obtained from Holzel Diagnostika (Cologne, Germany); canine serum albumin was obtained from Molecular Innovations (Novi, MI) ; and rabbit serum albumin was obtained from Fitzgerald Industries Intl. (Acton, MA). Various serum albumins are covalently immobilized via free amines on a carboxymethyldextran-coated CM5 sensor chip to achieve a surface density of approximately 100 (62-145) response units. Excessive reactive groups on the surfaces (flow cell 1 and 2) are deactivated by injecting 70 μl of 1 M ETA HCl-NaOH pH 8.5. RLN analogues from examples 1 to 14 are diluted in HBS-EP+ buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% Polysorbate 20) at concentrations of 1000, 333.3, 111.1, 37.04, 12.35, 4.12, 1.37, 0.457, 0.152, 0.051 and 0.017 nM. 150 μL of sample is individually injected sequentially through immobilized serum albumins on the chip surface and dissociated for 600 s at 50 μL/min at 25°C. The surface is regenerated by introducing 10 mM glycine-HCl pH 1.5 (BR-1003-54) at 50 μl/min for 100 s. The resulting sensorgrams are analyzed using Biacore 8K Insight Evaluation Software (version 2.0.15.12933), fitting a 1:1 binding kinetics model to calculate the binding kinetic parameters - association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant ( KD).

Table 1

Binding kinetics of the RLN analogue of Example 1 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd (Esec) _KD (M) S A Person 9.8E+05 Ι,ΙΕ-04 Ι,ΙΕ-10 S A Javanese Macaque Ι,ΙΕ+06 5,ZE-04 5.0E-10 S A Mice 1,ЗЭ+06 4.2E-03 3,ZE-09 S A Rats 1.2E+06 2.6E-03 2DE-09 SA Pigs 7.8E+05 5.5E-03 7DE-09 SA Dogs 1,ЗЭ+06 1.5E-03 1.2E-09 S A Cows 1.4E+06 5.0E-02 3.7E-08 SA Rabbit No binding

KDs are defined as 0.1, 0.5, 3.3, 2.1, 7.1, 1.2 and 37 nM for serum albumin binding

- 22 046279 human, cynomolgus macaque, mouse, rat, pig, dog and cow, respectively, with the RLN analogue from example 1.

table 2

Binding kinetics of the RLN analogue of Example 2 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd (Esec) _KD (M) Human SA 9.2E+05 9.5E-05 1.0E-10 S A Javanese Macaque 1.2E+06 5.2E-04 4.5E-10 S A Mice Ι,ΙΕ+06 4.2E-03 3.8E-09 S A Rats 9.8E+05 2.9E-03 3.0E-09 SA Pigs 6.9E+05 5.6E-03 8.0E-09 SA Dogs 1.0E+06 1.5E-03 1.4E-09 S A Cows 9.8E+05 4.9E-02 5.0E-08 SA Rabbit No binding

KDs are defined as 0.1, 0.45, 3.8, 3.0, 8.0, 1.4 and 50 nM for human, cynomolgus, mouse, rat, pig, dog and cow serum albumin binding, respectively. with the RLN analogue from example 2.

Table 3

Binding kinetics of the RLN analogue of Example 3 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd (Esec) _KD (M) Human SA 1.4E+06 Ι,ΙΕ-04 7.6E-11 S A Javanese Macaque 1.5E+06 5.4E-04 3.6E-10 S A Mice 1.7E+06 4.0E-03 2.4E-09 S A Rats 1.7E+06 2.7E-03 1.6E-09 SA Pigs Ι,ΙΕ+06 5, ZE-OZ 4.8E-09 SA Dogs 2.0E+06 1.4E-03 7.0E-10 S A Cows n/a (equilibrium state) 2.6E-08 SA Rabbit No binding

KDs are defined as 0.08, 0.36, 2.4, 1.6, 4.8, 0.7 and 26 nM for human, cynomolgus, mouse, rat, pig, dog and cow serum albumin binding, respectively. with the RLN analogue from example 3.

Table 4

Binding kinetics of the RLN analogue of Example 4 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd (1/sec) _KD (M) Human SA 1.2E+06 Ι,ΙΕ-04 8.9E-11 S A Javanese Macaque 1,ЗЭ+06 5,ZE-04 4.2E-10 S A Mice 1,ЗЭ+06 3.9E-03 3.0E-09 S A Rats 1.2E+06 2.6E-03 2DE-09 SA Pigs 8.6E+05 5,ZE-OZ 6.2E-09 SA Dogs 1.4E+06 1.4E-03 1.0E-09 S A Cows n/a (equilibrium state) 3.7E-08 SA Rabbit No binding

KDs are defined as 0.09, 0.42, 3.0, 2.1, 6.2, 1.0 and 37 nM for human, cynomolgus, mouse, rat, pig, dog and cow serum albumin binding, respectively. with analogue RLN from

- 23 046279 example 4.

Table 5

Binding kinetics of the RLN analogue of Example 5 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd (Esec) _KD (M) Human SA 5.8E+05 2.6E-04 4.5E-10 S A Javanese Macaque 8.2E+05 1.5E-03 1.8E-09 S A Mice 5.9E+05 8, ZE-OZ 1.4E-08 S A Rats 7.0E+05 7.2E-03 1.0E-08 SA Pigs 5.8E+05 1,ZE-02 2,ZE-08 SA Dogs 7.1E+05 3.4E-03 4.7E-09 S A Cows n/a (equilibrium state) 1.2E-07 SA Rabbit No binding

KDs are determined to be 0.45, 1.8, 14, 10, 23, 4.7 and 120 nM for binding human, cynomolgus, mouse, rat, pig, dog and bovine serum albumin, respectively, to the RLN analogue of Example 5 .

Table 6

Binding kinetics of the RLN analogue of Example 6 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd (Esec) _KD (M) Human SA 4.7E+05 2.4E-04 5.1E-10 S A Javanese Macaque 4,ЗЭ+05 1.0E-03 2,ZE-09 S A Mice 6,ЗЭ+05 7.6E-03 1.2E-08 S A Rats 4.9E+05 4.8E-03 9.7E-09 SA Pigs 4.1E+05 9.9E-03 2.4E-08 SA Dogs 4.8E+05 2.0E-03 4DE-09 S A Cows n/a (equilibrium state) 1,ZE-07 SA Rabbit No binding

KDs are determined to be 0.51, 2.3, 12, 9.7, 24, 4.1, and 130 nM for the binding of human, cynomolgus, mouse, rat, pig, dog, and bovine serum albumin, respectively, to the RLN analogue from example 6.

Table 7

Binding kinetics of the RLN analogue of Example 7 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd (1/sec) _KD (M) Human SA 1.9E+06 1.2E-04 6,ZE-11 S A Javanese Macaque 2.1E+06 5.9E-04 2.9E-10 S A Mice 2.0E+06 4DE-03 2DE-09 S A Rats 2.0E+06 2.8E-03 1.4E-09 SA Pigs 1.5E+06 5.4E-03 3.7E-09 SA Dogs 2.2E+06 1.5E-03 6.9E-10 S A Cows n/a (equilibrium state) 2.5E-08 SA Rabbit No binding

K _D are determined to be 0.06, 0.29, 2.1, 1.4, 3.7, 0.69 and 25 nM for binding human, cynomolgus, mouse, rat, pig, dog and cow serum albumin, respectively , with analogue RLN

- 24 046279 from example 7.

Table 8

Binding kinetics of the RLN analogue of Example 8 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd(1/sec) _KD (M) Human SA 1.5E+06 Ι,ΙΕ-04 7.8E-11 S A Javanese Macaque 1.9E+06 5.9E-04 3.2E-10 S A Mice 2.2E+06 3.9E-03 1.8E-09 S A Rats 1.6E+06 2.8E-03 1.7E-09 SA Pigs 1,ЗЭ+06 5.2E-03 4DE-09 SA Dogs 2,ЗЭ+06 1.5E-03 6.6E-10 S A Cows n/a (equilibrium state) 2DE-08 SA Rabbit No binding

KDs are determined to be 0.08, 0.32, 1.8, 1.7, 4.1, 0.66, and 21 nM for human, cynomolgus, mouse, rat, pig, dog, and cow serum albumin, respectively. with the RLN analogue from example 8.

Table 9

Binding kinetics of the RLN analogue of Example 9 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd (1/sec) _KD (M) Human SA 9.5E+05 1.8E-04 1.9E-10 S A Javanese Macaque Ι,ΙΕ+06 9.4E-04 8.6E-10 S A Mice 1.2E+06 Ι,ΙΕ-02 9.5E-09 S A Rats 9.1E+05 7.5E-03 8.2E-09 SA Pigs 8.7E+05 1.2E-02 1.4E-08 SA Dogs 9.5E+05 3.6E-03 3.8E-09 S A Cows n/a (equilibrium state) 8.4E-08 SA Rabbit No binding

KDs are determined to be 0.19, 0.86, 9.5, 8.2, 14, 3.8, and 84 nM when binding human, cynomolgus, mouse, rat, pig, dog, and bovine serum albumin, respectively, to the analog RLN from example 9.

Table 10

Binding kinetics of the RLN analogue of Example 10 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd (1/sec) _KD (M) Human SA 5.5E+05 2.0E-04 3.7E-10 S A Javanese Macaque 5.1E+05 7.7E-04 1.5E-09 S A Mice 7.4E+05 7.2E-03 9.7E-09 S A Rats 6,ЗЭ+05 4.9E-03 7.8E-09 SA Pigs 4.9E+05 9.6E-03 2.0E-08 SA Dogs 6,ЗЭ+05 2DE-03 3.4E-09 S A Cows n/a (equilibrium state) 1.0E-07 SA Rabbit No binding

KDs are determined to be 0.37, 1.5, 9.7, 7.8, 20, 3.4, and 100 nM when binding human, cynomolgus, mouse, rat, pig, dog, and bovine serum albumin, respectively, to the analog RLN from

- 25 046279 example 10.

Table 11

Binding kinetics of the RLN analogue of Example 11 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C.

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd (Esec) _KD (M) Human SA 1.2E+06 2DE-04 1.7E-10 S A Javanese Macaque 1,ЗЭ+06 6.4E-04 4.8E-10 S A Mice 1.4E+06 5,OE-O3 3.6E-09 S A Rats 2.1E+06 2.8E-03 1,ZE-09 SA Pigs 8.5E+05 6.2E-03 7,ZE-09 SA Dogs 1.4E+06 1.7E-03 1,ZE-09 S A Cows n/a (equilibrium state) 2.7E-08 SA Rabbit No binding

KDs are defined as 0.17, 0.48, 3.6, 1.3, 7.3, 1.3 and 27 nM for human, cynomolgus, mouse, rat, pig, dog and cow serum albumin binding, respectively. with the RLN analogue from example 11.

Table 12

Binding kinetics of the RLN analogue of Example 12 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd (Esec) _KD (M) Human SA 1.4E+06 E9E-04 E4E-10 S A Javanese Macaque 1.5E+06 7.0E-04 4.7E-10 S A Mice 1.8E+06 4.9E-03 2.6E-09 S A Rats 2.0E+06 3,ZE-OZ 1.7E-09 SA Pigs 1.4E+06 5.8E-03 4.2E-09 SA Dogs 2.1E+06 1.6E-03 7.7E-10 S A Cows n/a (equilibrium state) 3.0E-08 SA Rabbit No binding

K _D are determined to be 0.14, 0.47, 2.6, 1.7, 4.2, 0.77 and 30 nM for binding human, cynomolgus, mouse, rat, pig, dog and cow serum albumin, respectively , with the RLN analogue from example 12.

Table 13

Binding kinetics of the RLN analogue of Example 13 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°C

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd (1/sec) _KD (M) Human SA 1.4E+06 1.9E-04 1.4E-10 S A Javanese Macaque 1.4E+06 7.0E-04 4.9E-10 S A Mice 1.8E+06 4.8E-03 2.6E-09 S A Rats 1.7E+06 3,ZE-OZ 2.0E-09 SA Pigs 1.2E+06 6.0E-03 5.0E-09 SA Dogs 1.9E+06 1.7E-03 9.2E-10 S A Cows n/a (equilibrium state) 2.9E-08 SA Rabbit No binding

KDs are determined to be 0.14, 0.49, 2.6, 2.0, 5.0, 0.92, and 29 nM for human, cynomolgus, mouse, rat, pig, dog, and cow serum albumin, respectively. with analogue RLN

- 26 046279 from example 13.

Table 14

Binding kinetics of the RLN analogue of Example 14 to human, cynomolgus, mouse, rat, pig, dog, cow and rabbit serum albumin at 25°

Binding to Immobilized Serum Albumin (SA) k _a (1/Msec) kd(1/sec) _KD (M) S A Person 1.2E+06 1.9E-04 1.6E-10 S A Javanese Macaque Ι,ΙΕ+06 6.8E-04 6DE-10 S A Mice 1.5E+06 4.6E-03 ZDE-09 S A Rats 1.4E+06 3.2E-03 2.2E-09 SA Pigs 9.7E+05 5.8E-03 6.0E-09 SA Dogs 1.5E+06 1.7E-03 Ι,ΙΕ-09 S A Cows n/a (equilibrium state) 3.4E-08 SA Rabbit No binding

KDs are defined as 0.16, 0.61, 3.1, 2.2, 6.0, 1.1 and 34 nM for human, cynomolgus, mouse, rat, pig, dog and cow serum albumin binding, respectively. with the RLN analogue from example 14.

Example 18: In vitro efficacy of an RLN analogue on the RXFP1 HRXFP2 receptors.

Establishment of the CHO-CRE-Luc cell line: CHO-K1 cells (ATCC) are cultured in DMEM-F12 3:1 with 5% FBS with 20 mM GEPES, 40 μg/ml L-proline, 1x antibiotic solution and divided 1:5 every 2-3 days using TrypLE™ Express enzyme (Gibco). Cells were transfected with pGL4.29 plasmid DNA [luc2P/CRE/Hygro] (Promega) and Fugene HD (Promega) according to the manufacturer's instructions. Transfected cells are selected by selection on hygromycin B at a concentration of 1 mg/ml for 3-4 weeks. Clonal lines were generated by limited dilution cloning in 96-well plates and confirmed by forskolin response using a luciferase reporter gene assay with Bright-Glo reagent (Promega). Clones are propagated, collected, resuspended in cell freezing medium, aliquoted into cryovials, and stored in liquid nitrogen for long-term storage. The best cell line with strong antibody-producing properties was selected based on the best response to forskolin (signal to background ratio), clonal line no. 2B6, for subsequent transfection using human RXFP1 and RXFP2 receptors.

Creation of cell lines expressing human RXFP1 and human RXFP2: CHO-CRELuc cells line No. 2B6 are cultured in DMEM-F12 3:1 with 5% FBS with 20 mM GEPES, 40 μg/ml L-proline, 1x antibiotic solution, 1 mg/ ml of hygromycin B and divided 1:5 every 2-3 days using the TrypLE Express enzyme (Gibco). Cells were transfected with human RXFP1 receptor or human RXFP2 receptor plasmid DNA and Fugene FID (Promega) according to the manufacturer's instructions. Transfected cells are selected by selection on hygromycin B (1 mg/ml) and puromycin (6 μg/ml) for 3-4 weeks. Clonal lines are obtained by limited dilution cloning in 96-well plates. Clonal lines were confirmed by responding to human RLN2. Clones are propagated, collected, resuspended in cell freezing medium, aliquoted into cryovials, and stored frozen in liquid nitrogen for long-term storage. Clonal lines are selected based on the best response to human RLN2 (signal to background ratio) to validate the assay.

Human RXFP1 and RXFP2 receptor luciferase reporter gene assay: CHO cell lines expressing human RXFP1 or human RXFP2 are cultured in selective medium (DMEM-F12 3:1 with 5% FBS with 20 mM GEPES, 40 μg/ml L-proline, 1x antibiotic solution, 6 µg/ml puromycin, 1 mg/ml hygromycin B). On day -1 (the day before cAMP CRE luciferase reporter gene assay), cells were washed once with PBS and removed from flasks containing cell dissociation solution (enzyme-free cell dissociation solution, GIBCO catalog number 13151-014:TrypLE™ Express= 30:1) and resuspended in culture medium in bacteriological dishes (DMEM-F12 3:1 with 20 mM GEPES, 1x antibiotic solution, 0.5% FBS). Cells were seeded into a 96-well plate (Falcon, catalog number 353219) at a rate of 20,000 cells/0.1 ml/well. Cells were cultured in an incubator at 37°C, 5% CO2 overnight. On day 1 (cAMP CRE luciferase reporter gene assay day), the medium is removed and replaced with 90 μl of serum-free medium (DMEM-F12 3:1 with 20 mM GEPES, 1x antibiotic solution). The plates are incubated at 37°C for 2 h, then 10 μl of 10x ligand (RLN2, final 1x) is added. The plates are incubated for another 4 hours at 37°C. At the end of the incubation period, the dishes are brought to room temperature for 15 minutes. 50 µl of Bright-Glo™ is then added to each well and the plates are read using a Biotek Neo2 reader with

- 27 046279 Gen5 software. Statistical data analysis: Data are imported from a Biotek Neo2 reader into GraphPad Prism® software (GraphPad Software, LLC; La Jolla, CA; version 7). The EC ₅₀ value was obtained from a four-parameter dose-response curve with variable slope.

Table 15

In vitro efficacy of RLN analogues on RXPF1 HRXPF2 receptors

Compound SEQ ID NO: ECso hRXFPl, nM, Geometric Mean SEM (standard error of the mean) N ECso hRXFP2, nM, Geometric Mean SEM (standard error of the mean) N R2/R1 ratio hRLN2 5 and 6 0.15 0.012 4 1.7 0.45 8 AND Example 1 24 3.0 2.8 6 65 17 2 22 Example 2 25 1.1 0.64 6 79 6.2 2 74 Example 3 26 4.0 1.0 2 19 2.4 4 5 Example 4 27 2.9 0.2 2 24 6.0 4 8 Example 5 28 8.1 2.9 2 98 16 4 12 Example 6 29 15 h,o 2 39 4.9 4 3 Example 7 thirty 0.91 0.04 2 26 2.5 4 28 Example 8 31 2.0 1.2 2 32 14 4 16 Example 9 32 5.2 0.01 2 97 14 4 19 Example 10 33 14 0.33 2 32 3.9 4 2 Example I 34 3.2 0.69 2 207 31 4 65 Example 12 35 2.9 0.51 2 193 29 4 66 Example 13 36 10 0.8 2 407 17 3 42 Example 14 37 17 2.3 2 628 51 3 37 Example 15 38 1.7 0.13 5 23 3.6 4 14 Example 16 39 2.0 0.22 4 70 23 2 35

In vivo activity.

Example 19. Pharmacokinetics of RLN analogues in male Sprague-Dawley rats.

Male Sprague-Dawley rats are administered subcutaneously with a single dose of 200 nmol/kg of the RLN analogue in His-NaCl buffer (pH 6.0) in a volume of 1.0 ml/kg. Blood was collected at 3, 6, 12, 24, 48, 72, 96, 120, 144, 168, and 240 hours postdose for pharmacokinetic characterization.

Plasma concentrations of RLN analogues were determined using a validated LC/MS method at Eli Lilly and Company. Example compounds and an analogue internal standard were extracted from 100% rat plasma using anti-human RLN antibody, followed by N-terminal tryptic peptide detection using a Q-Exactive™ Orbitrap® mass spectrometer.

Data for the RLN analogues from examples 4 and 7 are presented below in table. 16.

Table 16

Mean plasma pharmacokinetic parameters for RLN analogues following a single 200 nmol/kg subcutaneous dose in male Sprague-Dawley rats

Compound Animal identification number 1½ (h) Tshakh (h) Stach (nmol/l) AUCo-inf (h* nmol/l) CL/F (ml/h/kg) Example 4 1 28.0 72 297 35510 5.63 2 28.8 48 368 37529 5.33 3 32.8 48 356 39220 5.10 Average 29.9 56 340 37419 5.35 SD 2.6 14 38 1857 0.27 Example 7 1 40.1 48 387 51939 3.85 2 37.9 72 487 57843 3.46 3 31.3 48 344 44409 4.50 Average 36.4 56 406 51397 3.94 SD 4.6 14 74 6734 0.53

Note: Abbreviations: t ¹ / ₂ = half-life, T _max = time of maximum concentration, C _max = maximum observed plasma concentration, AUC _0-inf = area under the curve from time point 0 h to infinity, CL/F = clearance/ bioavailability, N=3 animals/group/time point.

As presented in table. 16, the RLN analogues of Examples 4 and 7 demonstrate an enhanced pharmacokinetic profile in Sprague-Dawley rats.

Example 20. In vivo effect of RLN analogues on renal blood flow after intravenous administration to male Sprague-Dawley rats.

- 28 046279

Five-week-old male Sprague-Dawley rats (Charles River Laboratories, Inc.) were housed in a vivarium on a normal day/night cycle for one week prior to the start of the experiment. Rats were then randomized to the following treatment groups: vehicle (20 mM His/20 mM NaCl, pH 6.0 buffer) and RLN analogue from Example 7 based on body weight. The RLN analogue is administered at a dose of 2.44 mcg/kg via IV bolus followed by an IV infusion of 0.36 mcg/kg/h, at a dose of 8.13 mcg/kg via IV bolus followed by IV infusion of 1.2 mcg/kg/h, at a dose of 24.4 mcg/kg via IV bolus followed by IV infusion of 3.6 mcg/kg/h, and at a dose of 81.3 mcg /kg using an IV bolus followed by an IV infusion of 11.9 mcg/kg/hour.

To evaluate the effect of the RLN analogue on renal blood flow, rats were anesthetized using urethane (1.2 g/kg, i.p.) and prepared for abdominal/renal ultrasound scans and renal artery blood flow measurements using pulsed wave Doppler (VisualSonics, Ultrasound System , model Vevo 3100; Fujifilm). A long-term catheter is placed in the tail vein for IV bolus and infusion. After a 30-minute acclimation period, baseline renal blood flow is measured, and renal blood flow is measured 3 hours after dosing begins.

Table 17

Effect of IV administered RLN analogue on renal blood flow in healthy rats

Compound and Dose Time Renal Blood Flow (ml/min) Percentage Change from Media (%) Vehicle with an IV bolus of 1.6 ml/kg followed by an IV infusion of 0.475 ml/kg/h baseline 8.58 ±0.16 in 3 hours 8.56 ±0.18 — RLN analogue from Example 7 with IV bolus administration of 2.44 mcg/kg followed by IV infusion of 0.36 mcg/kg/h baseline 8.49 ±0.31 in 3 hours 8.59 ±0.33 0.4 RLN analogue from Example 7 with IV bolus administration of 8.13 mcg/kg followed by IV infusion of 1.2 mcg/kg/h baseline 8.63 ± 0.44 in 3 hours 10.84 ±0.53f* 26.3 RLN analogue from Example 7 with IV bolus administration of 24.4 mcg/kg followed by IV infusion of 3.6 mcg/kg/h baseline 8.54 ±0.23 in 3 hours 12.74 ±0.37f* 48.8 RLN analogue from Example 7 with IV bolus administration of 81.3 mcg/kg followed by IV infusion of 11.9 mcg/kg/h baseline 8.34 ±0.15 in 3 hours 13.73 ±0.47f* 60.4

Note: Results are expressed as mean ± standard deviation, f significantly different from baseline p < 0.001 ANOVA, * significantly different from vehicle ANOVA p < 0.001, N = 5 per treatment group.

As can be seen from table. 17, the RLN analogue from Example 7 significantly increases renal blood flow in groups with an IV bolus of 8.13 μg/kg followed by an IV infusion of 1.2 μg/kg/h, with an IV bolus of 24.4 μg /kg followed by an IV infusion of 3.6 mcg/kg/h, with an IV bolus injection of 81.3 mcg/kg followed by an IV infusion of 11.9 mcg/kg/h, at 26.6, 48.8 and 60.4% after 3-hour exposure.

Example 21. In vivo effect of RLN analogues on renal blood flow after subcutaneous administration to male Sprague-Dawley rats.

Five-week-old male Sprague-Dawley rats (Charles River Laboratories, Inc.) were housed in a vivarium on a normal day/night cycle for one week prior to the start of the experiment. Rats were then randomized to the following treatment groups: vehicle (20 mM His/20 mM NaCl, pH 6.0 buffer) and RLN analogue from Example 7 based on body weight. The RLN analogue is a 180 mcg/kg SC dose.

To evaluate the effect of the RLN analogue on renal blood flow, 48 hours after dosing, rats were anesthetized using urethane (1.2 g/kg, i.p.) and prepared for abdominal/renal ultrasound scans and pulsed wave measurements of renal artery blood flow. Dopplerography (VisualSonics, ultrasound system, model Vevo 3100; Fujifilm). After a 30-minute acclimation period, renal blood flow measurements are taken.

- 29 046279

Table 18

Effect of a subcutaneously administered RLN analogue on renal blood flow in healthy rats

Compound and Dose Renal Blood Flow (ml/min) Percentage Change from Media (%) Vehicle at a dose of 5 ml/kg 8.51 ±0.33 RLN analogue from Example 7 at a dose of 180 μg/kg 11.27 ± 0.81* 32.4

Note: Results are expressed as mean ± standard deviation, * significantly different from vehicle ANOVA p < 0.001, N = 10 per treatment group.

As can be seen from table. 18, the RLN analogue of Example 7 significantly increased renal blood flow after 48 hours of exposure when administered at a subcutaneous dose of 180 μg/kg.

Sequences

The following nucleic acid and/or amino acid sequences are included in the disclosure of this invention and are provided below for reference.

SEQ Ш NO:1 - human npo-RLNl

MPRLFLFHLLEFCLLLNQFSRAVAAKWKDDVIKLCGRELVRAQIAICGMSTWSKRSLSQ EDAPQTPRPVAEIVPSFINKDTETIIIMLEFIANLPPELKAALSERQPSLPELQQYVPALKDS NLSFEEFKKLIRNRQSEAADSNPSELKYLGLDTHSQKKRRPYVALFEKCCLIGCTKRSLA KYC

SEQ Ш NO:2 - Human A-chain RLN1

PYVALFEKCCLIGCTKRSLAKYC

SEQ Ш NO:3 - Human B-chain RLN1

VAAKWKDDVIKLCGRELLVRAQIAICGMSTWS

SEQ Ш NO:4 - human npo-RLN2

MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLSQ EDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVL KDSSLLFEEFKKLIRNRQSEAADSPSELKYLGLDTHSRKKRQLYSALANKCCHVGCTK RSLARFC

SEQ Ш NO:5 - Human A-chain RLN2

QLYSALANKCCHVGCTKRSLARFC

SEQ Ш NO:6 - Human RLN2 B-chain

DSWMEEVIKLCGRELLVRAQIAICGMSTWS

[0257] SEQ Ш NO:7 - human npo-RLN3

MARYMLLLLLAVWVLTGELWPGAEARAAPYGVRLCGREFIRAVIFTCGGSRWRRSDIL AHEAMGDTFPDADADEDSLAGELDEAMGSSEWLALTKSPQAFYRGRPSWQGTPGVLR GSRDVLAGLS S SCCKWGCSKSEIS SLC

- 30 046279

SEQ Ш NO:8 - Human A-chain RLN3

DVLAGLSSSCCKWGCSKSEISSLC

SEQ Ш NO:9 - Human RLN3 B-chain

RAAPYGVRLCGREFIRAVIFTCGGSRW

SEQ Ш NO: 10 - fragment VHH No. 1 (C22)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO: 11 - VHH fragment No. 2 (C22.43)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPP

SEQ Ш NO: 12 - fragment VHH No. 3 (C80)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO: 13 - fragment VHH No. 4 (C80.43)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPP

SEQ Ш NO: 14 - Li (main sequence (GGGGQ) _n ) GGGGQ

SEQ Ш NO: 15 - Li (main sequence (GGGQ) _n )

GGGQ

- 31 046279

SEQ Ш NO: 16 - Li (main sequence (GGGGS) _n ) GGGGS

SEQ Ш NO: 17 - Li (main sequence (PGPQ) _n ) PGPQ

SEQ Ш NO: 18 - Li (main sequence (PGPA) _n )

PGPA

SEQ ID NO: 19 - Li #1 ((GGGGQ)s)

GGGGQGGGGQGGGGQGGGGQGGGGQ

SEQ ID NO:20 - Li No.2 ((PGPQ)s)

PGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQ

SEQ ID NO:21 - Li No.3 (PGPA) ₈

PGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPA

SEQ ID NO:22 - L ₂ No. 1

GGGSGGSGGG

SEQ ID NO:23 - L ₂ No. 2

GGGSGGSGGSGGG

SEQ ID NO:24 - Analogue RLN2 No. 1 (C22-(G4Q) ₅ -A10B(desBl)RLN)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQQLYSALANKCCHVGCTKRSLAR FCGGGSGGSGGGSWMEEVIKLCGRELVRAQIA ICGMSTWS

- 32 046279

SEQ Ш NO:25 - Analogue RLN2 No. 2 (C22-(PGPA) ₈ -A10B(desBl)RLN)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAQLYSALANKCCHVGCTKR SLARFCGGGSGGSGGGSWMEEVIKLCGRELVRAQ IAICGMSTWS

SEQ Ш NO:26 - Analogue RLN2 No. 3 (C22-(G4Q) ₅ -B10A(desBl)RLN)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICG MSTWSGGGSGGSGGGQLYSALANKCCHVGCTK RSLARFC

SEQ Ш NO:27 - Analogue RLN2 No. 4 (C22-(PGPQ) ₈ -B10A(desBl)RLN)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSWMEEVIKLCGRELVRAQI AICGMSTWSGGGSGGSGGGQLY SALANKCCHVGCTKRSLARFC

SEQ Ш NO:28 - Analogue RLN2 No. 5 (B10A(desBl)RLN-(G4Q) ₅ -C22.43)

SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSL ARFCGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGR YIDETAVAWFRQAPGI<EREFVAGIGGVDITYYADSVI<GRFTISRDNSI<NTLYLQMNSL RPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPP

SEQ ID NO:29 - Analogue RLN2 No. 6 (B10A(desBl)RLN-(PGPQ) ₈ -C22.43)

SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSL ARFCPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGKEREFVAGIGGVDITYYADSVKGRFTISRDNSKNTLYLQM NSLRPEDTAVYYCAARPGRPLI TSKVADLYPYWGQGTLVTVSSPP

SEQ ID NO:30 - Analogue RLN2 No. 7 (C80-(G4Q) ₅ -B10A(desBl)RLN)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICG MSTWSGGGSGGSGGGQLYSALANKCCHVGCTK RSLARFC

- 33 046279

SEQ Ш NO:31 - Analogue RLN2 No. 8 (C80-(PGPQ)s-B10A(desBl)RLN)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQSWMEEVIKLCGRELVRAQI AICGMSTWSGGGSGGSGGGQLY SALANKCCHVGCTKRSLARFC

SEQ Ш NO:32 - Analogue RLN2 No. 9 (B10A(desBl)RLN-(G4Q) ₅ -C80.43) SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSL ARFCGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGR YIDETAVAWFRQAPGI<GREFVA GIGGGVDITYYADSVI<GRFTISRDNSI<NTLYLQMNSL RPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

SEQ Ш NO:33 - Analogue RLN2 No. 10 (B10A(desBl)RLN-(PGPQ)s-C80.43) SWMEEVIKLCGRELVRAQIAICGMSTWSGGGSGGSGGGQLYSALANKCCHVGCTKRSL ARFCPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETA VAWFRQAPGI<GREFVAGIGGVDITYYADSVI<GRFTISRDNSI<NTLYLQM NSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

SEQ Ш NO:34 - Analogue RLN2 No. 11 (C22-(G4Q) ₅ -B13A(desBl, desAl-4)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS GGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICG MSTWSGGGSGGSGGSGGGALANKCCHVGCTKRSLARFC

SEQ Ш NO:35 - Analogue RLN2 No. 12 (C80-(G4Q) ₅ -B13A(desBl, desAl-4)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS GGGGQGGGGQGGGGQGGGGQGGGGQSWMEEVIKLCGRELVRAQIAICG MSTWSGGGSGGSGGSGGGALANKCCHVGCTKRSLARFC

SEQ Ш NO:36 - Analogue RLN2 No. 13 (C80-(G4Q) ₅ -A10B(desBl, desAl-4)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

- 34 046279

QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQALANKCCHVGCTKRSLARFCGG GSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS

SEQ Ш NO:37 - Analogue RLN2 No. 14 (C80-(PGPQ)s-A10B(desBl, desAl-4)RLN) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSP GPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQALANKCCHVGCTKRSLAR FCGGGSGGSGGGSWMEEVIKLCGRELVRAQIAICGMSTWS

SEQ Ш NO:38 - Analogue RLN2 No. 15 (C22-(G4Q) ₅ -B10ARLN)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQDSWMEEVIKLCGRELVRAQIAIC GMSTWSSGGGGSGGGGQLYSALANKCCHVGCTK RSLARFC

SEQ Ш NO:39 - Analogue RLN2 No. 16 (C22-(PGPA)8-B10ARLN)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPADSWMEEVIKLCGRELVRA QIAICGMSTWSSGGGGSGGGGQLYSALANKCCHVGCT KRSLARFC

SEQ Ш NO:40 - human RXFP1 receptor

MTSGSVFFYILIFGKYFSHGGGQDVKCSLGYFPCGNITKCLPQLLHCNGVDDCGNQADE DNCGDNNGWSLQFDKYFASYYKMTSQYPFEAETPECLVGSVPVQCLCQGLELDCDETN LRAVPSVSSNVTAMSLQWNLIRKLPPDCFKNYHDLQKLYLQNNKITSISIYAFRGLNSLT KLYLSHNRITFLKPGVFE DLHRLEWLIIEDNHLSRISPPTFYGLNSLILLVLMNNVLTRLPD KPLCQHMPRLHWLDLEGNHIHNLRNLTFISCSNLTVLVMRKNKINHLNENTFAPLQKLD ELDLGSNKIENLPPLIFKDLKELSQLNLSYNPIQKIQANQFDYLVKLKSLSLEGIEISNIQQ RMFRPLMNLSHIYFKKFQYCGYAPHVRSCKPNTDGISSLE H QSAITATEIRNQVKKEMILAKRFFFIVFTDALCWIPIFVVKFLSLLQVEIPGTITSWVVIFI LPINSALNPILYTLTTRPFI<EMIHRFWYNYRQRI<SMDSI<GQI<TYAPSFIWVEMWPLQEM PPELMKPDLFTYPCEMSLISQSTRLNSYS

- 35 046279

SEQ Ш NO:41 - human RXFP2 receptor

MIVFLVFKHLFSLRLITMFFLLHFIVLINVKDFALTQGSMITPSCQKGYFPCGNLTKCLPR AFHCDGKDDCGNGADEENCGDTSGWATIFGTVHGNANSVALTQECFLKQYPQCCDCK ETELECVNGDLKSVPMISNNVTLLSLKKNKIHSLPDKVFIKYTKLKKIFLQHNCIRHISRK AFFGLCNLQILYLN HNCITTLRPGIFKDLHQLTWLILDDNPITRISQRLFTGLNSLFFLSMV NNYLEALPKQMCAQMPQLNWVDLEGNRIKYLTNSTFLSCDSLTVLFLPRNQIGFVPEKT FSSLKNLGELDLSSNTITELSPHLFKDLKLLQKLNLSSNPLMYLHKNQFESLKQLQSLDLE RIEIPNINTRMFQPMKNLSHIYFKNFRY CSYAPHVRICMPLTDGISSFEDLLANNILRIFVW VIAFITCFGNLFVIGMRSFIKAENTTHAMSIKILCCADCLMGVYLFFVGIFDIKYRGQYQK YALLWMESVQCRLMGFLAMLSTEVSVLLLTYLTLEKFLVIVFPFSNIRPGKRQTSVILICI WMAGFLIAVIPFWNKDYFGNFYGKNGVCFPLYYDQTEDIGSKGYSLGIFLG VNLLAFLII VFSYITMFCSIQKTALQTTEVRNCFGREVAVANRFFFIVFSDAICWIPVFVVKILSLFRVEI PDTMTSWIVIFFLPVNSALNPILYTLTTNFFKDKLKQLLHKHQRKSIFKIKKKSLSTSIVWI EDSSSLKLGVLNKITLGDSIMKPVS

SEQ Ш NO:42 - human RXFP3 receptor

MQMADAATIATMNKAAGGDKLAELFSLVPDLLEAANTSGNASLQLPDLWWELGLELP DGAPPGHPPGSGGAESADTEARVRILISVVYWVVCALGLAGNLLVLYLMKSMQGWRK SSINLFVTTNLALTDFQFVLTLPFWAVENALDFKWPFGKAMCKIVSMVTSMNMYASVFF LTAMSVTRYHSVASALKSHRTRGHGRGDCCGRSLGDSCC FSAKALCVWIWALAALASL PSAIFSTTVKVMGEELCLVRFPDKLLGRDRQFWLGLYHSQKVLLGFVLPLGIIILCYLLLV RFIADRRAAGTKGGAAVAGGRPTGASARRLSKVTKSVTIVVLSFFLCWLPNQALTTWSI LIKFNAVPFSQEYFLCQVYAFPVSVCLAHSNSCLNPVLYCLVRREFRKALKSLLWRIASP SITSMRPFTA TTKPEHEDQGLQAPAPPHAAAEPDLLYYPPGWVYSGGRYDLLPSSSAY

SEQ Ш NO:43 - human RXFP4 receptor

MPTLNTSASPPTFFWANASGGSVLSADDAPMPVKFLALRLMVALAYGLVGAIGLLGNL AVLWVLSNCARRAPGPPSDTFVFNLALADLGLALTLPFWAAESALDFHWPFGGALCKM VLTATVLNVYASIFLITALSVARYWVVAMAAGPGTHLSLFWARIATLAVWAAAALVTV PTAVFGVEGEVCGVRLCLLRFPSRYWLGAYQLQRVVLA FMVPLGVITTSYLLLLAFLQR RQRRRQDSRVVARSVRILVASFFLCWFPNHVVTLWGVLVKFDLVPWNSTFYTIQTYVFP VTTCLAHSNSCLNPVLYCLLRREPRQALAGTFRDLRLRLWPQGGGWVQQVALKQVGR RWVASNPRESRPSTLLTNLDRGTPG

- 36 046279

SEQ Ш NO:44 - signal peptide

METDTLLLWVLLLWVPGSTG

SEQ Ш NO:45 - fragment VHH No. 5 (MC6.1)

EVQLLESGGGLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWG QGTLVTVSS

SEQ Ш NO:46 - fragment VHH No. 6 (MC6.1C6)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDSTAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSRVANLYPYWG QGTLVTVSS

SEQ Ш NO:47 - fragment VHH No. 7 (C22-G26Y)

EVQLLESGGGLVQPGGSLRLSCAASYRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:48 - fragment VHH No. 8 (C22-R27A)

EVQLLESGGGLVQPGGSLRLSCAASGAYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:49 - fragment VHH No. 9 (C22-I57E)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDET YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYW GQGTLVTVSS

SEQ Ш NO:50 - fragment VHH No. 10 (C22-I57Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDQT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYW GQGTLVTVSS

- 37 046279

SEQ Ш NO:51 - fragment VHH No. 11 (C22-Y59A)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITA YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO:52 - VHH fragment #12 (C22-Y59E)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITE YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:53 - fragment VHH No. 13 (C22-Y59Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITQ YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:54 - fragment VHH No. 14 (C22-Y59S)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITS YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:55 - fragment VHH No. 15 (C22-Y59T)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITT YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:56 - fragment VHH No. 16 (C22-R102K)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGKPLITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:57 - fragment VHH No. 17 (C22-R102Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGQPLITSKVADLYPYWG QGTLVTVSS

- 38 046279

SEQ Ш NO:58 - fragment VHH No. 18 (C22-R102S)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGSPLITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:59 - fragment VHH No. 19 (C22-P103E)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRELITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:60 - fragment VHH No. 20 (C22-P103Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRQLITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:61 - fragment VHH No. 21 (C22-P103S)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRSLITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:62 - fragment VHH No. 22 (C22-L104E)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPEITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:63 - fragment VHH No. 23 (C22-L104G)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPGITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:64 - fragment VHH No. 24 (C22-L104Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPQITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:65 - fragment VHH No. 25 (C22-L104T)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPTITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:66 - fragment VHH No. 26 (C22-S107E)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITEKVADLYPYWG QGTLVTVSS

SEQ Ш NO:67 - L ₂ №3

SGGGGSGGGG

Claims (23)

1. A compound containing the structure: VIHI-L-A-L-B. VIHI-L-B-L-A. A-L2-B-L1-VHH or B-L2-AL ₁ -VHH; where VHH contains an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 10, 11, 12 and 13, or a sequence having at least 90% similarity thereto; where A is a relaxin A chain containing an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 2, 5 and 8, or a sequence having at least 90% similarity thereto; where B represents a relaxin B chain containing an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 3, 6 and 9, or a sequence having at least 90% similarity thereto; where L ₁ is a first linker containing an amino acid sequence selected from the group consisting of (GGGGQ)n (SeQ ID NO: 14), (PGPQ)n (SEQ ID NO: 17) and (PGPA)n (SEQ ID NO: : 18), and where n can be from 1 to 10; and where L2 is a second linker containing an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 22, 23 and 67; or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, characterized in that A represents the sequence of SEQ ID NO: 2. 3. A compound according to claim 1 or 2, characterized in that B represents the sequence of SEQ ID NO: 3. 4. The compound according to claim 1, characterized in that A represents the sequence of SEQ ID NO: 5. 5. A compound according to claim 1 or 2, characterized in that B represents the sequence of SEQ ID NO: 6. 6. The compound according to claim 1, characterized in that A represents the sequence of SEQ ID NO: 5, in which the first four amino acids (desA1-4) are missing. 7. The compound according to claim 1, characterized in that B represents the sequence of SEQ ID NO: 6, in which the first amino acid (desB1) is missing. 8. The compound according to claim 1, characterized in that A represents the sequence of SEQ ID NO: 5, which lacks the first four amino acids (desA1-4), and B represents the sequence of SEQ ID NO: 6, which lacks first amino acid (desB1). 9. The compound according to claim 1, characterized in that A represents the sequence of SEQ ID NO: 8. 10. The compound according to claim 1 or 2, characterized in that B represents the sequence of SEQ ID NO: 9. 11. A compound according to any one of claims 1 to 10, characterized in that L1 is the sequence of SEQ ID NO: 19. 12. A compound according to any one of claims 1 to 10, characterized in that L1 is the sequence of SEQ ID NO: 20. 13. A compound according to any one of claims 1 to 10, characterized in that L ₁ represents the sequence of SEQ ID NO: 21. 14. A compound according to claim 1, containing an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 24-39, or a sequence having at least 90% similarity thereto, or a pharmaceutically acceptable salt thereof. 15. A compound according to claim 1, consisting of an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 24-39, or a sequence having at least 90% similarity thereto, or a pharmaceutically acceptable salt thereof. 16. A pharmaceutical composition containing a compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable buffer. 17. A method for treating pathological conditions, diseases and/or disorders of the heart, lungs and/or kidneys in an individual, comprising the step of: administering to the individual an effective amount of a compound according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16. 18. Use of a compound according to any one of claims 1-15 for the treatment of pathological conditions, diseases - 40 046279 diseases and/or disorders of the heart, lungs and/or kidneys. 19. Use of a compound according to any one of claims 1 to 15 for the production of a medicinal product for the treatment of pathological conditions, diseases and/or disorders of the heart, lungs and/or kidneys. 20. A compound containing the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS (SEQ Ш NO:10) or a sequence having at least 90% similarity thereto. 21. A compound containing the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPP (SEQ Ш NO: И) or a sequence having at least 90% similarity thereto. 22. A compound containing the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSS (SEQ Ш NO: 12) or a sequence having at least 90% similarity thereto. 23. A compound containing the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGT LVTVSSPP (SEQ Ш NO: 13) or a sequence having at least 90% similarity thereto.