Relaxin analogs and methods of use thereof

Abstract

The present invention relates to relaxin analogs and methods of use thereof. Relaxin (RLN) analogs are disclosed that include modifications as compared to native human RLN that increase half-life, maintain selectivity for the RXFP1 receptor, and provide stability in vitro and in vivo to improve druggability and reduce immunogenicity. Also disclosed are pharmaceutical compositions comprising one or more RLN analogs described herein in a pharmaceutically acceptable carrier. Also disclosed are methods of making and using the RLN analogs, particularly for the treatment of cardiovascular, pulmonary and / or renal conditions, diseases or disorders.

Description

[0001] This application is a divisional application of an invention patent application with an application date of July 31, 2020, application number 202080054330.2, and invention name “Relaxin analogs and methods of use thereof”. Technical Field

[0002] The present disclosure relates generally to biology and medicine, and more particularly it relates to relaxin (RLN) analogs, especially long-acting single-chain RLN analogs, which can bind to RLN / insulin-like family peptide (RXFP) receptors, such as RXFP1 receptors, thereby acting as RXFP receptor agonists. The present disclosure further relates to compositions comprising them and their use in treating cardiovascular, pulmonary and / or renal conditions, diseases or disorders. Background Art

[0003] Relaxin (RLN) is part of the insulin superfamily and includes seven peptides with high structural similarity but low sequence similarity in humans - RLN1 (H1RLX, RLXH1 or H1), RLN2 (H2RLX, RLXH2 or H2), RLN3 (RXN3, ZINS4 or H3), insulin-like (INSL) peptide 3 (INSL3), INSL4, INSL5 and INSL6. Of particular interest herein is RLN2, which is a heterodimer of two peptide chains (A chain and B chain) of 24 and 29 amino acids, respectively, linked by two disulfide bonds, wherein the A chain further has one intramolecular disulfide bond (see, Schwabe & McDonald (1977) Science 197: 914-915). RLN2 is generated from its prohormone (prorelaxin) by cleaving the C peptide therefrom.

[0004] Physiologically, RLN2 exhibits multiple functions, regulating cardiovascular, hepatic, neural, pancreatic, pulmonary, and renal adaptations such as vasodilation, 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-containing GPCRs LGR7 and LGR8, now known as 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 poses a challenge when using it as a therapeutic agent. In fact, natural RLN2 has a t of several minutes in vivo. 1 / 2. Therefore, clinicians administer RLN2 by continuous intravenous infusion, which often results in inconvenience and short-term effectiveness for the individual receiving the RLN compound.

[0005] There are many improved t 1For example, International Patent Application Publication No. WO 2018 / 148419 describes analogs that include non-natural amino acid residues (such as p-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 analogs that are fusions of the A chain and the B chain with a linker of at least fifteen amino acids and a half-life extending portion to improve t 1 / 2. International Patent Application Publication No. WO 2017 / 201340 describes analogs that are fusions of the A and B chains with variable light chain fragments to improve t 1 / 2. International Patent Application Publication No. WO 2015 / 067791 describes analogs that are prodrugs linked to carriers, particularly PEG-based carriers, to improve t 1 / 2 (see also WO 2012 / 024452 for additional PEG-linked analogs). International Patent Application Publication No. WO 2014 / 102179 describes analogs that are fusions of the A and B chains with an Fc portion of IgG2 or IgG4 to improve t 1 / 2. International Patent Application Publication No. WO2013 / 004607 describes analogs that are fusions of the A chain and the B chain with a linker of at least five amino acids but less than fifteen amino acids to improve t 1 / 2, or they are fusions of A and B chains with the Fc domain of an antibody to improve t 1 / 2.

[0006] Considering the increased understanding of the various physiological roles of RLNs, there remains a need for improved t 1 / 2 long-acting RLN analog. Summary of the invention

[0007] To meet this need, the present disclosure first describes a single-chain RLN analog that has primary activity on the RXFP1 receptor (i.e., acts as an RXFP1 receptor agonist). Such an RXFP1 receptor agonist includes the following basic structure from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus):

[0008] VHH-L1-A-L2-B,

[0009] VHH-L1-B-L2-A,

[0010] A-L2-B-L1-VHH, or

[0011] B-L2-A-L1-VHH,

[0012] wherein VHH is a portion that can act as a pharmacokinetic enhancer, A is the RLNA chain, B is the RLN B chain, L1 is the first linker, and L2 is the second linker.

[0013] In some cases, the VHH portion can have an amino acid sequence of SEQ ID NO: 10, 11, 12 or 13, in particular SEQ ID NO: 10 or 12. In other cases, the VHH portion can have one or more additions, deletions, insertions or substitutions such that the VHH portion has an amino acid sequence having at least about 90% to about 99% sequence similarity to any one of SEQ ID NO: 10, 11, 12 or 13 (see, e.g., SEQ ID NO: 45-66).

[0014] In some cases, the A chain may have an amino acid sequence of SEQ ID NO: 2, 5 or 8, in particular SEQ ID NO: 5. In other cases, the A chain may have 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 one of SEQ ID NO: 2, 5 or 8. For example, the A chain may be des1-4 of SEQ ID NO: 5.

[0015] In some cases, the B chain may have an amino acid sequence of SEQ ID NO: 3, 6 or 9, in particular SEQ ID NO: 6. In other cases, the B chain may have one or more additions, deletions, insertions or substitutions such that the B chain has an amino acid sequence having at least about 90% to about 99% sequence similarity to any one of SEQ ID NO: 3, 6 or 9. For example, the B chain may be des1 of SEQ ID NO: 6.

[0016] In some cases, L1 can have (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), wherein n can be 1-10, in particular about 5 to about 8. In other cases, L1 can have an amino acid sequence of SEQ ID NO: 19, 20 or 21. In other cases, L1 can have one or more additions, deletions, insertions or substitutions such that L1 has an amino acid sequence having at least about 90% to about 99% sequence similarity to any one of SEQ ID NOs: 14 to 21.

[0017] In some cases, L2 may have the amino acid sequence of SEQ ID NO: 22, 23, or 67. In other cases, L2 may have one or more additions, deletions, insertions, or substitutions.

[0018] In some cases, the RLN analog may have an amino acid sequence comprising: VHH of SEQ ID NO: 10, 11, 12 or 13; A chain of SEQ ID NO: 2, 5 or 8; B chain of SEQ ID NO: 3, 6 or 9; L1 of SEQ ID NO: 19, 20 or 21; and L2 of SEQ ID NO: 22, 23 or 67. Alternatively, the RLN analog may have an amino acid sequence having at least about 90% to about 99% sequence similarity to an amino acid sequence comprising an amino acid sequence comprising: VHH of SEQ ID NO: 10, 11, 12 or 13; A chain of SEQ ID NO: 2, 5 or 8; B chain of SEQ ID NO: 3, 6 or 9; L1 of SEQ ID NO: 19, 20 or 21; and L2 of SEQ ID NO: 22, 23 or 67.

[0019] In certain instances, the RLN analog may have an amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39, in particular SEQ ID NO: 26, 27, 30, 31, 34 or 35. Alternatively, the RLN analog may have an amino acid sequence having at least about 90% to about 99% sequence similarity to an amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39, in particular SEQ ID NO: 26, 27, 30, 31, 34 or 35.

[0020] In some cases, the binding affinity of the RLN analog to the RXFP1 receptor is comparable to the binding affinity of native human RLN2 (SEQ ID NOs: 5 and 6). In other cases, the binding affinity of the RLN analog to the RXFP1 receptor is greater than the binding affinity of native human RLN2 (SEQ ID NOs: 5 and 6). In other cases, the binding affinity of the RLN analog to the RXFP1 receptor is less than the binding affinity of native human RLN2 (SEQ ID NOs: 5 and 6).

[0021] In certain instances, when administered to humans, the t 1 / 2 than the natural human RLN2 (SEQ ID NO: 5 and 6) t 1 / 2 longer, including up to about 20 days to about 30 days longer.

[0022] Alternatively, the above composition may be a nucleic acid sequence encoding the amino acid sequence described herein, as well as a vector for expressing the RLN analog described herein and a host cell comprising the same.

[0023] Secondly, pharmaceutical compositions are described that include at least one RLN analog described herein or a pharmaceutically acceptable salt thereof (e.g., trifluoroacetate, acetate, or hydrochloride) and a pharmaceutically acceptable carrier. In some cases, the pharmaceutically acceptable carrier is a buffer, for example, normal 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 composition may further include a carrier, a diluent, and / or an excipient.

[0024] In addition, the pharmaceutical composition may include at least one additional therapeutic agent, for example, an agent used as a standard of care for cardiovascular, pulmonary and / or renal conditions, diseases or disorders. In some cases, at least one additional therapeutic agent may be an anticoagulant, angiotensin-converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB), ARB neprilysin inhibitor (ARNI), beta-blocker, diuretic, digitalis, digoxin, hydralazine / isosorbide dinitrate, mineralocorticoid receptor antagonist (MRA; or aldosterone antagonist), sodium-glucose cotransporter-2 (SGLT2) inhibitor, statin and / or anti-glycemic agent.

[0025] Third, methods of using the RLN analogs described herein, in particular methods of using the RLN analogs to treat cardiovascular, pulmonary and / or renal conditions, diseases or disorders are described. 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.

[0026] In some cases, the RLN analog can be administered via any standard route of administration, e.g., intramuscularly, intravenously, parenterally, subcutaneously, or transdermally. In some cases, the RLN analog is administered subcutaneously (SQ), intramuscularly (IM), or intravenously (IV). In certain cases, the RLN analog can be administered to a subject subcutaneously or intravenously.

[0027] Likewise, and in some cases, the RLN analog can be administered daily, every other day, three times a week, twice a week, once a week (i.e., weekly), every two weeks (i.e., every other week), once a month (i.e., monthly), every two months (i.e., every other month), or even every three months. In some cases, the RLN analog can be administered subcutaneously every other day, three times a week, twice a week, once a week, every other week, or once a month. In certain cases, the RLN analog is administered subcutaneously once a week (QW).

[0028] Alternatively, the RLN analog can be administered intravenously to the individual. As above, the RLN analog can be administered daily, every other day, three times a week, twice a week, once a week (i.e., every week), every two weeks (i.e., every other week), or monthly. In some cases, the RLN analog can be administered intravenously every other day, intravenously three times a week, intravenously twice a week, intravenously once a week, intravenously every other week, or intravenously once a month. In certain cases, the RLN analog is administered intravenously once a week.

[0029] The method may also include the step of administering the RLN analog in combination with an effective amount of at least one additional therapeutic agent. In short, the standard of care for many of the conditions / diseases / disorders described herein include anticoagulants, ACE inhibitors, ARBs, ARNIs, beta-blockers, diuretics, digitalis, digoxin, hydralazine / isosorbide dinitrate, MRA or other aldosterone antagonists, SGLT2 inhibitors, statins and / or anti-glycemic agents, as well as other therapeutic agents for controlling comorbidities (including, but not limited to, high cholesterol, hypertension, atrial fibrillation and diabetes). In some cases, additional therapeutic agents may be administered simultaneously, separately or sequentially with the RLN analog.

[0030] For example, the additional therapeutic agent may be administered at the same frequency as the RLN analog (i.e., every other day, twice a week, weekly, or even monthly). In other cases, the additional therapeutic agent may be administered at a different frequency than the RLN analog. In other cases, the additional therapeutic agent may be administered subcutaneously or intravenously. In other cases, the RLN analog is administered subcutaneously and the additional therapeutic agent may be administered orally or intravenously. Alternatively, the RLN analog is administered intravenously and the additional therapeutic agent is administered subcutaneously.

[0031] In certain instances, the individual in need thereof is a diabetic, a hypertensive patient with impaired renal function, and / or an obese patient.

[0032] The method may also include steps such as measuring or obtaining blood pressure and comparing the value so obtained to one or more baseline values ​​or previously obtained values ​​to assess the effectiveness of the treatment / therapy.

[0033] The methods may also be combined with diet and exercise and / or may be combined with additional therapeutic agents other than those discussed above.

[0034] Fourth, uses comprising at least one RLN analog described herein are described. For example, the RLN analog described herein can be provided for treatment, particularly for the treatment of cardiovascular, pulmonary and / or renal conditions, diseases or disorders. The RLN analog can optionally be administered simultaneously, separately or sequentially (i.e., in combination) with at least one additional therapeutic agent. Similarly, uses of the RLN analog described herein in the preparation of a medicament for the treatment of cardiovascular, pulmonary and / or renal conditions, diseases or disorders are provided, wherein the medicament optionally can further include one or more additional therapeutic agents as described above.

[0035] Fifth, a compound is provided, which comprises the following amino acid sequence:

[0036]

[0037] In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:10.

[0038] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0039]

[0040] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:11.

[0041] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0042]

[0043] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:12.

[0044] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0045]

[0046] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:13.

[0047] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0048]

[0049] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:45.

[0050] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0051]

[0052] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:46.

[0053] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0054]

[0055] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:47.

[0056] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0057]

[0058] In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:48.

[0059] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0060]

[0061] In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:49.

[0062] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0063]

[0064] In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:50.

[0065] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0066]

[0067] In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:51.

[0068] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0069]

[0070] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:52.

[0071] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0072]

[0073] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:53.

[0074] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0075]

[0076] In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:54.

[0077] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0078]

[0079] In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:55.

[0080] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0081]

[0082] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:56.

[0083] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0084]

[0085] In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:57.

[0086] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0087]

[0088] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:58.

[0089] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0090]

[0091] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:59.

[0092] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0093]

[0094] In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:60.

[0095] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0096]

[0097] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:61.

[0098] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0099]

[0100] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:62.

[0101] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0102]

[0103] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:63.

[0104] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0105]

[0106] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:64.

[0107] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0108]

[0109] In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:65.

[0110] Alternatively, a compound is provided which comprises the following amino acid sequence:

[0111]

[0112] In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:66.

[0113] One advantage of the RLN analogs described herein is that they can be chemically or recombined into single-chain polypeptides (i.e., monomers) and therefore do not require endoproteolytic processing to obtain biological activity. However, it is contemplated that in some cases, the VHH portion can be conjugated not only to single-chain RLNs, but also to double-chain RLNs (e.g., natural). On the VHH portion, conjugation can be made not only to the N- and C-termini, but also to any surface-exposed amino acid of the VHH (provided that such conjugation does not completely abolish albumin binding).

[0114] One advantage of the RLN analogs described herein is that the VHH portion can be used not only with native A and B chain sequences, but also with modified sequences thereof. In addition, the VHH portion can be further modified to have enhanced or additional functionality by linking other peptide / protein fusions or small molecules to the VHH portion.

[0115] One advantage of the RLN analogs described herein is that the VHH portion provides a prolonged duration of action in mammals, such as humans, and can have a t of about 20 to about 30 days compared to native human RLN, particularly native human RLN2 (SEQ ID NOs: 5 and 6). 1 / 2, thereby allowing administration at least weekly or biweekly, which may improve compliance.

[0116] One advantage of the RLN analogs described herein is that they have similar or better selectivity, affinity and / or potency for the RXFP1 receptor than for the RXFP2 receptor compared to native human RLN2 (SEQ ID NOs: 5 and 6). In other words, the RLN analogs described herein result in sufficient activity for the RXFP1 receptor and reduced or insufficient activity for one or more of the RXFP2, RXFP3 and RXFP4 receptors.

[0117] One advantage of the RLN analogs described herein is that they have tunable pharmacokinetics by varying the albumin affinity of the VHH portion.

[0118] One advantage of the RLN analogs described herein is that they have improved stability in stored formulations compared to native human RLN2 (SEQ ID NOs: 5 and 6) or RLN analogs with Fc fusions.

[0119] Furthermore, an advantage of VHH moieties is that they bind equally not only to human serum albumin, but also to dog, monkey, mouse, pig and rat serum albumin, making pharmacodynamic, pharmacokinetic and toxicology studies more easily translatable from these species to humans.

[0120] One advantage of VHH portions is that they can be used not only to improve the t of the RLN analogs described herein compared to native human RLN2 (SEQ ID NOs: 5 and 6), but also to improve the t of the RLN analogs described herein. 1 / 2, and can be used to improve the t of other biologically active peptides and proteins (e.g., insulin, growth differentiation factor 15 (GDF-15) or glucose-dependent insulinotropic peptide 1 (GLP-1)). 1 / 2. DETAILED DESCRIPTION OF THE INVENTION

[0122] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of RLN analogs, pharmaceutical compositions, and methods, preferred methods and materials are described herein.

[0123] Furthermore, reference to an element with the indefinite article "a / an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one and only one of the element is present. Thus, the indefinite article "a / an" generally means "at least one".

[0124] definition

[0125] As used herein, "about" means within a statistically significant range of one or more values, such as a stated concentration, length, molecular weight, pH, sequence similarity, time range, temperature, volume, etc. Such values ​​or ranges can be within an order of magnitude, usually within 20%, more usually within 10%, and even more usually within 5% of a given value or range. The permissible variations encompassed by "about" will depend on the particular system under study and can be readily appreciated by those skilled in the art.

[0126] As used herein and with respect to one or more RXFP receptors, "activity," "activation," "activation," and the like refer to the ability of a compound, such as the RLN analogs described herein, to bind to its receptor and induce a response at its receptor as measured using assays known in the art, such as the in vitro assays described below.

[0127] As used herein, "amino acid" refers to a molecule that, from a chemical point of view, is characterized by the presence of one or more amine groups and one or more carboxylic acid groups and may contain other functional groups. As is known in the art, there is a set of twenty amino acids that are designated as standard amino acids and that can be used as building blocks for most peptides / proteins produced by any organism. The amino acid sequences in the present disclosure contain the standard single-letter or three-letter codes for the twenty standard amino acids.

[0128] As used herein, an "analog" refers to a compound, such as a synthetic peptide or polypeptide, that activates a target receptor and elicits at least one in vivo or in vitro effect elicited by a natural agonist of that receptor.

[0129] "Conservative substitution" as used herein refers to a variant of a reference peptide or polypeptide that is identical to a reference molecule except that it has one or more conservative amino acid substitutions in its amino acid sequence. In general, conservatively modified variants include amino acid sequences that have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the reference amino acid sequence. More specifically, conservative substitutions represent substitutions of an amino acid with an amino acid that has similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.) and has minimal effect on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitutions of functionally similar amino acids are well known in the art and therefore do not need to be described in detail herein.

[0130] As used herein, an "effective amount" refers to an amount or dosage of one or more RLN analogs described herein or a pharmaceutically acceptable salt thereof, which, after administration of a single dose or multiple doses to an individual in need thereof, provides the desired effect in such an individual undergoing diagnosis or treatment (i.e., can produce a clinically measurable difference in the individual's condition, e.g., increased angiogenesis in the heart, kidney, liver or lung, increased vascular compliance, increased cardiac blood flow, increased liver blood flow, increased pulmonary blood flow, increased renal blood flow, increased glomerular filtration rate, reduced blood pressure, reduced (or prevented) inflammation and / or reduced (or prevented) fibrosis). An effective amount can be readily determined by one skilled in the art using known techniques and by observing the results obtained under similar circumstances. In determining the effective amount for an individual, a number of factors are taken into consideration including, but not limited to, the species of mammal, its size, age, and general health, the particular disease or disorder involved, the extent or involvement or severity of the disease or disorder, the response of the individual, the particular RLN analog administered, the mode of administration, the bioavailability characteristics of the administered formulation, the dosage regimen selected, the use of concomitant medications, and other relevant circumstances.

[0131] As used herein, "extended duration of action" means that the binding affinity and activity of the RLN analogs described herein persist for a longer period of time than native RLN, especially native human RLN2 (SEQ ID NOs: 5 and 6), thereby allowing for less frequent dosing, such as at least once a day or even three times a week, twice a week, or once a week. The time-action characteristics of RLN analogs can be measured using known pharmacokinetic test methods, such as those used in the following examples.

[0132] As used herein, “half-life” or “t 1"t / 2" refers to the time it takes for half the amount of a compound (such as a native RLN or a RLN analog described herein) to be removed from a subject's fluid or other physiological space (such as serum or plasma) by biological processes. Alternatively, t 1 / 2 may also refer to the time it takes for a certain amount of such a compound to lose half of its pharmacological, physiological or radiological activity.

[0133] As used herein, “half maximal effective concentration” or “EC 50 ” refers to the concentration of compound that results in 50% activation / stimulation of an assay endpoint such as a dose-response curve (e.g., cAMP, PI3K-Akt, NFκβ, VEGF and / or nitric oxide (NO) signaling pathways).

[0134] As used herein, "in combination with" refers to the administration of at least one RLN analog described herein with one or more additional therapeutic agents simultaneously, sequentially, or in a single combined formulation.

[0135] As used herein, "a subject in need thereof" refers to a mammal, such as a human, having a condition, disease, disorder or symptom for which treatment or therapy is desired, including, for example, those listed herein. In particular, the subject to be treated is a human.

[0136] As used herein, "long-acting" means that the binding affinity and activity of the RLN analogs described herein persist for a longer period of time than native human RLN2 (SEQ ID NOs: 5 and 6), thereby allowing for less frequent dosing, such as at least once a day or even three times a week, twice a week, once a week, or once a month. The time-action characteristics of RLN analogs can be measured using known pharmacokinetic test methods, such as those described in the following examples.

[0137] As used herein, "non-standard amino acids" refer to amino acids that may be naturally present in cells but do not participate in peptide synthesis. Non-standard amino acids may be components of peptides and are often produced by modifying standard amino acids in peptides (i.e., by post-translational modifications). Non-standard amino acids may include D-amino acids, which have an absolute chirality opposite to that of the above-mentioned standard amino acids.

[0138] As used herein, "pharmaceutically acceptable buffer" refers to any of the standard pharmaceutical buffers known to those skilled in the art.

[0139] As used herein, "RLN" refers to relaxin obtained or derived from any species, such as a mammalian species, especially humans, wherein the native form is a heterodimeric peptide having two peptide chains (e.g., A chain and B chain) linked by two disulfide bonds, and wherein the A chain further has a single intramolecular disulfide bond. RLNs include native RLNs (i.e., full length) and variants thereof (i.e., additions, deletions, insertions and / or substitutions of native RLNs). In humans, there are three native RLN subtypes - RLN1, RLN2 and RLN3. RLN processing starts with preprorelaxin, which is processed into prorelaxin (including A chain, B chain and C peptide; native RLN has the structure of BCA), wherein the sequence of native human proRLN1 is shown in SEQ ID NO: 1 (see also, UniProt / SwissProt database accession number P04808), the sequence of native human proRLN2 is shown in SEQ ID NO: 4 (see also, UniProt / SwissProt database accession number P04090), and the sequence of native human proRLN3 is shown in SEQ ID NO: 7 (see also, UniProt / SwissProt database accession number Q8WXF3). Prorelaxin undergoes further processing, wherein the C peptide is cleaved to give RLN. The sequences of the A chain of native human RLN1, RLN2 and RLN3 are shown in SEQ ID NO: 2, 5 and 8, respectively. Likewise, the sequences of the B chains of native human RLN1, RLN2, and RLN3 are shown in SEQ ID NOs: 3, 6, and 9, respectively.

[0140] In humans, there are four RLN receptors - RXFP1 (SEQ ID NO: 40; see also UniProt / SwissProt database accession number Q9HBX9), RXFP2 (SEQ ID NO: 41; see also UniProt / SwissProt database accession number Q8WXD0), RXFP3 (SEQ ID NO: 42; see also UniProt / SwissProt database accession number Q9NSD7) and RXFP4 (SEQ ID NO: 43; see also UniProt / SwissProt database accession number Q8TDU9) - they act as GPCRs (see, Halls et al. (2007) Br. J. Pharmacol. 150: 677-691). Of interest herein are the RXFP1 and RXFP2 receptors, both of which can bind to RLN1 and RLN2. RXFP1 receptors have been found in the brain, blood cells, bone, heart, kidney, lung, liver, and vasculature, while RXFP2 receptors are more restricted and have been found in bone and gubernaculum. Stimulation of RXFP1 and RXFP2 receptors activates a signaling network involving adenylate cyclase, protein kinase A, protein kinase C, phosphatidylinositol 3-kinase, and / or extracellular signal-regulated kinase (Erk1 / 2).

[0141] As used herein, "RLN analogs" and the like refer to compounds, such as peptides or polypeptides, that elicit one or more effects of a natural RLN at one or more RXFP receptors, but are altered in some way in terms of amino acid sequence compared to the natural RLN. RLN analogs may also include variants of these compounds that are functionally equivalent to RLN but have a sequence as a fragment, or a complete sequence but with additions, deletions, insertions and / or substitutions. Unless otherwise indicated, all references to amino acid positions in the unmodified or modified RLNs described herein are based on the corresponding positions in the A chain of SEQ ID NO: 5 or the B chain of SEQ ID NO: 6 of the natural human RLN2. In some cases, the RLN analogs described herein may bind to RXFP with a higher or lower affinity than the natural RLN, especially the natural human RLN2 (SEQ ID NO: 5 and 6), but exhibit a longer t in vivo or in vitro. 1 / 2. In this manner, the RLN analogs described herein are synthetic compounds that act as agonists of the RXFP receptor.

[0142] As used herein, "sequence similarity" refers to a quantitative property of two or more nucleic acid sequences or amino acid sequences of biological compounds, for example, the correspondence of two or more sequences over the entire length or comparison window. Sequence similarity can be measured by (1) percent identity or (2) percent similarity. Percent identity measures the percentage of identical residues between two biological compounds divided by the length of the shortest sequence; whereas percent similarity measures identity and, in addition, includes sequence gaps and residue similarities in the evaluation. Methods and algorithms for determining sequence similarity are well known in the art and, therefore, need not be described in detail herein. The specified percentage of identical nucleotide or amino acid positions is at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0143] As used herein, "single-chain relaxin," "scRLN," and the like refer to RLN polypeptides in which the A chain and the B chain are linked to each other by a linker (i.e., L2), such as in A-L2-B or B-L2-A. In addition, the scRLN may include at least one of the native inter-chain and / or intra-chain disulfide bonds to maintain the correct structural folding.

[0144] As used herein, "two-chain relaxin," "tcRLN," and the like refer to RLN polypeptides in which the A chain and the B chain are linked to each other by one or more interchain and / or intrachain disulfide bonds, but not by any linker, to maintain the correct structural folding, such as native RLN.

[0145] As used herein, "treatment" refers to the management and care of an individual with a condition, disease, disorder, or symptom for which the administration of a RLN analog is indicated to achieve the purpose of attenuating, inhibiting, reversing, slowing down, or stopping the progression or severity of the condition, disease, disorder, and / or symptom. Treatment includes administering to an individual a RLN analog as described herein or a composition containing a RLN analog as described herein to prevent the onset of symptoms or complications, alleviate symptoms or complications, or eliminate the condition, disease, disorder, or symptom. Treatment includes administering to an individual a RLN analog or a composition containing a RLN analog as described herein to cause, for example, increased angiogenesis, increased vascular compliance, increased cardiac blood flow, increased liver blood flow, increased pulmonary blood flow, increased renal blood flow, increased glomerular filtration rate, reduced blood pressure, reduced (or prevented) inflammation, and / or reduced (or prevented) fibrosis in the heart, kidney, liver, or lung. The individual to be treated is a mammal, particularly a human.

[0146] As used herein, "individual," "patient," and "subject" are used interchangeably and refer to a mammal, particularly a human. In certain instances, the individual is further characterized as having a condition, disease, disorder, and / or symptom that would benefit from administration of a RLN analog described herein.

[0147] As used herein, "VHH" or "VHH portion" refers to a form of a single domain antibody, particularly an antibody fragment of a single monomer variable region of a heavy chain antibody (HcAb), which has a very small size of about 15 kDa. It has been found herein that the VHH portion can be used as a pharmacokinetic enhancer to extend the duration of action of the RLN analogs described herein and / or improve their t 1 / 2. The VHH portion herein binds serum albumin; however, the VHH portion can be used to bind IgG (including the Fc domain), neonatal Fc receptor (FcRn) or other persistent serum proteins. Although the VHH portion herein is used to improve the t 1 / 2, but they can also be used to improve the t of other bioactive peptides / proteins such as insulin, GDF-15 or GLP-1 1 / 2.

[0148] Certain abbreviations are defined as follows: “ACR” denotes urine albumin / urine creatinine ratio; “amu” denotes atomic mass unit; “AUC” denotes area under the curve; “Boc” denotes tert-butyloxycarbonyl; “cAMP” denotes cyclic adenosine monophosphate; “CMV” denotes cytomegalovirus; “CV” denotes column volume; “DNA” deoxyribonucleic acid; “DMF” denotes dimethylformamide; “DMSO” denotes dimethyl sulfoxide; “EDC” denotes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; “EDTA” denotes ethylenediaminetetraacetic acid; “EIA / RIA” denotes enzyme immunoassay / radioimmunoassay; “ETA” denotes ethanolamine; “GS” denotes glutamine synthetase; “HIC” denotes hydrophobic interaction chromatography; “hr” denotes hour; “HTRF” denotes homogeneous time-resolved fluorescence; “IV” denotes intravenous "IP" means intravenous; "kDa" means kilodaltons; "LC / MS" means liquid chromatography-mass spectrometry; "min" means minutes; "MS" means mass spectrometry; "MSX" means methionine sulfoximine; "NaOAc" means sodium acetate; "NHS" means N-hydroxysuccinimide; "OtBu" means O-tert-butyl; "Pbf" means NG-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; "PEI" means polyethyleneimine; "RP-HPLC" means reversed-phase high-performance liquid chromatography; "RU" means resonance units; "sec" means seconds; "SPR" means surface plasmon resonance; "SQ" means subcutaneous; "SEC" means size exclusion chromatography; "SEM" means standard error of the mean; "TFA" means trifluoroacetic acid; and "Trt" means trityl.

[0149] RLN analogs

[0150] The RLN analogs described herein have structural similarities to natural human RLNs, but have many structural differences. For example, compared to natural human RLN2 (SEQ ID NOs: 5 and 6), the RLN analogs lack one or more amino acids present in natural human RLN2, including a peptide linker between the A chain and the B chain, and include a VHH portion that binds to albumin. The RLN analogs result in sufficient activity against the RXFP1 receptor and reduced or insufficient activity against one or more of the RXFP2, RXFP3, and RXFP4 receptors. Likewise, the RLN analogs have beneficial properties associated with their developability as therapeutic treatments, including improved solubility in aqueous solution, improved chemical and physical formulation stability, extended pharmacokinetic properties (which can be adjusted based on the affinity of the VHH for serum albumin) and / or minimized immunogenicity potential.

[0151] Briefly, the RLN analogs described herein include an amino acid sequence having one of the following structures from the N-terminus to the C-terminus:

[0152] VHH-L1-A-L2-B,

[0153] VHH-L1-B-L2-A,

[0154] A-L2-B-L1-VHH, or

[0155] B-L2-A-L1-VHH,

[0156] wherein VHH is the moiety that acts as a pharmacokinetic enhancer, A is the RLN A chain, B is the RLN B chain, L1 is a first peptide linker and L2 is a second peptide linker, wherein L1 and L2 are different from each other (ie, each has a different amino acid sequence).

[0157] Regarding the A chain, it can be a native RLN A chain, such as a native human RLN1 A chain (SEQ ID NO: 2); a native human RLN2 A chain (SEQ ID NO: 5); or a native human RLN3 A chain (SEQ ID NO: 8). Alternatively, the A chain can be a variant thereof. For example, an A chain variant can have an amino acid sequence lacking residues 1-4 of SEQ ID NO: 5 (i.e., des1-4 human RLN2 A chain or desA1-4).

[0158] Similarly, with respect to the B chain, it can be a native RLN B chain, such as native human RLN1 B chain (SEQ ID NO: 3); native human RLN2 B chain (SEQ ID NO: 6); or native human RLN3 B chain (SEQ ID NO: 9). Alternatively, the B chain can be a variant thereof. For example, a B chain variant can have an amino acid sequence lacking residue 1 of SEQ ID NO: 6 (i.e., des1 human RLN2 B chain or desB1).

[0159] In some cases, the A chain can be a natural human RLN1 A chain (SEQ ID NO: 2) and the B chain can be a natural human RLN1 B chain (SEQ ID NO: 3); the A chain can be a natural human RLN2 A chain (SEQ ID NO: 5) and the B chain can be a natural human RLN2 B chain (SEQ ID NO: 6); the A chain can be a natural human RLN3 A chain (SEQ ID NO: 8) and the B chain can be a natural human RLN3 B chain (SEQ ID NO: 9); the A chain can be a natural human RLN1 A chain (SEQ ID NO: 2) and the B chain can be a natural human RLN2 B chain (SEQ ID NO: 6); the A chain can be a natural human RLN1 A chain (SEQ ID NO: 2) and the B chain can be a natural human RLN3 B chain (SEQ ID NO: 9); the A chain can be a natural human RLN2 A chain (SEQ ID NO: 5) and the B chain can be a natural human RLN1 B chain (SEQ ID NO: 3); the A chain can be a natural human RLN2 A chain (SEQ ID NO: 5) and the B chain can be a natural human RLN3 B chain (SEQ ID NO:9); the A chain can be native human RLN3 A chain (SEQ ID NO:8) and the B chain can be native human RLN1 B chain (SEQ ID NO:3); or the A chain can be native human RLN3 A chain (SEQ ID NO:8) and the B chain can be native human RLN2 B chain (SEQ ID NO:6).

[0160] In some cases, the A chain can be a RLN2 A chain variant lacking residues 1-4 (desA1-4) and the B chain can be any native B chain. In other cases, the A chain can be any native A chain and the B chain can be a RLN2 B chain variant lacking residue 1 (desB1). In still other cases, the A chain can be a RLN2 A chain variant lacking residues 1-4 (desA1-4) and the B chain can be a RLN2 B chain variant 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.

[0161] Other A and B chains that can be used in the RLN analogs described herein are described, for example, in International Patent Application Publication Nos. 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 / 024452; and U.S. Patent Application Publication No. US2011 / 0243942. See also, Chan et al. (2012) J. Biol. Chem. 287:41152-41164; Claasz et al. (2002) Eur. J. Biochem. 269:6287-6293; Hossain et al. (2015) Org. Biomol. Chem. 13:10895-10890; Hossain et al. (2016) Chem. Sci. 7:3805-3819; Park et al. (2008) J. Biol. Chem. 283:32099-32109 and Wilkinson et al. (2005) BMC E vol. Biol. 5:14.

[0162] Regarding L1, it can be a peptide of about 1 amino acid to about 50 amino acids. Alternatively, L1 can be about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50 amino acids. Also alternatively, L1 can be about 5 amino acids to about 10 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 45 amino acids or about 45 amino acids to about 50 amino acids. In some cases, L1 can be omitted so that the A chain or the B chain is directly conjugated to the VHH portion. In some cases, L1 can include (GGGGQ) n (SEQ ID NO: 14), wherein n may be from about 1 to about 10, particularly 5 (i.e., (GGGGQ)5; SEQ ID NO: 19). In other cases, L1 may include (PGPQ) n(SEQ ID NO: 17), wherein n may be from about 1 to about 10, particularly 8 (i.e., (PGPQ)8; SEQ ID NO: 20). In other cases, L1 may include (PGPA) n (SEQ ID NO: 18), wherein n may be from about 1 to about 10, in particular 8 (ie, (PGPA) 8; SEQ ID NO: 21).

[0163] Other linkers that can be used as L1 for RLN analogs include, but are not limited to (GGGQ) n (SEQ ID NO: 15) or (GGGGS) n (SEQ ID NO: 16).

[0164] Regarding L2, it can be a peptide of about 1 amino acid to about 15 amino acids. Alternatively, L2 can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 or about 15 amino acids. Also alternatively, L2 can be about 1 amino acid to about 5 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 15 amino acids, especially 10 amino acids to 15 amino acids. In some cases, L2 can include a mixture of Ala / A, Gln / Q, Gly / G, Pro / P and Ser / S residues. In other cases, L2 can be SEQ ID NO:22, 23 or 67.

[0165] Regarding VHH, it may be a polypeptide of about 50 amino acids to about 200 amino acids, particularly about 125 amino acids to about 150 amino acids, which may bind to serum albumin or have a long t 1 In some cases, the VHH may be any one of SEQ ID NOs: 10 to 13. Alternatively, the VHH may be any one of SEQ ID NOs: 45-66. The structural features of these VHH portions result in a longer t compared to native RLNs, particularly native human RLN2 (SEQ ID NOs: 5 and 6). 1 / 2. Given that the VHH portion of this article targets serum albumin, it can be expected that the t of the RLN analog 1 / 2 is similar to the t of serum albumin of the species to which the RLN analog is administered 1 / 2 (taking into account any target-mediated drug disposition).

[0166] In addition to the changes described in this disclosure, the RLN analogs described herein may include one or more additional amino acid modifications, particularly conservative substitutions, provided, however, that the RLN analog is still able to bind to and activate the RXFP1 receptor.

[0167] In summary, exemplary RLN analogs are as follows:

[0168] RLN analog 1, which includes the VHH portion (underlined), (G4Q)5L1 (italics), the A chain of RLN2, 10 residues of L2 (bold), and the B chain of RLN2 (desB1), has the following amino acid sequence:

[0169]

[0170] or a pharmaceutically acceptable salt thereof;

[0171] RLN analog 2, which includes the VHH portion (underlined), (PGPA)8L1 (italics), the A chain of RLN2, 10 residues of L2 (bold), and the B chain of RLN2 (desB1), has the following amino acid sequence:

[0172]

[0173] or a pharmaceutically acceptable salt thereof;

[0174] RLN analog 3, which includes the VHH portion (underlined), (G4Q)5L1 (italics), the B chain of RLN2 (desB1), 10 residues of L2 (bold) and the A chain of RLN2, has the following amino acid sequence:

[0175]

[0176] or a pharmaceutically acceptable salt thereof;

[0177] RLN analog 4, which includes the VHH portion (underlined), (PGPQ)8L1 (italics), the B chain of RLN2 (desB1), 10 residues of L2 (bold) and the A chain of RLN2, has the following amino acid sequence:

[0178]

[0179] or a pharmaceutically acceptable salt thereof;

[0180] RLN analog 5, which includes the B chain (desB1) of RLN2, 10 residues of L2 (bold), the A chain of RLN2, (G4Q)5L1 (italics) and the VHH portion (underlined), has the following amino acid sequence:

[0181]

[0182] or a pharmaceutically acceptable salt thereof;

[0183] RLN analog 6, which includes the B chain (desB1) of RLN2, 10 residues of L2 (bold), the A chain of RLN2, (PGPQ)8L1 (italics) and the VHH portion (underlined), has the following amino acid sequence:

[0184]

[0185] or a pharmaceutically acceptable salt thereof;

[0186] RLN analog 7, which includes the VHH portion (underlined), (G4Q)5L1 (italics), the B chain of RLN2 (desB1), 10 residues of L2 (bold), and the A chain of RLN2, has the following amino acid sequence:

[0187]

[0188] or a pharmaceutically acceptable salt thereof;

[0189] RLN analog 8, which includes the VHH portion (underlined), (PGPQ)8-L1 (italics), the B chain of RLN2 (desB1), 10 residues of L2 (bold) and the A chain of RLN2, has the following amino acid sequence:

[0190]

[0191] or a pharmaceutically acceptable salt thereof;

[0192] RLN analog 9, which includes the B chain (desB1) of RLN2, 10 residues of L2 (bold), the A chain of RLN2, (G4Q)5L1 (italics) and the VHH portion (underlined), has the following amino acid sequence:

[0193]

[0194] or a pharmaceutically acceptable salt thereof;

[0195] RLN analog 10, which includes the B chain (desB1) of RLN2, 10 residues of L2 (bold), the A chain of RLN2, (PGPQ)8L1 (italics) and the VHH portion (underlined), has the following amino acid sequence:

[0196]

[0197] or a pharmaceutically acceptable salt thereof;

[0198] RLN analog 11, which includes the VHH portion (underlined), (G4Q)5L1 (italics), the B chain of RLN2 (desB1), 13 residues of L2 (bold), and the A chain of RLN2 (desA1-4), has the following amino acid sequence:

[0199]

[0200] or a pharmaceutically acceptable salt thereof;

[0201] RLN analog 12, which includes the VHH portion (underlined), (G4Q)5L1 (italics), the B chain of RLN2 (desB1), 13 residues of L2 (bold), and the A chain of RLN2 (desA1-4), has the following amino acid sequence:

[0202]

[0203] or a pharmaceutically acceptable salt thereof;

[0204] RLN analog 13, which includes the VHH portion (underlined), (G4Q)5L1 (italics), the A chain of RLN2 (desA1-4), 10 residues of L2 (bold), and the B chain of RLN2 (desB1), has the following amino acid sequence:

[0205]

[0206] or a pharmaceutically acceptable salt thereof;

[0207] RLN analog 14, which includes the VHH portion (underlined), (PGPQ)8L1 (italics), the A chain of RLN2 (desA1-4), 10 residues of L2 (bold), and the B chain of RLN2 (desB1), has the following amino acid sequence:

[0208]

[0209] or a pharmaceutically acceptable salt thereof;

[0210] RLN analog 15, which includes the VHH portion (underlined), (G4Q)5L1 (italics), the B chain of RLN2 (native), 10 residues of L2 (bold), and the A chain of RLN2 (native), has the following amino acid sequence:

[0211]

[0212] or a pharmaceutically acceptable salt thereof; and

[0213] RLN analog 16, which includes the VHH portion (underlined), (PGPA)8L1 (italics), the B chain of RLN2 (native), 10 residues of L2 (bold), and the A chain of RLN2 (native), has the following amino acid sequence:

[0214]

[0215] or a pharmaceutically acceptable salt thereof.

[0216] The half-life of the RLN analogs described herein can be measured using methods known in the art, including, for example, those described in the Examples below. Similarly, the affinity of the RLN analogs for albumin of different species can be measured using methods known in the art for measuring binding affinity, such as those described in the Examples below, and is generally expressed as the equilibrium dissociation constant (K D ) value. In addition, the activity of RLN analogs at each RXFP receptor can be measured using methods known in the art, including, for example, the in vitro activity assays described below, and is generally expressed as EC 50 value.

[0217] As a result of the above modifications, when administered to mammals, especially humans, the RLN analogs described herein have a longer t than natural RLN, especially natural human RLN2 (SEQ ID NOs: 5 and 6). 1 / 2. As noted above, the VHH portion herein targets serum albumin; therefore, it is expected that the t 1 / 2 with the serum albumin of the species to which the RLN analogue is administered 1 In some cases, the RLN analogs may have a t of about 1 day to about 31 days, about 5 days to about 25 days, about 10 days to about 20 days, or even about 15 days. 1 / 2. In other cases, the RLN analogs can have a t of about 1 day to about 5 days, about 6 days to about 10 days, about 11 days to about 15 days, about 16 days to about 20 days, about 21 to about 25 days, or even about 26 to about 31 days. 1 In other cases, the RLN analogs can have a t of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, or even about 31 days. 1 In certain cases, when administered to humans, the RLN analogs may have a t of about 20 days.1 / 2.

[0218] Likewise, when administered to humans, the RLN analogs described herein are within about 10-fold to about 100-fold more potent than native human RLN2 (SEQ ID NOs: 5 and 6) at the RXFP1 receptor, for example.

[0219] Pharmaceutical compositions and kits

[0220] The RLN analogs described herein can be formulated into pharmaceutical compositions that can be administered parenterally (e.g., intravenously, intraperitoneally, intramuscularly, subcutaneously, or transdermally). Such pharmaceutical compositions and techniques 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 certain cases, RLN analogs are administered subcutaneously or intravenously. Alternatively, however, RLN analogs can be formulated into forms for other pharmaceutically acceptable routes, e.g., tablets or other solids for oral administration, timed-release capsules, and any other forms currently used, including creams, lotions, inhalants, and the like.

[0221] As noted above, and to improve their in vivo compatibility and effectiveness, the RLN analogs described herein can be reacted with any number of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and common techniques for preparing them are well known in the art (see, e.g., Stahl et al., "Handbook of Pharmaceutical Salts: Properties, Selection and Use" (2nd revised edition, Wiley-VCH, 2011)). Pharmaceutically acceptable salts for use herein include sodium salts, trifluoroacetates, hydrochlorides, and acetates.

[0222] The RLN analogs described herein can be administered by a physician or self-administered using an injection. It should be understood that the gauge size and amount of injection volume can be easily determined by one skilled in the art. However, the amount of injection volume can be ≤ about 2 mL or even ≤ about 1 mL, and the needle gauge can be ≥ about 27 G or even ≥ about 29 G.

[0223] The present disclosure also provides and therefore encompasses novel intermediates and methods that can be used to synthesize the RLN analogs described herein or pharmaceutically acceptable salts thereof. Intermediates and RLN analogs can be prepared by a variety of techniques well known in the art. For example, methods using recombinant synthesis are illustrated in the following examples. The specific steps of each of the techniques described can be combined in different ways to prepare RLN analogs. Reagents and starting materials are readily available to those skilled in the art.

[0224] The RLN analogs described herein are generally effective over a wide dosage range. Exemplary dosages of RLN analogs or pharmaceutical compositions comprising them can be milligrams (mg) or micrograms (μg), nanograms (ng) or picograms (pg) per kilogram (kg) of individual. In this manner, a daily dose can be from about 1 μg to about 100 mg.

[0225] Here, the effective amount of the RLN analog in the pharmaceutical composition can be a dosage of about 0.25 mg to about 5.0 mg. However, those skilled in the art understand that in some cases, the effective amount (i.e., dosage / dosage) may be lower than the lower limit of the above range and more than enough, while in other cases, the effective amount may be a larger dosage and can be used with acceptable side effects.

[0226] In addition to the RLN analogs described herein, the pharmaceutical compositions may also include at least one additional therapeutic agent, especially therapeutic agents that are generally used as standard of care for cardiovascular, pulmonary and renal conditions, diseases and disorders.

[0227] In this manner, a pharmaceutical composition can include an effective amount of at least one RLN analog described herein, a pharmaceutically acceptable carrier, and optionally at least one additional therapeutic agent. For example, the pharmaceutical composition may include an effective amount of a RLN analog of SEQ ID NO:24 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:25 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:26 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:27 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:28 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:29 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:30 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:31 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:32 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:33 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:34 and a pharmaceutically acceptable carrier, an effective amount of NO:35 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:36 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:37 and a pharmaceutically acceptable carrier, an effective amount of a RLN analog of SEQ ID NO:38 and a pharmaceutically acceptable carrier, or an effective amount of a RLN analog of SEQ ID NO:39 and a pharmaceutically acceptable carrier.

[0228] Alternatively, the RLN analogs described herein can be provided as part of a kit. In some cases, the kit includes a device for administering at least one RLN analog (and optionally at least one additional therapeutic agent) to an individual. In some cases, the kit includes a syringe and needle for administering at least one RLN analog (and optionally at least one additional therapeutic agent). In certain cases, the RLN analog (and optionally at least one additional therapeutic agent) is preformulated in an aqueous solution within a syringe.

[0229] Methods of making and using RLN analogs

[0230] The RLN analogs described herein can be prepared by any number of standard recombinant DNA methods or standard chemical peptide synthesis methods known in the art. With respect to recombinant DNA methods, a polynucleotide having a nucleic acid sequence encoding an amino acid sequence of a RLN analog can be constructed using standard recombinant techniques, incorporated into a recombinant expression vector, and introduced into a host cell such as bacteria, yeast, and mammalian cells to produce the RLN analog. See, e.g., Green & Sambrook, "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 4th edition, 2012).

[0231] About recombinant DNA method, can be by using recombinant DNA technology to produce albumen or precursor protein molecule to prepare compound herein.DNA, including cDNA and synthetic DNA, can be double-stranded or single-stranded, and wherein the coding sequence encoding compound herein may change due to the redundancy or degeneracy of genetic code.In short, the DNA sequence encoding compound herein is introduced into host cell to produce compound or its precursor.Host cell can be bacterial cell such as K12 or B strain of Escherichia coli, fungal cell such as yeast cell, or mammalian cell such as Chinese hamster ovary (CHO) cell.

[0232] The appropriate host cell is transiently or stably transfected or transformed with an expression system such as an expression vector to produce the compounds herein or their precursors. The expression vector can usually be replicated in the host organism as an episome or as an integral part of the host chromosomal DNA. Typically, the expression vector will contain a selection marker such as tetracycline, neomycin, G418 and dihydrofolate reductase to allow selection of those cells transformed with the desired DNA sequence.

[0233] The specific biosynthetic or synthetic steps for each step described herein may be used, not used, or combined in various ways to prepare the compounds herein.

[0234] For chemical peptide synthesis methods, standard manual or automated solid phase synthesis procedures can be used. For example, automated peptide synthesizers are commercially available from, for example, Applied Biosystems (Foster City, CA) and Protein Technologies Inc. (Tucson, AZ). Reagents for solid phase synthesis are readily available from commercial sources. A solid phase synthesizer can be used according to the manufacturer's instructions to block interfering groups, protect amino acids during the reaction, couple, deprotect, and cap unreacted amino acids. Additional details for preparing synthetic RLNs can be found in U.S. Patent Nos. 4,835,251 and 5,166,191.

[0235] One use of the RLN analogs described herein is for treating cardiovascular conditions, diseases and / or disorders. Exemplary cardiovascular conditions, diseases and disorders include, but are not limited to, acute heart failure, chronic heart failure, atherosclerosis, coronary artery disease, diabetes, stroke, hypercholesterolemia, hypertension, ischemia, vasoconstriction and ventricular hypertrophy.

[0236] Another use of the RLN analogs described herein is for treating pulmonary conditions, diseases and / or disorders.Exemplary pulmonary conditions, diseases and disorders include, but are not limited to, pulmonary hypertension and chronic obstructive pulmonary disease (COPD).

[0237] Another use of the RLN analogs described herein is for treating renal conditions, diseases and / or disorders.Exemplary renal conditions, diseases and disorders include, but are not limited to, chronic kidney disease and diabetic nephropathy.

[0238] The method may include the steps described herein, and these steps may, but not necessarily, be performed in the order described. However, other orders are also conceivable. In addition, single or multiple steps may be performed in parallel and / or overlapping in time and / or individually or in a step that is repeated multiple times. In addition, the method may include additional, unspecified steps.

[0239] Thus, such methods may include selecting an individual who has a cardiovascular condition, disease or disorder or is susceptible to them. Alternatively, the method may include selecting an individual who has a pulmonary condition, disease or disorder or is susceptible to them. Alternatively, the method may include selecting an individual who has a renal condition, disease or disorder or is susceptible to them. In some cases, the method may include selecting an individual who is a diabetic, a hypertensive patient with renal impairment, and / or an obese patient.

[0240] The method may also include administering to the individual an effective amount of at least one RLN analog described herein, which may be in the form of a pharmaceutical composition also described herein. In some cases, the RLN analog / pharmaceutical composition may contain additional therapeutic agents such as anticoagulants, ACE inhibitors, ARBs, ARNIs, beta-blockers, diuretics, digitalis, digoxin, hydralazine / isosorbide dinitrate, MRA or other aldosterone antagonists, SGLT2 inhibitors, statins and / or anti-glycemic agents, as well as other therapeutic agents for controlling comorbidities (including, but not limited to, high cholesterol, hypertension, atrial fibrillation and diabetes).

[0241] Concentrations / doses / amounts of RLN analogs and optional additional therapeutic agents are discussed elsewhere herein.

[0242] Regarding the route of administration, the RLN analog or a pharmaceutical composition comprising the same can be administered according to known methods, such as orally; by injection (i.e., intraarterial, intravenous, intraperitoneal, intracerebral, intraventricular, intramuscular, intraocular, intraportal or intralesional); by a sustained release system, or by an implant device. In some cases, the RLN analog or a pharmaceutical composition comprising the same can be administered subcutaneously by rapid bolus injection or continuously.

[0243] Regarding the frequency of administration, the RLN analog or a pharmaceutical composition comprising the same can be administered daily, every other day, three times a week, twice a week, once a week (i.e., every week), every two weeks (i.e., every other week), or monthly. In some cases, the RLN analog or a pharmaceutical composition comprising the same is administered subcutaneously every other day, three times a week, twice a week, once a week, every other week, or monthly. In certain cases, the RLN analog or a pharmaceutical composition comprising the same is administered subcutaneously once a week (QW).

[0244] For those instances where a RLN analog or a pharmaceutical composition comprising the same is administered in combination with an effective amount of at least one additional therapeutic agent, the additional therapeutic agent may be administered simultaneously, separately or sequentially with the RLN analog or a pharmaceutical composition comprising the same.

[0245] In addition, the additional therapeutic agent may be administered at the same frequency as the RLN analog or pharmaceutical composition comprising the same (i.e., every other day, twice a week, or even weekly). Alternatively, the additional therapeutic agent may be administered at a different frequency than the RLN analog or pharmaceutical composition comprising the same. In other cases, the additional therapeutic agent may be administered subcutaneously. In other cases, the additional therapeutic agent may be administered intravenously. In other cases, the additional therapeutic agent may be administered orally.

[0246] It is further contemplated that the methods may be combined with diet and exercise, and / or may be combined with additional therapeutic agents other than those described above. Example

[0247] The following non-limiting examples are offered for purposes of illustration and not limitation.

[0248] Peptide expression

[0249] Example 1: Recombinant expression of RLN analog 1

[0250] Example 1 is a RLN analog having the following amino acid sequence:

[0251]

[0252] Here, the RLN analog of SEQ ID NO: 24 was produced in a mammalian cell expression system using a CHOK1 cell derivative. The cDNA sequence encoding SEQ ID NO: 24 was subcloned into an expression plasmid backbone containing GS (pEE12.4-based plasmid). The cDNA sequence was 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. The expression was driven by a viral CMV promoter.

[0253] To generate RLN analogs by transient transfection, CHOK1 cells were transfected with recombinant expression plasmids using a PEI-based method. Briefly, 4 x 10 6 An appropriate volume of CHOK1 suspension cells at 10 cells / mL was transferred to a shake flask, and PEI and recombinant plasmid DNA were added to the cells. The cells were incubated at 32°C for 6 days in suspension culture. At the end of the incubation period, the cells were removed by low-speed centrifugation, and the RLN analog protein was purified from the conditioned medium.

[0254] Alternatively, and to produce RLN analogs by stable transfection, CHOK1 cells are stably transfected using electroporation and an appropriate amount of the recombinant expression plasmid, and the transfected cells are maintained in suspension culture at an appropriate cell density. Selection of transfected cells is accomplished by growing in serum-free medium containing 25 μM MSX and incubating at about 35°C-37°C and about 5%-7% CO2.

[0255] RLN analogs are secreted from CHO cells into the culture medium and purified by protein A affinity chromatography and subsequent ion exchange and hydrophobic interaction chromatography or size exclusion chromatography. Specifically, RLN analogs from the harvested culture medium are captured onto Mab Select Protein A resin (GE). The resin is then briefly washed with a running buffer such as phosphate-buffered saline (PBS; pH 7.4) or a buffer containing Tris to remove non-specifically bound substances. The protein is eluted from the resin with a low pH solution, such as 10 mM citric acid pH 3. The fractions containing the RLN analogs are combined and can be kept at a low pH to inactivate potential viruses. The pH can be neutralized by adding a base such as 0.1 M Tris pH 8.0. The RLN analogs can be further purified by ion exchange chromatography using a resin such as Poros 50HS (ThermoFisher). The RLN analog was eluted from the column using a 0-500 mM NaCl gradient in 20 mM NaOAc (pH 5.0) over 15 column volumes.

[0256] The RLN analogs can be further purified by hydrophobic interaction chromatography using a Capto Phenyl ImpRes HIC column (GE Healthcare). Purification is performed by adjusting the column loading solution to about 0.5 M sodium sulfate and eluting with a gradient from 0.5 M to 0 M sodium sulfate in 20 mM Tris pH 8 solution for 10 CVs. After HIC, the RLN analogs can be further purified by SEC, where the concentrated Capto Phenyl ImpRes pool is loaded onto a Superdex 200 (GE Healthcare) and isocratically eluted in PBS pH 7.4 or in 20 mM histidine, 50 mM NaCl pH 6.0.

[0257] Purified RLN analogs can be passed through a virus retaining filter, such as a Planova 20N (Asahi Kasei Medical), and then concentrated / diafiltered into 20 mM histidine, 20 mM NaCl pH 6 using tangential flow ultrafiltration on a regenerated cellulose membrane (Millipore).

[0258] Thus, RLN analogs are prepared in this manner or in a similar manner readily determined by one skilled in the art.

[0259] Example 2: Recombinant expression of RLN analog 2

[0260] Example 2 is a RLN analog having the following amino acid sequence:

[0261]

[0262] Here, the RLN analog of SEQ ID NO:25 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:25 was used in the expression plasmid.

[0263] Example 3: Recombinant expression of RLN analog 3

[0264] Example 3 is a RLN analog having the following amino acid sequence:

[0265]

[0266] Here, the RLN analog of SEQ ID NO:26 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:26 was used in the expression plasmid.

[0267] Example 4: Recombinant Expression of RLN Analog 4

[0268] Example 4 is a RLN analog having the following amino acid sequence:

[0269]

[0270] Here, the RLN analog of SEQ ID NO:27 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:27 was used in the expression plasmid.

[0271] Example 5: Recombinant expression of RLN analog 5

[0272] Example 5 is a RLN analog having the following amino acid sequence:

[0273]

[0274] Here, the RLN analog of SEQ ID NO:28 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:28 was used in the expression plasmid.

[0275] Example 6: Recombinant expression of RLN analog 6

[0276] Example 6 is a RLN analog having the following amino acid sequence:

[0277]

[0278] Here, the RLN analog of SEQ ID NO:29 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:29 was used in the expression plasmid.

[0279] Example 7: Recombinant expression of RLN analog 7

[0280] Example 7 is a RLN analog having the following amino acid sequence:

[0281]

[0282] Here, the RLN analog of SEQ ID NO:30 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:30 was used in the expression plasmid.

[0283] Example 8: Recombinant expression of RLN analog 8

[0284] Example 8 is a RLN analog having the following amino acid sequence:

[0285]

[0286] Here, the RLN analog of SEQ ID NO:31 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:31 was used in the expression plasmid.

[0287] Example 9: Recombinant Expression of RLN Analog 9

[0288] Example 9 is a RLN analog having the following amino acid sequence:

[0289]

[0290] Here, the RLN analog of SEQ ID NO:32 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:32 was used in the expression plasmid.

[0291] Example 10: Recombinant Expression of RLN Analog 10

[0292] Example 10 is a RLN analog having the following amino acid sequence:

[0293]

[0294] Here, the RLN analog of SEQ ID NO:33 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:33 was used in the expression plasmid.

[0295] Example 11: Recombinant expression of RLN analog 11

[0296] Example 11 is a RLN analog having the following amino acid sequence:

[0297]

[0298] Here, the RLN analog of SEQ ID NO:34 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:34 was used in the expression plasmid.

[0299] Example 12: Recombinant Expression of RLN Analog 12

[0300] Example 12 is a RLN analog having the following amino acid sequence:

[0301]

[0302] Here, the RLN analog of SEQ ID NO:35 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:35 was used in the expression plasmid.

[0303] Example 13: Recombinant expression of RLN analog 13

[0304] Example 13 is a RLN analog having the following amino acid sequence:

[0305]

[0306] Here, the RLN analog of SEQ ID NO:36 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:36 was used in the expression plasmid.

[0307] Example 14: Recombinant Expression of RLN Analog 14

[0308] Example 14 is a RLN analog having the following amino acid sequence:

[0309]

[0310] Here, the RLN analog of SEQ ID NO:37 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:37 was used in the expression plasmid.

[0311] Example 15: Recombinant Expression of RLN Analog 15

[0312] Example 15 is a RLN analog having the following amino acid sequence:

[0313]

[0314] Here, the RLN analog of SEQ ID NO:38 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:38 was used in the expression plasmid.

[0315] Example 16: Recombinant expression of RLN analog 16

[0316] Example 16 is a RLN analog having the following amino acid sequence:

[0317]

[0318] Here, the RLN analog of SEQ ID NO:39 was generated essentially as described with respect to Example 1, except that the cDNA sequence encoding SEQ ID NO:39 was used in the expression plasmid.

[0319] In vitro function

[0320] Example 17: RLN Analog Albumin Binding by SPR

[0321] In vitro binding of RLN analogs to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin was determined by SPR. Specifically, the affinities of the RLN analogs of Examples 1-14 for serum albumin of these species are summarized in Tables 1-14 below.

[0322] Binding of the RLN analogs of Examples 1-14 to various serum albumins was performed on a Biacore 8K instrument. Serum albumin was immobilized to the surface of a S-series sensor chip CM5 according to the manufacturer's instructions (Amine Coupling Kit BR-1000-50). Briefly, the carboxyl groups on the sensor chip surface (flow cells 1 and 2) were activated by injecting 70 μL of a mixture containing 75 mg / mL EDC and 11.5 mg / mL NHS at 10 μL / min. Human, cynomolgus monkey, mouse, rat, porcine, dog, bovine, and rabbit serum albumins were diluted at 0.5, 0.5, 2.2, 0.6, 0.6, 0.8, 0.6, and 0.3 μg / mL in 10 mM sodium acetate, pH 4.0 (BR-1003-49) and then injected at 10 μL / min onto the activated chip surface (flow cell 2, channels 1-8) for 180 sec (human, mouse, rat, porcine, and bovine serum albumins were obtained from Sigma Aldrich (St. Louis, MO); cynomolgus monkey serum albumin was obtained from Diagnostika (Cologne, Germany); dog serum albumin was obtained from Molecular Innovations (Novi, MI); and rabbit serum albumin was obtained from Fitzgerald Industries Intl. (Acton, MA). Each serum albumin was covalently immobilized via free amines onto a carboxymethyl dextran coated sensor chip CM5 with a target surface density of approximately 100 (62-145) RU. Excess reactive groups on the surface (flow cells 1 and 2) were inactivated by injection of 70 μL 1 M ETA HCl-NaOH pH 8.5.

[0323] The RLN analogs of Examples 1-14 were diluted in HBS-EP+ buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20) 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 was injected individually and sequentially over serum albumin immobilized on the chip surface and dissociated at 25° C. for 600 seconds at a flow rate of 50 μL / min. The surface was regenerated by injecting 10 mM glycine-HCl pH 1.5 (BR-1003-54) at 50 μL / min for 100 seconds. The sensorgrams obtained were analyzed using the Biacore 8K Insight evaluation software (version 2.0.15.12933) 1:1 binding kinetic model fitting analysis to calculate the binding kinetic parameters association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ).

[0324] Table 1: Binding kinetics of RLN analogs of Example 1 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0325]

[0326] Using the RLN analog of Example 1, K D They were determined to be 0.1, 0.5, 3.3, 2.1, 7.1, 1.2 and 37 nM respectively.

[0327] Table 2: Binding kinetics of RLN analogs of Example 2 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0328]

[0329] Using the RLN analog of Example 2, K D They were determined to be 0.1, 0.45, 3.8, 3.0, 8.0, 1.4 and 50 nM respectively.

[0330] Table 3: Binding kinetics of RLN analogs of Example 3 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0331]

[0332] Using the RLN analog of Example 3, K D They were determined to be 0.08, 0.36, 2.4, 1.6, 4.8, 0.7 and 26 nM respectively.

[0333] Table 4: Binding kinetics of RLN analogs of Example 4 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0334]

[0335] Using the RLN analog of Example 4, K D They were determined to be 0.09, 0.42, 3.0, 2.1, 6.2, 1.0 and 37 nM respectively.

[0336] Table 5: Binding kinetics of RLN analogs of Example 5 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0337]

[0338] Using the RLN analog of Example 5, K D They were determined to be 0.45, 1.8, 14, 10, 23, 4.7 and 120 nM respectively.

[0339] Table 6: Binding kinetics of RLN analogs of Example 6 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0340]

[0341] Using the RLN analog of Example 6, K D They were determined to be 0.51, 2.3, 12, 9.7, 24, 4.1 and 130 nM respectively.

[0342] Table 7: Binding kinetics of RLN analogs of Example 7 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0343]

[0344] Using the RLN analog of Example 7, K DThey were determined to be 0.06, 0.29, 2.1, 1.4, 3.7, 0.69 and 25 nM respectively.

[0345] Table 8: Binding kinetics of RLN analogs of Example 8 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0346]

[0347] Using the RLN analog of Example 8, K D They were determined to be 0.08, 0.32, 1.8, 1.7, 4.1, 0.66 and 21 nM respectively.

[0348] Table 9: Binding kinetics of RLN analogs of Example 9 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0349]

[0350] Using the RLN analog of Example 9, K D They were determined to be 0.19, 0.86, 9.5, 8.2, 14, 3.8 and 84 nM respectively.

[0351] Table 10: Binding kinetics of RLN analogs of Example 10 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0352]

[0353] Using the RLN analog of Example 10, K D They were determined to be 0.37, 1.5, 9.7, 7.8, 20, 3.4 and 100 nM respectively.

[0354] Table 11: Binding kinetics of RLN analogs of Example 11 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0355]

[0356] Using the RLN analog of Example 11, K D They were determined to be 0.17, 0.48, 3.6, 1.3, 7.3, 1.3 and 27 nM respectively.

[0357] Table 12: Binding kinetics of RLN analogs of Example 12 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0358]

[0359] Using the RLN analog of Example 12, K D They were determined to be 0.14, 0.47, 2.6, 1.7, 4.2, 0.77 and 30 nM respectively.

[0360] Table 13: Binding kinetics of RLN analogs of Example 13 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0361]

[0362] Using the RLN analog of Example 13, K D They were determined to be 0.14, 0.49, 2.6, 2.0, 5.0, 0.92 and 29 nM respectively.

[0363] Table 14: Binding kinetics of RLN analogs of Example 14 to human, cynomolgus monkey, mouse, rat, pig, dog, bovine and rabbit serum albumin at 25°C

[0364]

[0365] Using the RLN analog of Example 14, K D They were determined to be 0.16, 0.61, 3.1, 2.2, 6.0, 1.1 and 34 nM respectively.

[0366] Example 18: In vitro potency of RLN analogs at RXFP1 and RXFP2 receptors

[0367] Generation of CHO-CRE-Luc cell line: CHO-K1 cells (ATCC) were cultured in DMEM-F123:1 containing 5% FBS and 20 mM HEPES, 40 μg / mL L-proline, 1x antibiotics, and treated with TrypLE every 2-3 days. TMExpress (Gibco) 1:5 split. According to the manufacturer's instructions, cells were transfected with plasmid DNA of pGL4.29 [luc2P / CRE / Hygro] (Promega) and Fugene HD (Promega). Transfected cells were selected for 3-4 weeks with 1 mg / mL of hygromycin B. Clones were obtained by limiting dilution cloning into 96-well plates and confirmed by forskolin response using luciferase assay with Bright-Glo Reagent (Promega). The clones were expanded, harvested, resuspended in freezing medium, aliquoted into freezing vials, and stored in liquid nitrogen for long-term storage. The top responder clone #2B6 with the best forskolin response (signal to background ratio) was selected for subsequent transfection of human RXFP1 and RXFP2 receptors.

[0368] Generation of CHO human RXFP1 and human RXFP2 expressing cell lines: CHO-CRE-Luc line #2B6 cells were cultured in DMEM-F123:1 containing 5% FBS and 20mM HEPES, 40μg / mL L-proline, 1x antibiotics, 1mg / mL hygromycin B and split 1:5 every 2-3 days with TrypLE Express (Gibco). Cells were transfected with plasmid DNA of human RXFP1 receptor or human RXFP2 receptor and Fugene HD (Promega) according to the manufacturer's instructions. Transfected cells were selected with hygromycin B (1mg / mL) and puromycin (6μg / mL) for 3-4 weeks. Clonal lines were obtained by limiting dilution cloning into 96-well plates. Clonal lines were confirmed by human RLN2 response. Clones were expanded, harvested, resuspended in freezing medium, aliquoted into freezing vials, and cryopreserved under liquid nitrogen for long-term storage. The clonal lines with the best response (signal to background ratio) to human RLN2 were selected for assay validation.

[0369] Human RXFP1 and RXFP2 receptor luciferase assay: CHO cell lines expressing human RXFP1 or human RXFP2 were cultured with selection medium (DMEM-F123:1, containing 5% FBS and 20mM HEPES, 40μg / mL L-proline, 1x antibiotics, 6μg / mL puromycin, 1mg / mL hygromycin B). On day 1 (one day before cAMP CRE luciferase reporter assay), cells were washed once with PBS and lysed with cell dissociation solution (cell dissociation solution without enzymes, GIBCO catalog number 13151-014: TrypLE TMExpress=30:1) was removed from the flask and resuspended in plating medium (DMEM-F123:1, containing 20mM HEPES, 1x antibiotics, 0.5% FBS). The cells were plated at 20,000 cells / 0.1mL / well in a 96-well plate (Falcon catalog number 353219). The cells were cultured overnight at 37°C 5% CO2. On day 1 (the day of the cAMP CRE luciferase reporter assay), the medium was removed and replaced with 90μL serum-free medium (DMEM-F123:1, containing 20mM HEPES, 1x antibiotics). The plate was incubated at 37°C for 2 hours, and then 10μL of 10x ligand (RLN2, final 1x) was added. The plate was incubated at 37°C for another 4 hours. After the incubation period, the plate was allowed to reach room temperature for 15min. Then, 50μL Bright-Glo TM Add to each well and read the plate on a Biotek Neo2 reader using Gen5 software.

[0370] Statistical analysis of data: Importing data from the Biotek Neo2 reader into GraphPad EC was generated by variable slope-four-parameter dose-response curve analysis. 50 value.

[0371] Table 15: In vitro potency of RLN analogs at RXPF1 and RXPF2 receptors

[0372]

[0373] Function in the body

[0374] Example 19: Pharmacokinetics of RLN Analogs in Male Sprague Dawley Rats

[0375] Male Sprague Dawley rats were administered a single subcutaneous dose of 200 nmol / kg of RLN analogs in His-NaCl buffer (pH 6.0) at a volume of 1.0 mL / kg. Blood was collected for pharmacokinetic characterization at 3, 6, 12, 24, 48, 72, 96, 120, 144, 168, and 240 hours after dosing.

[0376] Plasma concentrations of RLN analogs were determined by a qualified LC / MS method at Eli Lilly and Company. Example compounds and analog internal standards were extracted from 100% rat plasma using human RLN antibody and subsequently purified using Q-Exactive TM N-terminal tryptic peptides were detected by mass spectrometry.

[0377] Data for the RLN analogs of Examples 4 and 7 are provided in Table 16 below.

[0378] Table 16: Mean plasma pharmacokinetic parameters of RLN analogs following a single 200 nmol / kg subcutaneous dose in male Sprague Dawley rats

[0379]

[0380] Note: Abbreviation: t 1 / 2 = half-life, T max = time to reach maximum concentration, C max = maximum observed plasma concentration, AUC 0-inf = area under the curve from time 0 hours to infinity, CL / F = clearance / bioavailability, N = 3 animals / group / time point.

[0381] As shown in Table 16, the RLN analogs of Examples 4 and 7 exhibited prolonged pharmacokinetic properties in Sprague Dawley rats.

[0382] Example 20: In vivo effects of RLN analogs on renal blood flow following intravenous administration in male Sprague Dawley rats

[0383] Prior to the start of the experiment, male 5-week-old Sprague Dawley rats (Charles River Laboratories, Inc.) were housed in an animal house with a normal light / dark cycle for 1 week. Rats were then randomized based on body weight into the following treatment groups: vehicle (20 mM His / 20 mM NaCl, pH 6.0 buffer) and the RLN analog of Example 7. The RLN analogs were administered as follows: 2.44 μg / kg IV bolus followed by 0.36 μg / kg / hr IV infusion, 8.13 μg / kg IV bolus followed by 1.2 μg / kg / hr IV infusion, 24.4 μg / kg IV bolus followed by 3.6 μg / kg / hr IV infusion, and 81.3 μg / kg IV bolus followed by 11.9 μg / kg / hr IV infusion.

[0384] To measure the effects of RLN analogs on renal blood flow, rats were anesthetized with urethane (1.2 g / kg, intraperitoneal) and prepared for abdominal / renal ultrasound imaging and renal artery pulsed wave Doppler blood flow measurement (VisualSonics, Model Vevo 3100 ultrasound system; Fujifilm). A chronic tail vein catheter was placed for intravenous bolus and infusion administration. After a 30-min acclimation period, renal blood flow measurements were obtained at baseline and 3 hours after the start of dosing.

[0385] Table 17: Effects of intravenously administered RLN analogs on renal blood flow in healthy rats

[0386]

[0387] Note: The results are expressed as mean ± SD. Significantly different from baseline p<0.001 ANOVA, *Significantly different from vehicle p<0.001 ANOVA, N=5 / treatment group.

[0388] As seen in Table 17, the RLN analog of Example 7 significantly increased renal blood flow by 26.6%, 48.8% and 60.4% after 3 hours of exposure in the groups treated with 8.13 μg / kg IV push followed by 1.2 μg / kg / hr IV infusion, 24.4 μg / kg IV push followed by 3.6 μg / kg / hr IV infusion, and 81.3 μg / kg IV push followed by 11.9 μg / kg / hr IV infusion.

[0389] Example 21: In vivo effects of RLN analogs on renal blood flow following subcutaneous administration in male Sprague Dawley rats

[0390] Male 5-week-old Sprague Dawley rats (Charles River Laboratories, Inc.) were housed in an animal house with a normal light / dark cycle for 1 week prior to the start of the experiment. Rats were then randomized based on body weight into the following treatment groups: vehicle (20 mM His / 20 mM NaCl, pH 6.0 buffer) and the RLN analog of Example 7. The RLN analog was administered subcutaneously at 180 μg / kg.

[0391] To measure the effects of RLN analogs on renal blood flow, 48 hours after dosing, rats were anesthetized with urethane (1.2 g / kg, intraperitoneal) and prepared for abdominal / renal ultrasound imaging and renal artery pulsed wave Doppler blood flow measurement (VisualSonics, Model Vevo 3100 ultrasound system; Fujifilm). After a 30-min acclimatization period, renal blood flow measurements were obtained.

[0392] Table 18: Effects of subcutaneously administered RLN analogs on renal blood flow in healthy rats

[0393]

[0394] Note: Results are expressed as mean ± SD, *Significantly different from vehicle p < 0.001 ANOVA, N = 10 / treatment group.

[0395] As seen in Table 18, the RLN analog of Example 7 significantly increased renal blood flow after 48 hours of exposure to a subcutaneous dose of 180 μg / kg.

[0396] sequence

[0397] The following nucleic acid and / or amino acid sequences are referred to in the present disclosure and are provided below for reference.

[0398] SEQ ID NO: 1 - Human RLN1 proto

[0399]

[0400] SEQ ID NO:2—Human RLN1 A chain

[0401]

[0402] SEQ ID NO:3—Human RLN1 B chain

[0403]

[0404] SEQ ID NO:4—Human RLN2 proto

[0405]

[0406] SEQ ID NO:5—Human RLN2 A chain

[0407]

[0408] SEQ ID NO:6—Human RLN2 B chain

[0409]

[0410] SEQ ID NO:7—Human RLN3 proto

[0411]

[0412] SEQ ID NO:8—Human RLN3 A chain

[0413] SEQ ID NO:9—Human RLN3 B chain

[0414] SEQ ID NO:10—VHH portion #1 (C22)

[0415]

[0416] SEQ ID NO: 11 - VHH part #2 (C22.43)

[0417]

[0418] SEQ ID NO:12 - VHH part #3 (C80)

[0419]

[0420] SEQ ID NO:13 - VHH section #4 (C80.43)

[0421]

[0422] SEQ ID NO:14-L1((GGGGQ) n The basic sequence of

[0423] GGGGQ

[0424] SEQ ID NO:15-L1((GGGQ) n The basic sequence of

[0425] GGG

[0426] SEQ ID NO:16-L1((GGGGS) n The basic sequence of

[0427] GGGGS

[0428] SEQ ID NO:17-L1((PGPQ) n The basic sequence of

[0429] PQ

[0430] SEQ ID NO:18-L1((PGPA) n The basic sequence of

[0431] PGPA

[0432] SEQ ID NO:19-L1#1((GGGGQ)5)

[0433]

[0434] SEQ ID NO:20-L1#2((PGPQ)8)

[0435]

[0436] SEQ ID NO:21-L1#3(PGPA)8

[0437]

[0438] SEQ ID NO:22-L2#1

[0439] GGGSGGSGGG

[0440] SEQ ID NO:23-L2#2

[0441] GGGSGGSGGSGGG

[0442] SEQ ID NO:24 - RLN2 analog #1 (C22-(G4Q)5-A10B(desB1)RLN)

[0443]

[0444] SEQ ID NO:25 - RLN2 Analog #2 (C22-(PGPA)8-A10B(desB1)RLN)

[0445]

[0446] SEQ ID NO:26 - RLN2 analog #3 (C22-(G4Q)5-B10A(desB1)RLN)

[0447]

[0448] SEQ ID NO:27 - RLN2 Analog #4 (C22-(PGPQ)8-B10A(desB1)RLN)

[0449]

[0450] SEQ ID NO:28 - RLN2 analog #5 (B10A (desB1) RLN-(G4Q)5-C22.43)

[0451]

[0452] SEQ ID NO:29 - RLN2 Analog #6 (B10A (desB1) RLN-(PGPQ)8-C22.43)

[0453]

[0454] SEQ ID NO:30 - RLN2 analog #7 (C80-(G4Q)5-B10A(desB1)RLN)

[0455]

[0456] SEQ ID NO:31 - RLN2 analog #8 (C80-(PGPQ)8-B10A(desB1)RLN)

[0457]

[0458] SEQ ID NO:32 - RLN2 analog #9 (B10A (desB1) RLN-(G4Q)5-C80.43)

[0459]

[0460] SEQ ID NO:33 - RLN2 Analog #10 (B10A (desB1) RLN-(PGPQ)8-C80.43)

[0461]

[0462] SEQ ID NO:34 - RLN2 analog #11 (C22-(G4Q)5-B13A (desB1, desA1-4) RLN)

[0463]

[0464] SEQ ID NO:35-RLN2 analog #12 (C80-(G4Q)5-B13A (desB1, desA1-4) RLN)

[0465]

[0466] SEQ ID NO:36 - RLN2 analog #13 (C80-(G4Q)5-A10B (desB1, desA1-4) RLN)

[0467]

[0468] SEQ ID NO:37 - RLN2 Analog #14 (C80-(PGPQ)8-A10B (desB1, desA1-4) RLN)

[0469]

[0470] SEQ ID NO:38 - RLN2 Analog #15 (C22-(G4Q)5-B10ARLN)

[0471]

[0472] SEQ ID NO:39 - RLN2 Analog #16 (C22-(PGPA)8-B10ARLN)

[0473]

[0474] SEQ ID NO:40—Human RXFP1 receptor

[0475] SEQ ID NO:41—Human RXFP2 receptor

[0476] SEQ ID NO:42—Human RXFP3 receptor

[0477] SEQ ID NO:43—Human RXFP4 receptor

[0478] SEQ ID NO:44—Signal peptide

[0479] SEQ ID NO:45—VHH section #5 (MC6.1)

[0480]

[0481] SEQ ID NO:46—VHH section #6 (MC6.1C6)

[0482]

[0483] SEQ ID NO:47 - VHH section #7 (C22-G26Y)

[0484]

[0485] SEQ ID NO:48—VHH section #8 (C22-R27A)

[0486]

[0487] SEQ ID NO:49—VHH section #9 (C22-I57E)

[0488]

[0489] SEQ ID NO:50—VHH section #10 (C22-I57Q)

[0490]

[0491] SEQ ID NO:51 - VHH section #11 (C22-Y59A)

[0492]

[0493] SEQ ID NO:52 - VHH section #12 (C22-Y59E)

[0494]

[0495] SEQ ID NO:53 - VHH section #13 (C22-Y59Q)

[0496]

[0497] SEQ ID NO:54—VHH section #14 (C22-Y59S)

[0498]

[0499] SEQ ID NO:55 - VHH section #15 (C22-Y59T)

[0500]

[0501] SEQ ID NO:56—VHH section #16 (C22-R102K)

[0502]

[0503] SEQ ID NO:57 - VHH section #17 (C22-R102Q)

[0504]

[0505] SEQ ID NO:58—VHH section #18 (C22-R102S)

[0506]

[0507] SEQ ID NO:59—VHH section #19 (C22-P103E)

[0508]

[0509] SEQ ID NO:60—VHH section #20 (C22-P103Q)

[0510]

[0511] SEQ ID NO:61 - VHH section #21 (C22-P103S)

[0512]

[0513] SEQ ID NO:62—VHH section #22 (C22-L104E)

[0514]

[0515] SEQ ID NO:63 - VHH section #23 (C22-L104G)

[0516]

[0517] SEQ ID NO:64 - VHH section #24 (C22-L104Q)

[0518]

[0519] SEQ ID NO:65—VHH section #25 (C22-L104T)

[0520]

[0521] SEQ ID NO:66—VHH section #26 (C22-S107E)

[0522]

[0523] SEQ ID NO:67-L2#3

[0524] SGGGGSGGGG.

Claims

1. A compound comprising the following amino acid sequence or a sequence having at least 90% sequence similarity thereto:

2. A compound comprising the following amino acid sequence or a sequence having at least 90% sequence similarity thereto:

3. A compound comprising the following amino acid sequence or a sequence having at least 90% sequence similarity thereto:

4. A compound comprising the following amino acid sequence or a sequence having at least 90% sequence similarity thereto:

Images