Natriuretic peptide analog and uses thereof

Abstract

The present invention relates to natriuretic peptide analogues with enhanced stability and a pharmaceutical composition comprising same. Exhibiting a prolonged in vivo half-life, high bioavailability, and a high bone growth rate as well as being relatively short sequence length, a C-type natriuretic peptide analogue according to an embodiment of the present invention has advantages of being easy to synthesize and reducing production costs, and thus is expected to be effectively used in compositions for promoting growth and in the prevention or treatment of bone-related diseases.

Description

Sodium diuretic peptide analogs and uses thereof

[0001] The present invention relates to natriuretic peptide analogs and uses thereof, and more particularly to natriuretic peptide analogs with increased stability and pharmaceutical compositions comprising the same.

[0002]

[0003] Natriuretic peptides, found in various animals including mammals, are generally divided into three types: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP).

[0004] CNP is an autocrine and paracrine regulatory mediator released by endothelial cells, cardiomyocytes, and fibroblasts, and acts through two cognate receptors, natriuretic peptide receptor B (NPR-B) and natriuretic peptide receptor C (NPR-C), which activate respective signaling pathways that mediate complementary but distinct cellular responses. Furthermore, CNP specifically binds to NPR-B, activates anilyl cyclase-B, and modulates intracellular cGMP levels, resulting in various physiological effects, such as natriuretic function, homeostasis, and blood pressure regulation.

[0005] However, CNP is rapidly degraded due to its very short half-life in plasma of 2.6 minutes. This is because CNP is primarily degraded by cleavage by neutral endopeptidase (NEP) or by internalization by the natriuretic peptide receptor C (NPR-C). Therefore, the development of CNP analogues with a longer half-life than wild-type CNP is required for use as pharmaceuticals.

[0006] Currently, C-type natriuretic peptide analogs under development for commercialization include BMN111 (BioMarin Pharmaceutical Inc.) and ASB20123 (Daiichi Sankyo Company, Limited). Their half-lives are known to be approximately 20 and 30 minutes, respectively. Given that BMN111 is commercially available as a once-daily formulation, there is still a need for CNP analogs with improved half-life, bioavailability, and efficacy, thereby reducing dosing frequency and improving administration route convenience.

[0007]

[0008] Accordingly, the inventors of the present invention developed a novel C-type natriuretic peptide analog (CNP analog) with improved in vivo stability, increased half-life and bioavailability, and completed the present invention.

[0009] Therefore, the object of the present invention is to provide a novel C-type natriuretic peptide analogue with increased half-life and bioavailability.

[0010] Another object of the present invention is to provide a growth promoting composition comprising the above C-type natriuretic peptide analogue.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating growth disorders comprising the above C-type natriuretic peptide analogue.

[0012] Another object of the present invention is to provide a method for promoting growth using the above C-type natriuretic peptide analogue.

[0013] Another object of the present invention is to provide a growth promoting use of the above C-type natriuretic peptide analogue.

[0014]

[0015] The present invention provides C-type natriuretic peptide analogues having a structure represented by the following general formula 1:

[0016] [General Formula 1]

[0017] (P1)-PPKKGPPNG-(P2)

[0018] In the above general formula 1,

[0019] P1 is a peptide consisting of a sequence of 0 to 4 amino acids.

[0020] P2 is a peptide consisting of an amino acid sequence of SEQ ID NO: 2 or 3 or a peptide comprising one or more amino acid substitution mutations in the amino acid sequence of SEQ ID NO: 2 or 3.

[0021] The present invention also provides a growth promoting composition comprising the above C-type natriuretic peptide analogue.

[0022] The present invention also provides a pharmaceutical composition for preventing or treating growth disorders, comprising the C-type natriuretic peptide analogue.

[0023] The present invention also provides a method for promoting growth in a non-human subject, comprising administering to the non-human subject a C-type natriuretic peptide analogue.

[0024] The present invention also provides a growth promoting use of the C-type natriuretic peptide analogue described above.

[0025]

[0026] A C-type natriuretic peptide analogue according to an example of the present invention exhibits increased stability, a long half-life in vivo, and thus high bioavailability, and exhibits a high bone growth effect, and thus can be used for growth promotion or for the prevention or treatment of growth disorders.

[0027]

[0028] Figure 1 is a graph showing the residual amount measured after administering a C-type natriuretic peptide analogue according to an example of the present invention to rats.

[0029] Figure 2 is a graph showing the half-life calculated by measuring the residual amount after administering a C-type natriuretic peptide analogue according to an example of the present invention to rats.

[0030] Figure 3 is a graph showing the results of pharmacokinetic evaluation after intravenous administration of a C-type natriuretic peptide analogue according to an example of the present invention to a rat.

[0031] Figure 4 is a graph showing the results of pharmacokinetic evaluation after subcutaneous administration of a C-type natriuretic peptide analogue according to an example of the present invention to a rat.

[0032] FIG. 5 is a graph showing the bioavailability (BA) of a C-type natriuretic peptide analogue according to an example of the present invention.

[0033] Figure 6 is a graph showing the results of evaluating the platelet aggregation inhibition effect of a C-type natriuretic peptide analog according to an example of the present invention.

[0034] Figure 7a is a graph showing changes in blood pressure after administering a C-type natriuretic peptide analogue according to an example of the present invention to rats.

[0035] Figure 7b is a graph showing changes in heart rate after administering a C-type natriuretic peptide analogue according to an example of the present invention to a rat.

[0036] Figure 8 is a graph confirming NEP resistance after administering a C-type natriuretic peptide analogue according to an example of the present invention to rats.

[0037] FIGS. 9a to 9c are graphs showing the results of growth in body weight (FIG. 9a), body length (FIG. 9b), and tail length (FIG. 9c) after administering a C-type natriuretic peptide analogue according to an example of the present invention to rats.

[0038] FIGS. 10a to 10c are graphs showing the results of growth in body weight (FIG. 10a), body length (FIG. 10b), and tail length (FIG. 10c) after cross-administration of a C-type natriuretic peptide analogue according to an example of the present invention to rats.

[0039] Figure 11a is a graph showing changes in body weight when a C-type natriuretic peptide analogue according to an example of the present invention is administered at various doses.

[0040] Figure 11b is a graph showing the results of trunk length growth when a C-type natriuretic peptide analogue according to an example of the present invention is administered at various doses.

[0041] Figure 11c is a graph showing the results of tail length growth when a C-type natriuretic peptide analogue according to an example of the present invention is administered at various doses.

[0042] Figure 11d is a photograph measuring mouse growth when various doses of a C-type natriuretic peptide analogue according to an example of the present invention were administered.

[0043]

[0044] The present invention relates to a C-type natriuretic peptide analogue having increased half-life and bioavailability, and to its use in preventing, improving or treating growth disorders and promoting growth.

[0045] The C-type natriuretic peptide analog of the present invention has a long half-life in vivo, high bioavailability due to this, and a high bone growth rate, and has a relatively short length, so that the synthesis of the peptide drug is easy and the synthesis cost can be reduced, and thus it can be effectively used as a composition for promoting growth or for treating or preventing bone-related diseases.

[0046]

[0047] (1) Half-life advantage

[0048] The half-life of a C-type natriuretic peptide analog (CNP analog) according to an example of the present invention may be increased compared to the wild-type, and is not limited thereto, but the half-life of the CNP analog according to the present invention may be increased by 1 to 200 times or more compared to the wild-type CNP. This half-life advantage corresponds to an important factor in determining the dosing frequency of a peptide drug, and thus can be advantageously utilized when developing a peptide drug.

[0049]

[0050] (2) Superior bioavailability (BA, %)

[0051] The bioavailability of a C-type natriuretic peptide analog (CNP analog) according to an example of the present invention may be improved compared to wild-type CNP, and is not limited thereto, but the bioavailability of the CNP analog according to the present invention may be increased by about 10 times or more compared to wild-type CNP as a control. This increase in bioavailability can be advantageously utilized in the development of pharmaceuticals.

[0052]

[0053] (3) Superiority in activity and effectiveness

[0054] When a C-type natriuretic peptide analog (CNP analog) according to an example of the present invention is administered to normal mice, a bone growth rate may be more than twice as high as that of wild-type CNP for 1 to 2 weeks after administration. This may be due to the high bioavailability characteristic resulting from the prolonged half-life of the C-type natriuretic peptide analog according to an example of the present invention. Preferably, the C-type natriuretic peptide analog according to an example of the present invention may have improved activity and efficacy compared to conventional techniques.

[0055] Specifically, the C-type natriuretic peptide analog (CNP analog) of the present invention can be manufactured by introducing one or more mutations selected from the group consisting of substitutions, deletions, and additions to amino acids constituting CNP in order to improve the efficacy of CNP, thereby producing a CNP analog candidate. In the present invention, a CNP analog candidate with improved efficacy was selected by performing resistance evaluation of neprilysin, a neutral endopeptidase that degrades CNP to cause it to lose its function, pharmacokinetic evaluation to verify in vivo stability, platelet aggregation inhibition effect evaluation, cardiovascular safety (hemodynamics) evaluation, and mouse growth measurement. However, any method for evaluating activity and efficacy that can be commonly employed by a person skilled in the art can be used, and there are no limitations thereto.

[0056]

[0057] A C-type natriuretic peptide analogue according to one embodiment of the present invention comprises at least one amino acid mutation selected from the group consisting of at least one amino acid substitution mutation, at least one amino acid addition mutation at the N-terminus and / or C-terminus, and at least one amino acid deletion mutation in wild-type CNP. A C-type natriuretic peptide analogue according to one embodiment of the present invention may have increased stability and an increased half-life in vivo while maintaining or increasing the physiological activity of wild-type CNP.

[0058] As an example, the C-type natriuretic peptide analogues of the present invention may have a structure represented by the following general formula 1:

[0059] [General Formula 1]

[0060] (P1)-PPKKGPPNG-(P2)

[0061] In the above general formula 1, P1 is a peptide consisting of 0 to 4 amino acid sequences, and may include the following amino acid sequence.

[0062] P1 is (X1)-(X2)-(X3)-(X4),

[0063] X1 is Gly(G), Pro(P), Thr(T), Ala(A), Ser(S), or absent,

[0064] X2 is Asp(D), Gly(G), Lys(K), Ser(S), Asn(N), Glu(E), Gln(Q), or absent,

[0065] X3 is Asn(N), Lys(K), Asp(D), Ser(S), Gln(Q), or absent,

[0066] X4 is Lys(K), Thr(T), Ser(S), Arg(R), Glu(E), Gln(Q), His(H), or absent.

[0067]

[0068] Also, more preferably, as an example, in the general formula 1, P1 is a peptide consisting of 0 to 4 amino acid sequences and may include the following amino acid sequence.

[0069] P1 is (X1)-(X2)-(X3)-(X4)

[0070] X1 is Gly (G), Pro (P), or absent,

[0071] X2 is Asp(D), Gly(G), Ser(S), or absent,

[0072] X3 is Asn(N), Lys(K), or absent,

[0073] X4 may contain an amino acid sequence of Lys (K), Ser (S), or absent.

[0074]

[0075] Additionally, P2 may be a peptide consisting of an amino acid sequence of SEQ ID NO: 2 or 3 or a peptide comprising one or more amino acid substitution mutations in the amino acid sequence of SEQ ID NO: 2 or 3.

[0076] Specifically, in the general formula 1, P2 may be a peptide consisting of / including an amino acid sequence of SEQ ID NO: 2, a peptide including one or more amino acid substitution mutations in the amino acid sequence of SEQ ID NO: 2, or a peptide in which the 4th amino acid, the 6th amino acid, and / or the 12th amino acid in the amino acid sequence of SEQ ID NO: 2 is substitutionally mutated.

[0077] For example, in the general formula 1 above, P2 may be represented by the following general formula 2 (SEQ ID NO: 5):

[0078] [General Formula 2]

[0079] CFG-(X5)-K-(X6)-DRIGS-(X7)-SGLGC

[0080] In the above general formula 2,

[0081] X5 and X7 are selected from amino acids having a cationic or hydrophobic side chain, and X6 can be selected from amino acids having a cationic or hydrophobic side chain except Ile (I).

[0082] As an example, in the general formula 2, X5 may be Leu(L) or His(H), X6 may be Leu(L), and X7 may be Met(M) or His(H), specifically,

[0083] X5 is Leu(L), X6 is Leu(L), X7 is Met(M);

[0084] X5 is Leu(L), X6 is Leu(L), X7 is His(H);

[0085] X5 is His (H), X6 is Leu (L), X7 is Met (M); or

[0086] X5 can be His(H), X6 can be Leu(L), and X7 can be His(H), but is not limited thereto.

[0087] Additionally, in the general formula 2, the first cysteine ​​(C) and the last cysteine ​​(C) can form a ring through a CC bond.

[0088]

[0089] A more preferred example of the C-type natriuretic peptide analogue of the present invention is:

[0090] P1 of the above general formula 1 is (X1)-(X2)-(X3)-(X4),

[0091] X1 is Gly(G), Pro(P), Thr(T), Ala(A), Ser(S), or absent,

[0092] X2 is Asp(D), Gly(G), Lys(K), Ser(S), Asn(N), Glu(E), Gln(Q), or absent,

[0093] X3 is Asn(N), Lys(K), Asp(D), Ser(S), Gln(Q), or absent,

[0094] X4 is Lys(K), Thr(T), Ser(S), Arg(R), Glu(E), Gln(Q), His(H), or absent, and at the same time, P2 can be represented by the following general formula 2 (SEQ ID NO: 5):

[0095] [General Formula 2]

[0096] CFG-(X5)-K-(X6)-DRIGS-(X7)-SGLGC

[0097] In the above general formula 2,

[0098] X5 and X7 are each selected from amino acids containing a cationic or hydrophobic side chain, and X6 can be selected from amino acids containing a cationic or hydrophobic side chain except Ile (I).

[0099] In a more specific embodiment of the present invention, P2 in the general formula 2,

[0100] X5 is Leu(L) or His(H),

[0101] X6 can be Leu(L), and X7 can be Met(M) or His(H),

[0102] At the same time, the above P1 is more specifically (X1)-(X2)-(X3)-(X4).

[0103] X1 is Gly (G), Pro (P), or none

[0104] X2 is Asp(D), Gly(G), Ser(S), or none

[0105] X3 is Asn(N), Lys(K), or none

[0106] X4 may contain an amino acid sequence of Lys(K), Ser(S), or none.

[0107]

[0108] According to an example of the present invention, a C-type natriuretic peptide analogue may include a mutation in which an acetyl group and / or a fatty acid (e.g., 8-amino-3,6-dioxaoctanoic acid, miniPEG, miniPEG2, caprylic acid, capric acid, lauric acid, stearic acid, palmitic acid, etc.) or a derivative thereof is introduced into at least one amino acid, or some amino acids are substituted with Aib (Aminoisobutyric acid), or some amino acids are substituted with D-type amino acids, etc.

[0109] A C-type natriuretic peptide analogue according to an example of the present invention may have at least one amino acid in the amino acid sequence of general formula 1 acetylated. For example, a C-type natriuretic peptide analogue according to an example of the present invention may have an N-terminal amino acid acetylated.

[0110] According to an example of the present invention, a C-type natriuretic peptide analogue may be one in which one or more (for example, one, two, or three) repeating units of a fatty acid or a derivative thereof are introduced directly or via a linker to at least one amino acid among the 1st to 5th amino acids from the N-terminus, for example, the first amino acid from the N-terminus. In this case, the fatty acid may be a fatty acid having 3 to 21 carbon atoms, but is not limited thereto. Specifically, the fatty acid or a derivative thereof may be introduced as one or more repeating units to one or more of the 1st to 5th or 1st to 4th amino acids from the N-terminus of the C-type natriuretic peptide analogue. As an example, the fatty acid or a derivative thereof may be introduced to the first or third amino acid, and as an example, the fatty acid may be introduced to the first or third amino acid.

[0111] The above fatty acid may be introduced into a C-type natriuretic peptide analogue according to an example of the present invention to increase stability. For example, the fatty acid or derivative thereof may be a small PEG (miniPEG) comprising a small polyethylene glycol moiety (PEG) comprising the structure [-O-CH2-CH2-]n (wherein n may be an integer from 2 to 16, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16), a functionalized miniPEG comprising one or more functional groups (including but not limited to, amine, hydroxyl, thiol, and carboxyl groups or any combination thereof), a fatty acid having 6 to 21 carbon atoms, a fatty acid having 13 to 21 carbon atoms, or a derivative of the fatty acid, as an example, 8-amino-3,6-dioxaoctanoic acid (trade name: miniPEG2), miniPEG, CH3(CH2) 14COOH (palmitic acid), caprylic acid, capric acid, lauric acid, or stearic acid.

[0112] The fatty acid or its derivative may be introduced directly or via a linker to at least one amino acid in the amino acid sequence of the C-type natriuretic peptide analog according to an example of the present invention. For example, the linker may be, but is not limited to, gamma glutamic acid, aminocaproic acid (AHX), PEG, etc.

[0113] A C-type natriuretic peptide analogue according to an example of the present invention has a structure of general formula 1 and may include one or more mutations selected from the group consisting of amino acid deletions, substitutions and additions, and specifically, a mutant CNP analogue according to an example of the present invention may have an amino acid sequence described in Table 1 below. In Table 1 below, (CC) means that cysteine ​​and cysteine ​​can combine to form a ring. Accordingly, a C-type natriuretic peptide analogue according to an example of the present invention may include one or more amino acid sequences selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 13.

[0114]

[0115] Sequence number Amino acid sequence 6GDNKPPKKGPPNGCFGLKLDRIGSMSGLGC (CC) 7GDNKPPKKGPPNGCFGHKLDRIGSMSGLGC (CC) 8GDNKPPKKGPPNGCFGHKLDRIGSHSGLGC (CC) 9GDNKPPKKGPPNGCFGLKLDRIGSHSGLGC (CC) 10PGNKPPKKGPPNGCFGLKLDRIGSHSGLGC (CC) 11GD-K(gamma Glu-Pal)-KPPKKGPPNGCFGHKLDRIGSHSGLGC (CC) 12Palmitoyl-GDNKPPKKGPPNGCFGHKLDRIGSHSGLGC (CC) 13GS-K(gamma Glu-Pal)-SPPKKGPPNGCFGHKLDRIGSHSGLGC (CC)

[0116] According to one embodiment of the present invention, a C-type natriuretic peptide analogue may have improved properties selected from the group consisting of in vivo half-life, bioavailability, and growth plate maintenance compared to wild-type CNP.

[0117] According to an example of the present invention, a C-type natriuretic peptide analogue may have a half-life that is greater than 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, or 60-fold greater than the in vivo half-life of wild-type CNP. At this time, even if the upper limit of the in vivo half-life is not specified, a person skilled in the art will be able to clearly understand the technical effect of the present invention in which the in vivo half-life is improved compared to wild-type CNP, but for example, the upper limit of the in vivo half-life may be 200 times or less, 150 times or less, 100 times or less, 80 times or less, or 70 times or less of the in vivo half-life of wild-type CNP, but is not limited thereto.

[0118] In one embodiment, the C-type natriuretic peptide analogue according to an example of the present invention may have an in vivo half-life that is greater than 1-fold, greater than 1.5-fold, greater than 2-fold, greater than 2.5-fold, greater than 3-fold, greater than 3.5-fold, greater than 4-fold, greater than 4.5-fold, greater than 5-fold, greater than 5.5-fold, or greater than 6-fold compared to BMN111. In this case, even if the upper limit of the in vivo half-life is not specified, a person skilled in the art will be able to clearly understand the technical effect of the present invention having improved in vivo half-life compared to BMN111. For example, the upper limit of the in vivo half-life may be, but is not limited to, 100-fold or less, 50-fold or less, 30-fold or less, 20-fold or less, 15-fold or less, or 10-fold or less of the in vivo half-life of BMN111.

[0119] The bioavailability (BA) of a C-type natriuretic peptide analogue according to an example of the present invention may be 1 to 20 times, 1 to 15 times, 1 to 10 times, 2 to 20 times, 2 to 15 times, 2 to 10 times, 5 to 20 times, 5 to 15 times, or 5 to 10 times, based on the bioavailability (BA) of wild-type CNP. For example, the bioavailability (BA) of a C-type natriuretic peptide analogue according to an example of the present invention may be increased by at least 2 times or more based on the bioavailability (BA) of wild-type CNP.

[0120]

[0121] A C-type natriuretic peptide analogue according to an example of the present invention may have a higher bone growth rate than wild-type CNP when administered in an amount equal to or less than wild-type CNP.

[0122] A C-type natriuretic peptide analogue according to an example of the present invention may be characterized by having resistance to neutral endopeptidase, and specifically, may have superior neutral endopeptidase resistance compared to wild-type CNP and BMN111. For example, when a C-type natriuretic peptide analogue according to an example of the present invention is treated with neutral endopeptidase, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more, or 100% or more may remain after 200 minutes, and the C-type natriuretic peptide analogue according to an example of the present invention may exhibit superior neutral endopeptidase resistance compared to the control group.

[0123] In another aspect, the present invention relates to a composition for promoting growth, or a composition for preventing and treating growth disorders, comprising the C-type natriuretic peptide analogue. The composition may be a pharmaceutical composition or a food composition.

[0124] The above-described growth promotion use may include, for example, increasing the growth rate and / or increasing the degree of growth of an individual. The target of the growth promotion may be a normal individual or an individual with a growth disorder, and preferably, a normal individual in need of growth promotion. Accordingly, one example of the present invention relates to a growth promotion composition comprising the C-type natriuretic peptide analog of the present invention. The above-described growth promotion use may include increasing the growth rate and / or increasing the degree of growth of an individual, and may include, for example, at least one selected from the group consisting of maintaining growth plates, expanding growth plates, improving growth plate shape, increasing bone length growth, and preventing and treating bone length growth disorders.

[0125] The above growth promoting composition, when administered to a subject, can promote the growth of the subject, and specifically, can increase the growth rate of the subject or increase the degree of growth of the subject. The subject is not limited by age, and can include, for example, both growing subjects (growing subjects) and subjects that have completed growth. For example, the growth promoting composition can promote the growth rate of a growing subject and increase the degree of growth, or can increase the growth of a subject that has completed growth.

[0126] The increase in growth rate or degree of the above entity may be assessed by measuring variables such as, but not limited to, height, sitting height, weight, head circumference, upper arm length, lower arm length, upper leg length, lower leg length, hand length, and / or foot length.

[0127] The increased growth rate of the subject may be an increased growth rate compared to the growth rate of the subject prior to administration of the growth promoting composition according to one embodiment of the present invention, or a baseline growth rate. The baseline growth rate may be determined from a population of subjects having the same general age, sex, and disease status as the subject. For example, the annual increase in growth rate may be in the range of 5% to 50% change from baseline in the individual, and specifically, the growth promoting composition according to one embodiment of the present invention may result in an annual increase in growth rate as measured by assessment of said variable (e.g., height, etc.) of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, or more, over baseline, and may result in an annual increase in growth rate as measured by height of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, or more, over baseline. Alternatively, it may result in an annualized increase in growth velocity as measured by sitting height, weight, head circumference, upper arm length, lower arm length, upper limb length, lower limb length (knee to toe), hand length (wrist to fingertip), or foot length of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater than baseline. The baseline may be an annualized growth velocity measured by a specific variable prior to administering the CNP according to the invention to the same individual, or may be a growth velocity measured from a population of individuals of the same general age, sex, and disease status as the individual to whom the CNP according to the invention is administered.

[0128] Another example of the present invention relates to a composition for preventing or treating growth disorders, comprising a C-type natriuretic peptide analog of the present invention, wherein an example of the growth disorder may be a bone-related disease or osteodysplasia. The composition may be a pharmaceutical composition or a food composition. The use for preventing or treating the growth disorder includes, for example, alleviating, improving, treating, or preventing the growth disorder in a subject. The subject of the prevention or treatment may be a subject with a growth disorder, and preferably, a subject with a bone-related disease or disorder.

[0129] The above bone-related diseases or bone dysplasias include achondroplasia, osteoarthritis, hypophosphatemic rickets, hypochondroplasia, short stature, dwarfism, osteochondrodysplasias, thanatophoric dysplasia, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, chondrodysplasia punctata, camptomelic dysplasia, congenital lethal hypophosphatasia, congenital Perinatal lethal type of osteogenesis imperfecta, short-rib polydactyly syndromes, hypochondroplasia, rhizomelic type of chondrodysplasia punctata, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenita, atelosteogenesis, diaspora dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia, Robinow syndrome,It may be at least one selected from the group consisting of Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, spondyloepimetaphyseal dysplasia, and Noonan syndrome.

[0130] In the present invention, "analogues" are peptide analogs, which may have one or more amino acids substituted, deleted, inverted or added compared to the wild type, may include extensions and / or truncations at the N-terminal or C-terminal positions, and may include one or more modified amino acids, such as glycosylated amino acids, pegylated amino acids, farnesylated amino acids, acetylated amino acids, acylated amino acids, biotinylated amino acids, unnatural amino acids, amino acids conjugated to a lipid moiety such as a fatty acid, or amino acids conjugated to an organic derivatization. In addition, the analogs may include derivatives or variants of the wild type peptide.

[0131] “Treatment” means preventive treatment, therapeutic treatment, or diagnostic treatment, and in certain specific embodiments, “treatment” means administering a composition to a subject for therapeutic, preventive, or diagnostic purposes.

[0132] A "prophylactic" treatment is one administered to a subject who does not exhibit signs of a disease, or who exhibits only early signs of the disease, with the aim of reducing the risk of developing the disease. A composition according to one embodiment of the present invention may be provided as a prophylactic treatment to reduce the likelihood of developing a disease, or, if it does develop, to minimize the severity of the disease.

[0133] A "therapeutic" treatment is a treatment administered to a subject exhibiting signs or symptoms of a pathological condition with the aim of reducing or eliminating the signs or symptoms. The signs or symptoms may be biochemical, cellular, histological, functional, or physical, subjective or objective. Furthermore, the composition of the present invention may be provided as a therapeutic treatment or for diagnostic purposes.

[0134] "Diagnosis" refers to the determination of the presence, extent, and / or nature of a pathological condition. Diagnostic methods vary in their specificity and selectivity. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it is sufficient if it provides clear indicators that aid in diagnosis.

[0135] Another example of the present invention relates to a pharmaceutical composition comprising a C-type natriuretic peptide analogue according to an example of the present invention.

[0136] A "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a subject animal, including humans and animals. The pharmaceutical composition comprises a therapeutically effective amount of a CNP variant, optionally another biologically active agent, and optionally a pharmaceutically acceptable excipient, carrier, or diluent. In one embodiment, the pharmaceutical composition comprises a composition comprising the active ingredient(s), and inactive ingredient(s) comprising a carrier, as well as any product resulting directly or indirectly from the combination, complexation, or aggregation of any two or more of these ingredients, or from any other type of reaction or interaction of one or more of these ingredients. Accordingly, another pharmaceutical composition according to one embodiment of the present invention comprises any composition prepared by mixing a C-type natriuretic peptide analog according to one embodiment of the present invention with a pharmaceutically acceptable excipient, carrier, or diluent.

[0137] "Pharmaceutically acceptable carrier" refers to standard pharmaceutical carriers, buffers, etc., such as phosphate buffered saline solution, 5% aqueous dextrose solution, and emulsions (e.g., oil / water or water / oil emulsions). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, glidants, sweeteners, flavoring agents, and coloring agents. The preferred pharmaceutical carrier depends on the intended route of administration of the active agent. Typical routes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection; or topical, transdermal, or transmucosal administration).

[0138] A "pharmaceutically acceptable salt" is a salt that can be formulated into a composition for pharmaceutical use, including, but not limited to, salts of metals (e.g., sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0139] "Pharmaceutically acceptable" or "pharmacologically acceptable" means a substance that is not biologically or otherwise undesirable, i.e., that the substance can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with components of a composition in which it is contained or with components present on or in the subject's body.

[0140] The term "subject" includes mammals and non-mammals. Examples of mammals include, but are not limited to, members of the class Mammalia, such as humans, mammals, primates, non-human primates, and non-human mammals such as chimpanzees, monkeys, and chimpanzee species; domestic animals such as cattle, horses, sheep, goats, and pigs; pets such as rabbits, dogs, and cats; and laboratory animals including rodents, such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, and the like.

[0141] The pharmaceutical composition may optionally further comprise other biologically active agents, optionally pharmaceutically acceptable excipients, carriers or diluents.

[0142] Another example of the present invention relates to a food composition comprising a C-type natriuretic peptide analogue according to an example of the present invention.

[0143] “Food composition” may be a functional food or a health functional food, and in particular, the above “health functional food” means a food manufactured and processed using raw materials or ingredients that have functionality useful to the human body, and “functionality” means consuming it for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological action.

[0144] The food or health functional food of the present invention can be manufactured and processed into pharmaceutical dosage forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, etc., or functional foods such as tea bags, infusions, beverages, candies, jellies, gums, etc. for the purpose of promoting growth. Another example of the present invention relates to a method for promoting growth, a method for preventing growth retardation, or a method for treating growth retardation, which comprises a step of administering a C-type natriuretic peptide analog of the present invention. The C-type natriuretic peptide analog of the present invention is as described above. The method for promoting growth, the method for preventing growth retardation, or the method for treating growth retardation of the present invention may be to administer the C-type natriuretic peptide analog of the present invention into a subject, and the method of administration may include, without limitation, any conventional method in the art. For example, the C-type natriuretic peptide analog of the present invention can be administered to a subject according to a known administration method via oral or parenteral route, and preferably can be administered parenterally such as intravenously, subcutaneously, intraperitoneally, or intraarticularly, for example, intracartilaginously. In addition, the C-type natriuretic peptide analog can be administered to a normal subject or a subject with a growth disorder, and preferably can be administered to a normal subject in need of growth promotion. The growth promotion, growth disorder, etc. are as described above.

[0145] A C-type natriuretic peptide analogue according to an example of the present invention may be administered according to standards known in the art depending on the condition of each patient. For example, the daily administration dose may be, but is not limited to, 0.01 to 500 nmol / kg. In addition, the dose may vary depending on various factors such as the age, health status, and complications of the subject to be administered, and since the C-type natriuretic peptide analogue according to an example of the present invention has a longer in vivo half-life than conventional C-type natriuretic peptides, it may be administered at a lower dose or with a lower frequency of administration than conventional C-type natriuretic peptides.

[0146] Hereinafter, examples will be described in detail to specifically explain the present specification. However, the embodiments according to the present specification may be modified in various different forms, and the scope of the present specification is not limited to the embodiments described below. The embodiments of the present specification are provided to more fully explain the present specification to those of ordinary skill in the art.

[0147]

[0148] Example 1. Preparation of a novel CNP analogue for improving stability in blood.

[0149] To improve stability in blood, novel CNP analogs represented by SEQ ID NOs: 6 to 10 were prepared by introducing a peptide sequence containing the amino acid sequence of SEQ ID NO: 1 (PPKKGPPNG) into variously modified CNP sequences. In addition, their half-lives were measured, and the results are shown in Table 2.

[0150] Classification Amino acid sequence MW (Cal. / Found) Retention time (min) Half-life (min) Control Wild type CNP (SEQ ID NO: 3) GLSKGCFGLKLDRIGSMSGLGC (CC) 2,196 / 2,1975.925 N / A SEQ ID NO: 6 GDNKPPKKGPPNGCFGLKLDRIGSMSGLGC (CC)-N / A 12.71 SEQ ID NO: 7 GDNKPPKKGPPNGCFGHKLDRIGSMSGLGC (CC) 3,066 / 3,0666.500 15.91 SEQ ID NO: 8 GDNKPPKKGPPNGCFGHKLDRIGSHSGLGC (CC) 3,071 / 3,0726.01 715.95 SEQ ID NO: 9 GDNKPPKKGPPNGCFGLKLDRIGSHSGLGC (CC)3,047 / 3,0486.66714.24 SEQ ID NO: 10PGNKPPKKGPPNGCFGLKLDRIGSHSGLGC (CC)3,029 / 3,0306.57512.57

[0151] The novel CNP analogs manufactured according to the present invention exhibited a half-life extension effect that was approximately 10 times longer than the half-life of 1.6 (minutes) of the wild-type CNP as a control.

[0152] This means that the half-life of the CNP analog manufactured in the present invention has increased and stability in blood has increased by adding the amino acid sequence of sequence number 1.

[0153]

[0154] Example 2. Pharmacokinetic evaluation of a novel CNP analogue (1): Confirmation of half-life

[0155] To evaluate the blood stability of the novel CNP analogs of SEQ ID NO: 6 to SEQ ID NO: 9 manufactured in Example 1, a pharmacokinetic evaluation was performed.

[0156] Specifically, peptides of SEQ ID NO: 6 to SEQ ID NO: 9 were administered intravenously to rats once at a concentration of 50 nmol / kg. As a control group (comparative example), wild-type CNP of SEQ ID NO: 3, which does not contain the amino acid of SEQ ID NO: 1, was used. In addition, BMN111 (SEQ ID NO: 14) was used as a positive control group.

[0157] Blood samples were collected 1, 3, 5, 10, 15, 30, and 60 minutes after administration, and serum was separated. The amount of candidate substances remaining in their intact form was measured using LC-MS / MS. The measurement results are shown in Figure 1, and the half-life calculated based on this is shown in Figure 2.

[0158] As shown in FIGS. 1 and 2, compared to the control group (comparative example), the half-life of BMN111 (SEQ ID NO: 14) was 13.7 minutes, and the half-lives of the novel CNP analogs, peptides of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, were 12.71 minutes, 15.91 minutes, 15.95 minutes, and 14.24 minutes, respectively. In particular, the peptides of SEQ ID NO: 7 and SEQ ID NO: 8, in which the sequence at position 17 was substituted with 'H (histidine),' showed a half-life that increased by about 10% or more than that of BMN111, confirming that novel CNP analogs that introduced a peptide containing the amino acid sequence of SEQ ID NO: 1 into the CNP sequence were more stable in the blood.

[0159] From these results, peptides of sequence number 7 and sequence number 8, which have improved half-lives compared to BMN111, were selected as candidate substances for subsequent experiments, and in particular, peptide of sequence number 8, which introduced 'H' (histidine) at position 25, was selected as a representative candidate substance for future experiments.

[0160]

[0161] Example 3. Derivation of a novel CNP analogue for improved stability in blood (2): Introduction of a palmitoyl group

[0162] Next, in order to further improve stability in blood, a novel CNP analogue was synthesized by introducing a palmitoyl group into the sequence of the peptide of SEQ ID NO: 8, which is a candidate substance, and a peptide having 0 to 4 amino acid substitutions or deletions at the N-terminus of the sequence, based on the results of Example 2. Afterwards, the EC, which is the half-effective concentration of the candidate substances, was calculated using GraphPad prism software. 50 The values ​​were measured. The sequence and EC of the novel CNP analogue 50 The value measurement results are shown in Table 3.

[0163] Amino acid sequenceMW (Cal. / Found)Retention time(min)EC 50 (nM) Wild-type CNP (SEQ ID NO: 3) GLSKGCFGLKLDRIGSMSGLGC (CC) 2,196 / 2,1975.92568.8 SEQ ID NO: 11 GD-K(gamma Glu-Pal)-KPPKKGPPNGCFGHKLDRIGSHSGLGC (CC) 3,454 / 3,4546.6420.3 SEQ ID NO: 12 Palmitoyl-GDNKPPKKGPPNGCFGHKLDRIGSHSGLGC (CC) 3,308 / 3,3096.4670.5 SEQ ID NO: 13 GS-K(gamma Glu-Pal)-SPPKKGPPNGCFGHKLDRIGSHSGLGC (CC) 3,383 / 3,3846.8080.1

[0164]

[0165] As shown in Table 3, EC 50 When the values ​​were calculated, the wild-type CNP showed 68 nM, but the peptides of SEQ ID NO. 11, SEQ ID NO. 12, and SEQ ID NO. 13, which are candidate substances that introduced a palmitoyl group to improve stability in blood, showed EC of 0.3 nM, 0.5 nM, and 0.1 nM, respectively. 50 The value was indicated.

[0166] This means that the novel CNP analogue according to the present invention has an EC of 100% compared to wild-type CNP. 50The results show that the material is significantly improved in value by approximately 150 to 700 times.

[0167]

[0168] Example 4. Pharmacokinetic evaluation of a novel CNP analogue (2): Intravenous administration

[0169] To evaluate the blood stability of the novel CNP analog (palmitoyl-CNP analog) manufactured in Example 3, a pharmacokinetic (PK) evaluation was performed according to intravenous administration.

[0170] Specifically, the peptides of SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 manufactured in Examples 1 and 3 were administered intravenously once to SD rats (5 rats, 8 weeks old) at a concentration of 50 nmol / kg. Blood was collected for up to 24 hours after administration, serum was separated, and the amount of candidate substances remaining in their original form was measured using LC-MS / MS, and is shown in Table 4 and Fig. 3.

[0171] Category C max T 1 / 2 AUC all(nM / L)(min)(min * nM / L) SEQ ID NO: 8240.017.12619.8 SEQ ID NO: 112232.2129.552208.8 SEQ ID NO: 121873.7179.540794.7 SEQ ID NO: 133132.4145.361988.2

[0172] As shown in Table 4 and Figure 3, the half-lives of the peptides of SEQ ID NO. 11, SEQ ID NO. 12, and SEQ ID NO. 13, which introduced a palmitoyl group, were increased by about 7.6-10 times or more compared to the peptide of SEQ ID NO. 8, which did not introduce a palmitoyl group, and the area under the curve (AUC) was confirmed to be increased by about 15-23 times or more.

[0173]

[0174] In summary of the above results, it can be seen that the C-type natriuretic peptide analogue according to the present invention, when administered intravenously, has a significantly increased half-life and excellent bioavailability compared to wild-type CNP through addition, substitution and / or deletion of amino acids to wild-type CNP and selective introduction of fatty acids.

[0175]

[0176] Example 5. Pharmacokinetic evaluation of novel CNP analogues (3): Subcutaneous administration

[0177] Next, in order to confirm whether the novel CNP analogue, whose blood stability was confirmed in Example 4, is a drug that can be administered subcutaneously, a pharmacokinetic evaluation according to subcutaneous administration was performed.

[0178] Specifically, the peptides of SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 manufactured in Examples 1 and 4 were administered subcutaneously once to SD rats (5 rats, 8 weeks old) at a concentration of 50 nmol / kg. Blood was collected for up to 48 hours after administration, serum was separated, and the amount of candidate substances remaining in the intact form was measured using LC-MS / MS, and the results are shown in Table 5 and Fig. 4.

[0179] PK parameter C max (pmol / ml) T max (min)AUC last (pmol*min / ml)T 1 / 2(min)BA(%)Wild type CNP(SEQ ID NO: 3)8.7(1.0)5.0(0.0)-3.2BMN111(SEQ ID NO: 14)95.6(41.0)5.0(0.0)6004.1(1345.0)38.3(25.3)14.0SEQ ID NO: 8171.2(61.2)10.0(7.1)11210.5(4344.7)47.7(27.2)25.4SEQ ID NO: 11657.2(40.9)480.0(169.7)527103.4(62238.6)246.9110.3SEQ ID NO 12607.4(4..3)360.0(169.7)456278.5(31788.2)202.5(14.4)127.8 Sequence number 13727.0(137.9)300.0(84.9)564614.3(41880.6)200.3(24.1)92.5

[0180] As shown in Table 5 and Figure 4, the pharmacokinetic evaluation results of the control group, wild-type CNP and BMN111, were similar to the results of a previous paper, confirming the reliability of this experiment.

[0181] In the case of the peptide with sequence number 8 without the palmitoyl group, the half-life was 47.7 minutes, which was about 1.2 times greater than the half-life of 38.3 minutes of the positive control, BMN111, and accordingly, the area under the curve increased by about 1.8 times.

[0182] In addition, it was confirmed that the peptides of sequence number 11, sequence number 12, and sequence number 13, which introduced palmitoyl groups, had a half-life that increased by about 5-6 times or more compared to the positive control group BMN111, and the area under the curve increased significantly by about 75-94 times or more.

[0183] These results show that the C-type natriuretic peptide analog of the present invention exhibits a significantly increased half-life when administered subcutaneously, similar to intravenous administration, not only compared to wild-type CNP but also compared to the positive control, BMN111. This demonstrates that the CNP analog of the present invention can be administered to a subject in various forms, including intravenous and subcutaneous administration.

[0184]

[0185] Example 6. Bioavailability (BA) analysis of novel CNP analogs

[0186] To confirm the bioavailability of the novel CNP analogs manufactured in Examples 1 and 3, the bioavailability was analyzed according to intravenous (IV) and subcutaneous (SC) administration at a dosage of 70.0 nml / kg with reference to Examples 4 and 5. The results are shown in Table 6 and Fig. 5.

[0187] PK parameters, dosage, and administration method C max T max AUC last T 1 / 2 BA abs PK parameters nmol / kg pmol / ml min pmol* min / ml min % Wild-type CNP (SEQ ID NO: 3) 70.0 IV 9 5.06 ± 24.4 0 1.00 ± 0.00 2 9 0.47 ± 80.01 1.59 ± 0.78 - BMN111 (SEQ ID NO: 14) 70.0 IV 4 10.53 ± 84.9 9 1.00 ± 0.00 4 76.37 ± 255.58 17.89 ± 1.45 - SEQ ID NO: 1370.0 IV 4 8 0 9.40 ± 841.16 1.00 ± 0.00 0 8 19 4.93 ± 59 36.89 109.24 ± 3.94 - Wild-type CNP (SEQ ID NO: 3) 70.0 SC 2.28 ± 0.523.00 ± 0.0014.49 ± 3.96-4.99 ± 1.36BMN111(SEQ ID NO: 14)70.0SC10.31 ± 2.838.60 ± 5.90341.78 ± 253.1138.10 ± 24.689.83 ± 7.28SEQ ID NO: 1370.0SC110.58 ± 28.66192.00 ± 65.7352803.82 ± 10118.05224.30 ± 26.6248.80 ± 9.35

[0188] As shown in Table 6 and Figure 5, when administered intravenously once at 70 nmol / kg, the in vivo half-life of the CNP analogue of sequence number 13 was measured to be approximately 109.2 minutes, which is a significant increase compared to the half-life of the wild-type CNP of sequence number 3, which is approximately 1.6 minutes, and the half-life of BMN111, which is approximately 18 minutes.

[0189] In addition, with regard to bioavailability, when administered subcutaneously once at 70 nmol / kg, the bioavailability of the positive control group, BMN111, was approximately 10%, but the bioavailability of the CNP analogue of sequence number 13 was approximately 48%. This means that the novel CNP analogue of sequence number 13 according to the present invention exhibits excellent physiological activity effects in vivo, with bioavailability increased by approximately 4 times or more compared to BMN111.

[0190]

[0191] Example 7. Evaluation of platelet aggregation inhibition activity of novel CNP analogs.

[0192] To determine whether novel CNP analogs have any effect on platelet aggregation, SD rat platelets were first isolated and then treated with agonists such as collagen and thrombin to induce platelet aggregation. Next, CNP analogs of SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 were treated, and the platelet aggregation was observed using a light transmission aggregation meter. The results are shown in Fig. 6.

[0193] As shown in Fig. 6, platelet aggregation was confirmed at a concentration of 10 μM collagen and thrombin, which is 20 times higher than the concentration used for CNP analogs according to an example of the present invention, and as a result, the CNP analogs according to an example of the present invention did not significantly cause platelet aggregation. This means that the CNP analogs according to the present invention are substances that do not have side effects on platelet aggregation.

[0194]

[0195] Example 8. Evaluation of Cardiovascular Safety (Hemodynamics) of Novel CNP Analogues

[0196] To evaluate the cardiovascular safety of CNP analogs according to an example of the present invention, blood pressure and heart rate were checked.

[0197] Specifically, a peptide having the sequence number 13, a CNP analog according to an example of the present invention, was administered subcutaneously to about 5 rats (n=5) at a concentration of 200 nmol / kg. Thereafter, blood samples were collected from 0 to 120 minutes, and changes in blood pressure (MAP change) and heart rate were measured for each individual. The changes in blood pressure are shown in Fig. 7a, and the changes in heart rate are shown in Fig. 7b.

[0198] According to FIGS. 7a and 7b, the CNP analogue of sequence number 13 showed smaller and more consistent changes in blood pressure and heart rate per individual than the positive control group, BMN111, confirming that it is a substance with cardiovascular safety.

[0199]

[0200] Example 9. Evaluation of neutral endopeptidase (NEP) resistance of novel CNP analogs

[0201] To evaluate the stability of CNP analogs according to an example of the present invention, resistance to in vitro cleavage by neutral endopeptidase (NEP) was confirmed.

[0202] CNP (SEQ ID NO: 3), BMN111 (SEQ ID NO: 14), and CNP analogs of SEQ ID NOs: 8, 11, 12, and 13 were prepared at a concentration of 4 μM using PBS containing 1% BSA, and mixed with 400 ng / ml of Neprilysin, a neutral endopeptidase, at a ratio of 1:1 (v / v). The mixtures were placed in a 37°C water bath for 0, 40, 80, 120, 160, and 200 min, respectively, and 10 mM EDTA, DTT, and EDTA were added to each sample and the reaction was stopped by placing them on ice. 1 / 10 (v / v) of 0.1% FA, 25% ACN (acetonitrile) (organic solvent for LC-MS / MS analysis) was added to each sample, and then analyzed by LC-MS / MS.

[0203] As a result, as shown in Table 7 and Figure 8 below, the control group, CNP, was completely decomposed after 160 minutes, and in the case of BMN111, it was confirmed that about 88% remained. However, it was confirmed that the CNP analogs of SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 according to an example of the present invention had higher NEP resistance than the control groups.

[0204] ClassificationMolecular weightNEP resistancekDa% IntactWild type CNP (SEQ ID NO: 3)2.20.0BMN111 (SEQ ID NO: 14)4.187.5±2.6SEQ ID NO: 83.096.5±2.4SEQ ID NO: 113.4103.5±2.9SEQ ID NO: 123.3100.7±0.7SEQ ID NO: 133.3116.0±1.1

[0205] Example 10. Evaluation of growth plate maintenance in normal mice (1)

[0206] Among the CNP analogs manufactured in Example 3, the CNP analog of sequence number 13 with an excellent half-life was used to evaluate the growth plate maintenance effect in normal mice.

[0207] Specifically, as shown in Table 8, wild-type CNP and a CNP analogue of SEQ ID NO: 13 were each dissolved in an excipient (5% mannitol in saline), and then vehicle, CNP (50 nmol / kg), and the peptide of SEQ ID NO: 13 (10, 25, 50 nmol / kg) were each subcutaneously injected into 3-week-old female ICR mice daily for 2 weeks. The body weight, body length, and tail length of the mice according to drug administration were measured, and are shown in Table 9 and Figures 9a to 9c.

[0208] Vehicle controlCon(-)Case 1Case 2Case 3Drug5% MannitolWild type CNP (SEQ ID NO. 3)SEQ ID NO. 13Dose (mg / kg)50 nmol / kg10 nmol / kg25 nmol / kg50 nmol / kgInjection volume5ml / kg5ml / kg5ml / kg5ml / kg5ml / kgAdministration routeSC,QDSC,QDSC,QDSC,QDNumber of animals (N=)86688

[0209] Dose (nmol / kg / day) Changes in body weight and height (length) Body weight (g) Height (mm) Tail length (mm) Vehicle 0 27.14 ± 2.46 86.88 ± 2.99 4.8 ± 3.28 CNP (SEQ ID NO: 3) 50 26.8 ± 1.95 86.5 ± 1.97 93.3 ± 1.86 SEQ ID NO: 13 50 28.06 ± 2.28 89.75 ± 2.25 104.5 ± 4.56 25 28.11 ± 2.56 88.4 ± 3.2 49 8.6 ± 3.66 10 28.95 ± 0.78 89.8 ± 3.87 96.8 ± 3.25

[0210] As shown in Table 9 and Figures 9a to 9c, when a CNP analog according to an example of the present invention was administered to mice, body weight, body length, and tail length showed concentration-dependent growth. In particular, the body length (body length (mm)) of normal mice increased by about 2.9 mm compared to the vehicle and about 3.3 mm compared to the wild-type CNP, and the tail length (tail length (mm)) increased by about 9.7 mm compared to the vehicle and about 11.2 mm compared to the wild-type CNP. That is, it can be seen that when the CNP analog of the present invention was administered in the same amount or a smaller amount compared to the wild-type CNP, the bone growth rate of the mice was significantly increased.

[0211]

[0212] Example 11. Evaluation of mouse growth plate maintenance by cross-administration (2)

[0213] Among the CNP analogs manufactured in Example 3, the peptide of sequence number 13 with an excellent half-life was used to evaluate growth plate maintenance in normal mice and growth plate maintenance by cross-administration.

[0214] Specifically, wild-type CNP (SEQ ID NO: 3) and the peptide of SEQ ID NO: 13 were dissolved in a vehicle (5% mannitol in saline), and then vehicle, CNP (50 nmol / kg), and the peptide of SEQ ID NO: 13 (50 nmol / kg) were injected subcutaneously daily for 2 weeks into 3-week-old female ICR mice. After washing out for 2 weeks, the CNP (SEQ ID NO: 3) (50 nmol / kg) group and the peptide administration group of SEQ ID NO: 13 were cross-administered for 2 weeks, and the body weight, trunk length, and tail length of the mice were measured, and the measurement results are shown in Table 10 and Figures 10a to 10c.

[0215] 1st treatment (Treatment) Washout 2nd treatment (Change medicine) Group 0~14 day 14~28 day Group 28~49 day Vehicle control 0.9±0.22 mm / day 0.4±0.14 mm / day Vehicle control 0.2±0.09 mm / day CNP 50 nmol / kg 0.8±0.26 mm / day 0.4±0.12 mm / day SEQ ID NO: 13 50 nmol / kg 0.4±0.16 *** mm / day sequence number 1350 nmol / kg 1.7±0.24 *** mm / day0.3±0.12 mm / dayCNP 50 nmol / kg0.1±0.08 mm / day

[0216] - ***: p < 0.001

[0217]

[0218] As shown in Table 10 and Figures 10a to 10c, in the case of the peptide of SEQ ID NO: 13, which is a CNP analog according to an example of the present invention, statistically significant differences in mouse trunk length and tail length growth were observed in the period of 0 to 14 days compared to the vehicle and wild-type CNP. In particular, while the average daily growth was 1.7 mm from the day after treatment with the peptide of SEQ ID NO: 13 to day 14, the wild-type CNP (SEQ ID NO: 3), which is the control group, showed only about 0.8 mm growth. In addition, even after cross-administration, the peptide of SEQ ID NO: 13 showed an average daily growth of 0.4 mm, and the control group, CNP, showed about 0.1 to 0.2 mm growth. It was confirmed that the CNP analog of the present invention showed a high bone growth rate even when cross-administered. This shows that the CNP analog of the present invention can be effectively used to treat bone-related diseases including achondroplasia and osteoarthritis or skeletal dysplasia.

[0219]

[0220] Example 12. Evaluation of mouse growth plate maintenance compared to the positive control group (ASB 20123) (3)

[0221] Among the CNP analogs manufactured in Example 3, the peptide with sequence number 13 having an excellent half-life was used to evaluate the growth plate maintenance and growth promotion effects in normal mice compared to the positive control group ASB 20123.

[0222] Specifically, ASB 20123 and the peptide of SEQ ID NO: 13 were each dissolved in a vehicle (5% mannitol in saline), and then vehicle, ASB20123 (50 nmol / kg), and the peptide of SEQ ID NO: 13 (5, 10, 15, 20 nmol / kg) were injected subcutaneously daily for 8 weeks into 3-week-old female ICR mice. Body weights were measured twice a week, and the lengths of the mouse trunk and tail were measured once a week, and the measurement results are shown in Table 11, Table 12, and Figures 11a to 11d. In Tables 11 and 12, each value represents the mean ± SD (average ± standard deviation) of 7 to 8 mice, and *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively, when compared to the vehicle control using Dunnett's test.

[0223] Group Height (mm) 7day 14day 21day 28day 35day 42day 49day 57day Negative control (Vehicle control) 76.4±1.85 83.3±3.0 684.8±3.0 588.1±2.9 789.3±3.3 392.9±3.1 892.9±3.1 894.5±3.2 1 Positive control ASB20123 (50 nmol / kg) 77.4±1.1 383.9±3.3 484.7±3.8 289.0±2.89 89.4±2.7 691.1±3.89 3.1±3.89 95.0±4.83 SEQ ID NO: 13 (5 nmol / kg)76.3±1.3883.7±1.1187.0±3.8388.6±4.0092.3±3.2591.7±3.6495.0±2.6595.3±1.80SEQ ID NO: 13 (10 nmol / kg)79.4±2.3385.0±1.9389.4±3.1691.4±2.9792.4±3.5095.3±3.41 * 96.3±4.0397.0±3.46Sequence number 13 (15nmol / kg)79.5±2.00 * 86.4±3.96 * 92.1±4.49 ** 95.8±3.73 *** 95.9±5.00 *** 97.1±4.55 ** 99.5±5.01**101.8±6.27 ** Sequence number 13 (20 nmol / kg) 82.1±3.83 *** 88.4±2.50 *** 94.1±3.83 *** 97.9±3.14 *** 100.8±2.43103.1±4.49 *** 104.3±4.89 *** 104.6±5.07 ***

[0224] Group Tail length (mm) 7day 14day 21day 28day 35day 42day 49day 57day Negative control (Vehicle control) 83.6±2.50 88.0±2.149 3.8±1.83 96.4±2.26 98.1±1.73 99.0±2.00 99.8±2.31 100.0±2.07 Positive control ASB20123 (50 nmol / kg) 84.0±1.63 91.4±2.15 98.1±3.44 102.0±3.79 * 104.4±3.99 * 107.4±3.69 ** 108.4±4.35 ** 109.1±4.81 *** SEQ ID NO: 13 (5 nmol / kg) 84.1±1.34 89.9±1.34 95.1±1.07 97.4±0.98 99.3±1.50 101.1±1.07 102.1±1.68 1002.7±1.60 SEQ ID NO: 13 (10 nmol / kg) 83.9±3.56 92.9±2.64 ** 98.3±3.54102.0±4.50 * 103.5±4.17 * 105.0±4.99 * 106.9±4.82 * 107.9±4.64 ** Sequence number 13 (15 nmol / kg) 85.4 ± 2.13 94.5 ± 3.50 *** 103.4±3.85 *** 108.9±3.18 *** 112.8±4.27 *** 114.6±2.45 *** 121.0±4.11 *** 123.5±4.31 *** Sequence number 13 (20 nmol / kg) 88.1 ± 2.95 ** 101.5±3.70 *** 113.3±5.12 *** 120.9±4.58 *** 126.0±5.83 *** 129.9±6.77 *** 135.4±7.07 *** 139.5±6.10 ***

[0225] As shown in Table 11, Table 12, and Figures 11a to 11d, in the case of the peptide of SEQ ID NO: 13, which is a CNP analog according to one embodiment of the present invention, as the administration period increased, the growth of mouse body weight, body length, and tail length increased compared to the negative control group (Vehicle) and the positive control group (ASB 20123), and it was confirmed that this showed a difference in growth rate in a concentration-dependent manner.

[0226] In particular, in the case of normal mice treated with the peptide of sequence number 13 at a concentration of 20 nmol / kg for 8 weeks (57 days), the body length (body length (mm)) increased by approximately 10.1 mm compared to the vehicle and approximately 9.6 mm compared to the positive control group ASB 20123, and the tail length (tail length (mm)) increased by approximately 39.5 mm compared to the vehicle and approximately 30.4 mm compared to the positive control group ASB 20123.

[0227] From these results, it was found that the novel CNP analog according to an example of the present invention has remarkably excellent effects in maintaining growth plates and promoting bone growth.

[0228]

[0229] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. C-type natriuretic peptide analogues having the structure of the following general formula 1: [General formula 1] (P1)-PPKKGPPNG-(P2) In the above general formula 1, P1 is a peptide consisting of a sequence of 0 to 4 amino acids. P2 is a peptide consisting of an amino acid sequence of SEQ ID NO: 2 or 3, or a peptide comprising one or more amino acid substitution mutations in the amino acid sequence of SEQ ID NO: 2 or 3.

2. In paragraph 1, The above P1 is a C-type natriuretic peptide analogue represented by the following amino acid sequence: P1 is (X1)-(X2)-(X3)-(X4), X1 is Gly (G), Pro (P), Thr (T), Ala (A), Ser (S), or absent, X2 is Asp(D), Gly(G), Lys(K), Ser(S), Asn(N), Glu(E), Gln(Q), or absent, X3 is Asn(N), Lys(K), Asp(D), Ser(S), Gln(Q), or absent, X4 is Lys (K), Thr (T), Ser (S), Arg (R), Glu (E), Gln (Q), His (H), or absent.

3. In paragraph 1, The above P2 is a C-type natriuretic peptide analogue represented by the following general formula 2 (SEQ ID NO: 5): [General formula 2] CFG-(X5)-K-(X6)-DRIGS-(X7)-SGLGC In the above general formula 2, X5 and X7 are each selected from amino acids containing cationic or hydrophobic side chains, X6 is selected from amino acids containing a cationic or hydrophobic side chain excluding Ile (I).

4. In paragraph 3, In the above general formula 2, X5 is Leu(L) or His(H), X6 is Leu(L), and X7 is a C-type natriuretic peptide analogue, either Met(M) or His(H).

5. In paragraph 1, A C-type natriuretic peptide analogue, wherein a fatty acid or a derivative thereof is introduced as one or more repeating units into at least one amino acid among the amino acid sequences of the above P1.

6. In paragraph 5, A C-type natriuretic peptide analogue, wherein the fatty acid has 3 to 21 carbon atoms and is introduced directly or through a linker into the 1st to 4th amino acid from the N-terminus of the C-type natriuretic peptide analogue.

7. In paragraph 6, A C-type natriuretic peptide analogue, wherein the fatty acid is selected from 8-amino-3,6-dioxaoctanoic acid, miniPEG, CH3(CH2)14COOH (palmitic acid), caprylic acid, capric acid, lauric acid, or stearic acid.

8. In paragraph 1, A C-type natriuretic peptide analogue comprising at least one amino acid sequence selected from the group consisting of sequence numbers 6 to 13.

9. In any one of paragraphs 1 to 8, The C-type natriuretic peptide analogue is characterized in that at least one selected from the group consisting of in vivo half-life, bioavailability, and growth plate maintenance is improved compared to the wild-type C-type natriuretic peptide.

10. A pharmaceutical composition for promoting growth, comprising a C-type natriuretic peptide analogue according to any one of claims 1 to 8.

11. A food composition for promoting growth, comprising a C-type natriuretic peptide analogue according to any one of claims 1 to 8.

12. A pharmaceutical composition for preventing or treating growth disorders, comprising a C-type natriuretic peptide analogue according to any one of claims 1 to 8.

13. In paragraph 12, A pharmaceutical composition for preventing or treating growth disorders, wherein the growth disorders include at least one selected from achondroplasia, dwarfism, bone-related diseases or bone dysplasia.

14. A method for promoting growth in a non-human organism, comprising administering to the non-human organism a C-type natriuretic peptide analogue according to any one of claims 1 to 8.

Images