CN118382462A

CN118382462A - Effective dose of CNP conjugate

Info

Publication number: CN118382462A
Application number: CN202280082042.7A
Authority: CN
Inventors: K·斯普罗格; M·J·凯杰拉德-汉森
Original assignee: Ascendis Pharma Growth Disorders AS
Current assignee: Ascendis Pharma Endocrinology Division AS
Priority date: 2021-12-13
Filing date: 2022-12-12
Publication date: 2024-07-23

Abstract

本发明涉及包含CNP缀合物或其药学上可接受的盐的单位剂型。The present invention relates to a unit dosage form comprising a CNP conjugate or a pharmaceutically acceptable salt thereof.

Description

Effective dose of CNP conjugate

The present invention relates to a unit dosage form comprising a CNP conjugate or a pharmaceutically acceptable salt thereof.

Achondroplasia (ACH) is a genetic disease that occurs due to an autosomal dominant mutation of the fibroblast growth factor receptor 3 (FGFR 3) gene, resulting in abnormal cartilage formation and dwarfism. Type C Natriuretic Peptide (CNP) is a hormone that binds to and activates peptide receptor B (NPR-B), thereby inhibiting FGFR3 downstream signaling. This in turn triggers endochondral growth and skeletal overgrowth, as observed in mice and humans over-expressing CNP. Overproduction of CNP in cartilage or sustained delivery of CNP by intravenous (iv) infusion normalizes dwarfism in hypoplastic mice, suggesting that administration of CNP at supraphysiological levels is a strategy for treating ACH. Human preproCNP consisting of 126 amino acids is further cleaved by furin to yield CNP-53.CNP-53 is biologically active, but is usually processed by an unknown mechanism into a 22 amino acid form that is biologically active in the circulation, CNP-22. The biological activity of CNP is tightly regulated and its clearance from plasma is very rapid. CNP administration to patients and animals with ACH has been shown to stimulate endochondral bone growth and improve the poor skeletal performance of the disease (Lorget et al.,Am.J.Hum.Genet.91,1108-1114,2012;Wendt et al.,J.Pharmacol.Exp.Ther.353,132-149,2015), recently, vosolin (vosoritide) (VOXZOGO ^TM) was approved for the treatment of achondroplasia.

The biology of CNP has been known for many years, but the significant drawbacks of natural CNP have limited its use as a pharmaceutical product. Natural CNP has a half-life in humans of only 2-3min and renal clearance and Neutral Endopeptidase (NEP) degradation make it difficult to maintain effective therapeutic drug exposure. The Fu Suoli peptide is a daily CNP analog with enhanced NEP resistance and a relevant half-life after subcutaneous administration of about 21.0 (+ -4.7) to 27.9 (+ -9.9) min (VOXZOGO ^TM prescription information, ref. ID:4891169, 2021, 11 months). Fu Suoli peptide was rapidly absorbed after administration and reached a peak concentration (C _max) of about 4.71 (+ -2.32) to 7.18 (+ -9.65) ng/ml (VOXZOGO ^TM prescription information, reference ID:4891169, 2021, month 11) about 15min after administration at a subcutaneous dose of 15 μg/kg. Cyclic guanosine monophosphate (cGMP) is a marker for NPR-B receptor binding, which is a pharmacological receptor for CNP. cGMP can be measured in blood and urine and is a biomarker for systemic CNP pharmacological activity. Following administration of Fu Suoli peptide to pediatric patients with achondroplasia, an increase in urinary cGMP concentration from the pre-dose baseline was observed within the first 4 hours post-dose and reached the highest level at 2 hours post-dose. Analysis of exposure response showed that Fu Suoli peptide activity, measured by urine cGMP, was near saturation at a daily dose of 15 μg/kg. (VOXZOGO ^TM prescription information, reference ID:4891169, month 11 of 2021). Furthermore, a temporary drop in blood pressure was also observed in the clinical study of Fu Suoli peptides. Thus, subjects suffering from severe heart or vascular disease and patients taking antihypertensive drugs are excluded from participation in clinical trials of the vosolidine peptide. Due to the risk of hypotension, caregivers and patients are informed that post-Fu Suoli peptide administration may lower blood pressure. In addition, prior to administration of the vosolidine, the patient should consume enough food and within one hour prior to administration, the patient should drink about 8-10 ounces (240-300 ml) of liquid (VOXZOGO ^TM prescription information, reference ID:4891169, 2021, 11 months).

In the clinical trial, a drop in blood pressure was observed in 8 (13%) of 60 subjects treated, with a total of 11 transient drop events in blood pressure, whereas in 61 subjects treated with placebo, a drop in blood pressure occurred in 3 (5%). 2 (3%) of the 60 subjects treated with the vosolidine had a symptomatic seizure with a decrease in blood pressure, with vomiting and/or dizziness, whereas of the 61 subjects treated with the placebo, this ratio was 0 (0%). Daily administration of Fu Suoli peptide also resulted in an increase in serum type X collagen marker (CXM), a endochondral ossification biomarker, from baseline levels and remained elevated after 24 months. This is consistent with the fact that: the difference in annual growth rate (AGV) from baseline change treatment was 1.57 cm/year after 52 weeks of treatment with the vosolidine compared to placebo (VOXZOGO ^TM prescription information, reference ID:4891169, 2021, 11). The hypotensive effect of CNP agonists is mediated through NPR-B receptors and is associated with peptide C _max. cGMP is a good biomarker for CNP target binding, and therefore it can also serve as a biomarker for risk of hypotension. In phase 2 studies of vosolidine, a transient dose-dependent increase in urinary cGMP concentration (a biomarker of systemic vosolidine pharmacological activity) was observed from pre-dose levels to at least 2 hours after dosing. A dose-dependent increase in median serum CXM concentration compared to pre-treatment levels was also observed and remained elevated until day 183. The median serum CXM concentrations were similar in all groups (cohort) and remained elevated from month 12 to month 24. This finding suggests that exposure to 15 μg/kg of vosolidine results in a sustained and maximal response of serum CXM concentration, and that this is consistent with the fact that the 15 μg/kg dose provides near maximal therapeutic effect in terms of annual growth rate of such daily CNP analogs (SAVARIRAYAN ET al, n.engl.j. Med.381,25-35,2019). Thus, higher doses are less likely to result in additional growth, but may be associated with higher systemic futuristic activity and therefore higher risk of hypotension.

It is therefore important to identify doses of sustained release CNP conjugates, which improve the pharmacological window and enable effective growth promotion based on the absence of hypotensive episodes, and with greatly reduced likelihood of hypotension.

It is therefore an object of the present invention to at least partially overcome the above-mentioned disadvantages.

This object is achieved with a unit dosage form comprising a therapeutically effective amount of a CNP conjugate, or a pharmaceutically acceptable salt thereof, wherein the CNP moiety is reversibly conjugated to the polymer moiety.

Within the meaning of the invention, the terms are used as follows.

As used herein, the term "about" is used in combination with a numerical value to denote a range, including the numerical value plus and minus a numerical value not exceeding 10% of the numerical value and therebetween; in certain embodiments, no more than 8% of this value; in certain embodiments, no more than 5% of this value; and in certain embodiments, no more than 2% of this value. For example, the phrase "about 200" is used to mean and include the values ranging from 200+/-10%, i.e., ranging from 180-220 and including 180 and 220; in certain embodiments, 200+/-8%, i.e., ranging from 184-216 and including 184 and 216; in certain embodiments, 200+/-5% and including the values, i.e., in the range of 190-210 and including 190 and 210; and in certain embodiments 200+/-2%, i.e., in the range of 196-204 and including 196 and 204. It is understood that a percentage of "about 20%" does not mean "20% +/-10%", i.e., ranges from 10% to 30% inclusive and including 10% and 30%; but "about 20%" means a range of 18% to 22% and includes 18% and 22%, i.e., a value of 20 plus or minus 10%.

As used herein, the term "antimicrobial agent" refers to a chemical, such as a chemical that kills or inhibits the growth of microorganisms (e.g., bacteria, fungi, yeasts, protozoa, mold) and/or destroys viruses.

As used herein, the term "buffer" or "buffer" refers to a compound that maintains pH within a desired range. Physiologically tolerable buffers are, for example, sodium phosphate, succinate, histidine, bicarbonate, citrate, acetate, sulfate, nitrate, chloride and pyruvate. Antacids such as Mg (OH) ₂ or ZnCO ₃ may also be used.

As used herein, the term "CNP" refers to all CNP polypeptides, in certain embodiments, from mammalian species, such as from human and mammalian species, particularly from human and murine species, as well as variants, analogs, orthologs, homologs and derivatives and fragments thereof, characterized by: regulate the growth, proliferation and differentiation of chondrocytes of the cartilage growth plate. The term "CNP" also includes all CNP variants, analogs, orthologs, homologs, derivatives and fragments. CNP variants, analogs, orthologs, homologs, derivatives and fragments disclosed in WO2009/067639A2 and WO2010/135541A2 are incorporated herein by reference.

As used herein, the term "CNP polypeptide variant" refers to a polypeptide from the same species but different from the reference CNP polypeptide. In general, differences are limited, so the amino acid sequences of the reference and variant are generally very similar and identical in many regions. In certain embodiments, the CNP polypeptide variant is at least 70%, 80%, 90% or 95% identical to the reference CNP polypeptide. By polypeptide having an amino acid sequence that is at least, for example, 95% "identical" to the query amino acid sequence, it is meant that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may contain up to 5 amino acid changes per 100 amino acids as compared to the query amino acid sequence. These changes in the reference sequence may occur at the amino-terminal (N-terminal) or carboxy-terminal (C-terminal) positions of the reference amino acid sequence, or anywhere between these terminal positions, interspersed either alone between residues in the reference sequence, or interspersed within the reference sequence in one or more contiguous groups. The query sequence may be the entire amino acid sequence of the reference sequence or any fragment specified as described herein. Such CNP polypeptide variants may be naturally occurring variants, such as naturally occurring allelic variants encoded by one of several alternatives to CNP occupying a given locus on a chromosome or organism, or isomers encoded by naturally occurring splice variants derived from a single primary transcript. Alternatively, a variant of a CNP polypeptide may be an unknown naturally occurring variant and may be made by mutagenesis techniques known in the art. It is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus of a biologically active peptide or protein without substantial loss of biological function. Such N-terminal and/or C-terminal deletions are also encompassed by the term CNP polypeptide variants.

One of ordinary skill in the art also recognizes that some amino acid sequences of CNP polypeptides may be altered without significantly affecting the structure or function of the peptide. Such mutants include deletions, insertions, inversions, repeats and substitutions selected according to general rules known in the art, thus having little effect on activity. For example, bowie et al (Science 247:1306-1310,1990), which is incorporated herein by reference in its entirety, provide guidance on how to perform phenotypically silent amino acid substitutions, in which the authors state that there are mainly two ways to study the tolerance of amino acid sequences to changes.

As used herein, the term "CNP analog" refers to a CNP of a different and unrelated organism that performs the same function in each organism, but that is not derived from an ancestral structure common to the ancestors of the organism. In contrast, similar CNPs appear alone, later evolving and performing the same or similar functions. In other words, a similar CNP polypeptide is a polypeptide that differs completely in amino acid sequence, but has the same biological activity, i.e., regulates the growth, proliferation and differentiation of chondrocytes of a cartilage growth plate.

As used herein, the term "CNP ortholog" refers to CNPs within two different species whose sequences are related to each other via a common homologous CNP in the ancestral species, but have evolved to be different from each other.

As used herein, the term "CNP homolog" refers to CNP of a different organism that performs the same function in each organism and that is derived from an ancestral structure that is common to the ancestors of that organism. In other words, a homologous CNP polypeptide is a polypeptide having a very similar amino acid sequence and the same biological activity, i.e. modulating the growth, proliferation and differentiation of chondrocytes of a cartilage growth plate. In certain embodiments, a CNP polypeptide homolog may be defined as a polypeptide that exhibits at least 40%, 50%, 60%, 70%, 80%, 90% or 95% identity to a reference CNP polypeptide.

Thus, a CNP polypeptide may be, for example: (i) A polypeptide in which at least one amino acid residue is substituted with a conserved or non-conserved amino acid residue (in certain embodiments a conserved amino acid residue), and such a substituted amino acid residue may or may not be an amino acid residue encoded by the genetic code; and/or (ii) a polypeptide wherein at least one amino acid residue comprises a substituent; and/or (iii) a polypeptide in which a CNP polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol); and/or (iv) polypeptides in which additional amino acids are fused to a CNP polypeptide, such as an IgG Fc fusion region peptide, or a leader or secretory sequence, or a sequence for purifying the above forms of the polypeptide, or a preprotein sequence.

As used herein, the term "CNP polypeptide fragment" refers to any peptide comprising a contiguous span of a portion of the amino acid sequence of a CNP polypeptide.

More specifically, a CNP polypeptide fragment comprises at least 6, such as at least 8, at least 10, or at least 17 consecutive amino acids of a CNP polypeptide. A CNP polypeptide fragment may also be described as a subgenera of a CNP polypeptide comprising at least 6 amino acids, where "at least 6" is defined as any integer from 6 to an integer representing the C-terminal amino acid of the CNP polypeptide. Further included are classes of CNP polypeptide fragments of at least 6 amino acids in length, which are further specified in terms of their N-terminal and C-terminal positions, as described above. In addition, as an individual species, the term "CNP polypeptide fragment" encompasses all CNP polypeptide fragments as described above, which are at least 6 amino acids in length, as described above, and can be specified by the positions of the N-and C-termini. That is, the length of each combination of the fragment at the N-terminal and C-terminal positions can occupy at least 6 consecutive amino acid residues on the amino acid sequence of any given CNP polypeptide.

Since the term CNP includes variants, analogs, orthologs, homologs, derivatives and fragments of the CNP described above, all references to a particular position within a reference sequence also include equivalent positions in variants, analogs, orthologs, homologs, derivatives and fragments of the CNP moiety, even if not explicitly mentioned.

Naturally occurring CNP-22 (SEQ ID NO: 1) has the following sequence:

GLSKGCFGLKLDRIGSMSGLGC，

Wherein the cysteines at positions 6 and 22 are linked by disulfide bonds.

In certain embodiments, the term "CNP" also refers to the following peptide sequences:

SEQ ID NO:2(CNP-53):

DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRI GSMSGLGC；

SEQ ID NO:3(G-CNP-53):

GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDR IGSMSGLGC；

SEQ ID NO:4(M-CNP-53):

MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDR IGSMSGLGC；

SEQ ID NO:5(P-CNP-53):

PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:6(CNP-53 M48N):

DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC；

SEQ ID NO:7(CNP-53Δ15-31):

DLRVDTKSRAAWARGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:8(CNP-52):

LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:9(CNP-51):

RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:10(CNP-50):

VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:11(CNP-49):

DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:12(CNP-48):

TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:13(CNP-47):

KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:14(CNP-46):

SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:15(CNP-45):

RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；SEQ ID NO:16(CNP-44):

AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；SEQ ID NO:17(CNP-44Δ14-22):

AAWARLLQEHPNAGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:18(CNP-44Δ15-22):

AAWARLLQEHPNARGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:19(CNP-43):

AWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:20(CNP-42):

WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:21(CNP-41):

ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:22(CNP-40):

RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:23(CNP-39):

LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:24(CNP-38):

LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC,

Wherein the cysteines at positions 22 and 38 are linked by disulfide bonds;

SEQ ID NO:25(CNP-37):

QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO. 26 (CNP-37Q 1pQ, where pQ=pyroglutamate):

pQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:27(G-CNP-37):

GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:28(P-CNP-37):

PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:29(M-CNP-37):

MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:30(PG-CNP-37):

PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:31(MG-CNP-37):

MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:32(CNP-37M32N):

QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC；

SEQ ID NO:33(G-CNP-37M32N):

GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC；

SEQ ID NO:34(G-CNP-37K14Q):

GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:35(G-CNP-37K14P):

GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:36(G-CNP-37K14Q,Δ15):

GQEHPNARKYKGANQGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:37(G-CNP-37 K14Q,K15Q):

GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:38(CNP-36):

EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:39(CNP-35):

HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:40(CNP-34):

PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:41(CNP-33):

NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:42(CNP-32):

ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:43(CNP-31):

RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:44(CNP-30):

KYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:45(CNP-29):

YKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:46(CNP-28):

KGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:47(GHKSEVAHRF-CNP-28):

GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:48(CNP-27):

GANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:49(CNP-27 K4Q,K5Q):

GANQQGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:50(CNP-27 K4R,K5R):

GANRRGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:51(CNP-27 K4P,K5R):

GANPRGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:52(CNP-27 K4S,K5S):

GANSSGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:53(CNP-27 K4P,K5R):

GANGANPRGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO:54(CNP-27 K4R,K5R,K9R):

GANRRGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO:55(CNP-27 K4R,K5R,K9R,M22N):

GANRRGLSRGCFGLKLDRIGSNSGLGC；

SEQ ID NO:56(P-CNP-27K4R,K5R,K9R):

PGANRRGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO:57(M-CNP-27K4R,K5R,K9R):

MGANRRGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO 58 (HSA fragment-CNP-27):

GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLG；

SEQ ID NO 59 (HSA fragment-CNP-27M 22N):

GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSNSGLGC；

SEQ ID NO. 60 (M-HSA fragment-CNP-27):

MGHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO. 61 (P-HSA fragment-CNP-27):

PGHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:62(CNP-26):

ANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:63(CNP-25):

NKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:64(CNP-24):

KKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:65(CNP-23):

KGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:66(R-CNP-22):

RGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:67(ER-CNP-22):

ERGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:68(R-CNP-22K4R):

RGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO:69(ER-CNP-22 4KR):

ERGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO:70(RR-CNP-22):

RRGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO:71 (HRGP fragment-CNP-22):

GHHSHEQHPHGANQQGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO. 72 (HRGP fragment-CNP-22):

GAHHPHEHDTHGANQQGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO 73 (HRGP fragment-CNP-22):

GHHSHEQHPHGANPRGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:74 (IgG ₁(F_c) fragment-CNP-22):

GQPREPQVYTLPPSGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO 75 (HSA fragment-CNP-22):

GQHKDDNPNLPRGANPRGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO 76 (HSA fragment-CNP-22):

GERAFKAWAVARLSQGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:77 (bone morphogenetic NPR C) inhibitor fragment-CNP 22):

FGIPMDRIGRNPRGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO. 78 (FGF 2 heparin binding domain fragment-CNP 22):

GKRTGQYKLGSKTGPGPKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO. 79 (IgG ₁(F_c) fragment-CNP-22K 4R):

GQPREPQVYTGANQQGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO. 80 (HSA fragment-CNP-22K 4R):

GVPQVSTSTGANQQGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO. 81 (fibronectin fragment-CNP-22K 4R):

GQPSSSSQSTGANQQGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO. 82 (fibronectin fragment-CNP-22K 4R):

GQTHSSGTQSGANQQGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO. 83 (fibronectin fragment-CNP-22K 4R):

GSTGQWHSESGANQQGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO. 84 (Zinc finger fragment-CNP-22K 4R):

GSSSSSSSSSGANQQGLSRGCFGLKLDRIGSMSGLGC；

SEQ ID NO:85(CNP-21):

LSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:86(CNP-20):

SKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:87(CNP-19):

KGCFGLKLDRIGSMSGLGC；

SEQ ID NO:88(CNP-18):

GCFGLKLDRIGSMSGLGC；

SEQ ID NO:89(CNP-17):

CFGLKLDRIGSMSGLGC；

SEQ ID NO. 90 (BNP fragment-CNP-17-BNP fragment):

SPKMVQGSGCFGLKLDRIGSMSGLGCKVLRRH；

SEQ ID NO:91(CNP-38L1G):

GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO 92 (Ac-CNP-37; wherein Ac=acetyl):

Ac-QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC；

SEQ ID NO:93:

QEHPNARX₁YX₂GANX₃X₄GLSX₅GCFGLX₆LDRIGSMSGLGC,

Wherein X ₁、X₂、X₃、X₄、X₅ and X ₆ are independently selected from: K. r, P, S, and Q, provided that at least one of X ₁、X₂、X₃、X₄、X₅ and X ₆ is selected from: r, P, S and Q; in certain embodiments, X ₁、X₂、X₃、X₄、X₅ and X ₆ are selected from: k and R, provided that at least one of X ₁、X₂、X₃、X₄、X₅ and X ₆ is R;

SEQ ID NO:94：

QEHPNARKYKGANX₁X₂GLSX₃GCFGLX₄LDRIGSMSGLGC，

Wherein X ₁、X₂、X₃ and X ₄ are independently selected from: K. r, P, S, and Q, provided that at least one of X ₁、X₂、X₃ and X ₄ is selected from: r, P, S and Q; in certain embodiments, X ₁、X₂、X₃ and X ₄ are selected from: k and R, provided that at least one of X ₁、X₂、X₃ and X ₄ is R;

SEQ ID NO:95：

QEHPNARKYKGANX₁X₂GLSKGCFGLKLDRIGSMSGLGC，

wherein X ₁X₂ is selected from: KR, RK, KP, PK, SS, RS, SR, QK, QR, KQ, RQ, RR, and QQ.

It will be appreciated that the cysteine equivalents at positions 22 and 38 in SEQ ID NO. 24 are also linked by disulfide bonds in SEQ ID NO. 2-95.

The term "CNP" also includes poly (amino acid) conjugates, such as depeptides, having a sequence as described above but having a backbone comprising amide and non-amide linkages (e.g., ester linkages). An depsipeptide is a chain of amino acid residues whose backbone comprises an amide (peptide) and an ester linkage. Thus, as used herein, the term "side chain" refers to the moiety attached to the α -carbon of an amino acid moiety if the amino acid moiety is attached via an amine linkage (as in a polypeptide), or to any carbon atom containing moiety attached to the backbone of a poly (amino acid) conjugate, as in the case of a depsipeptide. In certain embodiments, the term "CNP" refers to a polypeptide having a backbone formed by amide (peptide) bonds.

As used herein, the term "loop moiety" refers to an extension of a continuous amino acid residue of a CNP drug or moiety that is located between two cysteine residues that form an intramolecular disulfide bridge, or between homologous amino acid residues that are linked by a chemical linker. Preferably, the loop moiety is located between two cysteine residues that form an intramolecular disulfide bridge. These two cysteines correspond to the cysteines at positions 22 and 38 in the CNP-38 (SEQ ID NO: 24) sequence. Thus, if the CNP drug or moiety has the sequence of CNP-38, amino acids 23-37 are located in the loop moiety.

Regardless of the length of the CNP portion, the sequence of the loop portion of wild-type CNP is:

FGLKLDRIGSMSGLG(SEQ ID NO:96)。

Since the term CNP includes variants, analogs, orthologs, homologs, derivatives and fragments of the CNP described above, the term "loop portion" also includes the corresponding variants, analogs, orthologs, homologs, derivatives and fragments of the SEQ ID NO. 96 sequence. Thus, all references to specific positions within the reference sequence include equivalent positions in variants, analogues, orthologs, homologs, derivatives and fragments of the CNP moiety, even if not explicitly mentioned.

As used herein, the term "C _1-4 alkyl" alone or in combination means a straight or branched alkyl moiety having 1 to 4 carbon atoms. Examples of linear or branched C _1-4 alkyl groups if present at the molecular end are: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. When two moieties of a molecule are linked by a C _1-4 alkyl group then examples of such C _1-4 alkyl groups are ：-CH₂-、-CH₂-CH₂-、-CH(CH₃)-、-CH₂-CH₂-CH₂-、-CH(C₂H₅)- and-C (each hydrogen of the CH ₃)₂-.C_1-4 alkyl carbon may optionally be substituted with a substituent as defined above optionally C _1-4 alkyl may be interrupted by one or more moieties as defined below.

As used herein, the term "C _1-6 alkyl" alone or in combination means a straight or branched alkyl moiety having 1 to 6 carbon atoms. Examples of straight and branched C _1-6 alkyl groups, if present at the molecular end, are: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl and 3, 3-dimethylpropyl. When two moieties of a molecule are linked by a C _1-6 alkyl group then examples of such C _1-6 alkyl groups are ：-CH₂-、-CH₂-CH₂-、-CH(CH₃)-、-CH₂-CH₂-CH₂-、-CH(C₂H₅)- and-C (each hydrogen atom of the CH ₃)₂-.C_1-6 carbon may optionally be substituted by a substituent as defined above optionally C _1-6 alkyl may be interrupted by one or more moieties as defined below.

Thus, "C _1-10 alkyl", "C _1-20 alkyl", or "C _1-50 alkyl" respectively denote alkyl chains having 1 to 10, 1 to 20, or 1 to 50 carbon atoms, wherein each hydrogen atom of the C _1-10、C_1-20, or C _1-50 carbon may be optionally substituted with substituents as defined above. Optionally, the C _1-10 alkyl, C _1-20 alkyl, or C _1-50 alkyl may be interrupted by one or more moieties as defined below.

As used herein, the term "C _2-6 alkenyl" alone or in combination means a straight or branched hydrocarbon moiety having 2 to 6 carbon atoms containing at least one carbon-carbon double bond. If present at the molecular end, examples are: -ch=ch ₂、-CH＝CH-CH₃、-CH₂-CH＝CH₂、-CH＝CHCH₂-CH₃ and-ch=ch-ch=ch ₂. When two parts of the molecule are linked through a C _2-6 alkenyl group, one example of such a C _2-6 alkenyl group is-ch=ch-. Each hydrogen atom of the C _2-6 alkenyl moiety may be optionally substituted with substituents as defined above. Optionally, the C _2-6 alkenyl group may be interrupted by one or more moieties as defined below.

Thus, the term "C _2-10 alkenyl", "C _2-20 alkenyl", or "C _2-50 alkenyl", alone or in combination, means a straight or branched hydrocarbon moiety containing at least one carbon-carbon double bond and having 2 to 10, 2 to 20, or 2 to 50 carbon atoms. Each hydrogen atom of a C _2-10 alkenyl, C _2-20 alkenyl, or C _2-50 alkenyl group may be optionally substituted with substituents as defined above. Optionally, the C _2-10 alkenyl, C _2-20 alkenyl or C _2-50 alkenyl may be interrupted by one or more moieties as defined below.

As used herein, the term "C _2-6 alkynyl" alone or in combination means a straight or branched hydrocarbon moiety having 2 to 6 carbon atoms containing at least one carbon-carbon triple bond. If present at the molecular end, examples are: -C.ident.CH, -CH ₂-C≡CH、CH₂-CH₂ -C.ident.CH and CH ₂-C≡C-CH₃. When two parts of a molecule are linked by an alkynyl group, one example is-c=c-. Each hydrogen atom of the C _2-6 alkynyl group may be optionally substituted with substituents as defined above. Optionally, one or more double bonds may be present. Optionally, the C _2-6 alkynyl group may be interrupted by one or more moieties as defined below.

Thus, as used herein, the terms "C _2-10 alkynyl", "C _2-20 alkynyl" and "C _2-50 alkynyl", alone or in combination, mean a straight or branched hydrocarbon moiety containing at least one carbon-carbon triple bond having from 2 to 10, from 2 to 20 or from 2 to 50 carbon atoms, respectively. Each hydrogen atom of a C _2-10 alkynyl, C _2-20 alkynyl, or C _2-50 alkynyl group may be optionally substituted with substituents as defined above. Optionally, one or more double bonds may be present. Optionally, the C _2-10 alkynyl, C _2-20 alkynyl or C _2-50 alkynyl may be interrupted by one or more moieties as defined below.

As described above, C _1-4 alkyl, C _1-6 alkyl, C _1-10 alkyl, C _1-20 alkyl, C _1-50 alkyl, C _2-6 alkenyl, C _2-10 alkenyl, C _2-20 alkenyl, C _2-50 alkenyl, C _2-6 alkynyl, C _2-10 alkynyl, C _2-20 alkenyl, or C _2-50 alkynyl may be optionally interrupted by one or more moieties, in certain embodiments selected from the group consisting of:

Wherein the method comprises the steps of

The dashed line represents the remainder attached to the moiety or reagent; and

-R and-R ^a are independently from each other selected from the group: -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

As used herein, the term "C _3-10 cycloalkyl" means a cyclic alkyl chain having 3 to 10 carbon atoms, which may be saturated or unsaturated, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. Each hydrogen atom of the C _3-10 cycloalkyl carbon may be substituted with substituents as defined above. The term "C _3-10 cycloalkyl" also includes bridged bicyclic rings, such as norbornane or norbornene.

As used herein, the term "8-30 membered carbon polycyclic group" or "8-30 membered carbon polycyclic" means a cyclic moiety having two or more rings of 8-30 ring atoms, wherein two adjacent rings share at least one ring atom, and may contain no more than a maximum number of double bonds (fully, partially or unsaturated aromatic or non-aromatic). In certain embodiments, an 8-30 membered carbon multi-ring group means a cyclic moiety of two, three, four, or five rings, in certain embodiments two, three, or four rings.

As used herein, the term "3-10 membered heterocyclyl" or "3-10 membered heterocycle" means a ring having 3,4, 5, 6, 7, 8, 9 or 10 ring atoms which may contain no more than a maximum number of double bonds (fully, partially or unsaturated aromatic or non-aromatic rings) wherein at least one ring atom, up to 4 ring atoms, is substituted with a heteroatom selected from the group consisting of: sulfur (including-S (O) -, -S (O) ₂ -), oxygen, and nitrogen (including = N (O) -), and wherein the ring is attached to the remainder of the molecule via a carbon or nitrogen atom. Examples of 3-10 membered heterocycles include, but are not limited to: aziridine, oxirane, thiirane, azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepine, azepine, and homopiperazine. Each hydrogen atom of the 3-10 membered heterocyclic group or the 3-10 membered heterocyclic group may be substituted with a substituent as defined below.

As used herein, the term "8-11 membered heterobicyclic group" or "8-11 membered heterobicyclic" refers to a heterocyclic moiety having two rings of 8-11 ring atoms, wherein at least one ring atom is shared by both rings and may contain no more than a maximum number of double bonds (fully, partially or unsaturated aromatic or non-aromatic rings), wherein at least one ring atom, up to 6 ring atoms, is substituted with a heteroatom selected from the group consisting of: sulfur (including-S (O) -, -S (O) ₂ -), oxygen, and nitrogen (including = N (O) -), and wherein the ring is attached to the remainder of the molecule via a carbon or nitrogen atom. Examples of 8-11 membered heterobicyclic rings are: indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzoazepine, purine, and pteridine. The term "8-11 membered heterobicyclo" also includes spiro structures of two rings, such as 1, 4-dioxa-8-azaspiro [4.5] decane, or bridged heterocycles, such as 8-azabicyclo [3.2.1] octane. Each hydrogen atom of the 8-11 membered heterobicyclic group or the 8-11 membered heterobicyclic carbon may be substituted with a substituent as defined below.

Similarly, the term "8-30 membered heteropolycyclyl" or "8-30 membered heteropolycyclyl" means a heterocyclic moiety of two or more rings having 8-30 ring atoms, in certain embodiments three, four, or five rings, wherein two adjacent rings share at least one ring atom and may contain no more than a maximum number of double bonds (fully, partially, or unsaturated aromatic or non-aromatic rings), wherein at least one ring atom, up to 10 ring atoms, are replaced with heteroatoms selected from the group consisting of: sulfur (including-S (O) -, -S (O) ₂ -), oxygen, and nitrogen (including = N (O) -), and wherein the ring is attached to the remainder of the molecule via a carbon or nitrogen atom.

It is understood that the phrase "R ^x/R^y pair, taken together with the atoms to which they are attached, forms a C _3-10 cycloalkyl or 3-10 membered heterocyclyl":

meaning that R ^x and R ^y form the following structure:

Wherein R is C _3-10 cycloalkyl or 3-10 membered heterocyclyl.

It will also be appreciated that the phrase "R ^x/R^y pair, referring to the following moieties, taken together with the atoms to which they are attached forms a ring a":

meaning that R ^x and R ^y form the following structure:

As used herein, the term "dose" or "unit dose" refers to a predetermined amount of a drug (e.g., CNP) administered at one time to produce a degree of biological response in a patient. The dosage of the drug depends on its inherent efficacy, in which case it is a therapeutic dosage or unit dose.

As used herein, the term "dosage form" refers to a physical form comprising an active pharmaceutical ingredient and selected additional ingredients or excipients intended to be delivered to the site of action in the body by a variety of routes of drug administration. It also refers to the physical form of an exact mixture of the active pharmaceutical ingredient and excipients that aid in administration and delivery to the site of action to achieve rapid onset of action and to enhance bioavailability. As used herein, the term "unit dosage form" refers to a dosage form configured for single administration to a patient. For example, the unit dosage form may be a single vial (val) or a container containing a quantity of drug suitable for a single administration.

As used herein, the term "dosing regimen" is a combination of the dosage and frequency of administration of a drug. The dosing regimen may also include a route of administration (e.g., subcutaneously) and/or a duration of administration (e.g., until the patient reaches 18 years of age or epiphyseal closure). Administration of the dosing regimen may maintain a steady state serum concentration of CNP wherein the peak, trough and area under the curve over a defined time interval remain within a defined fluctuation range and/or the ratio of peak to trough does not exceed a defined threshold.

As used herein, the term "drug" refers to a substance used in the treatment, cure, prevention, or diagnosis of a disease or for enhancing physical or mental health. If a drug (e.g., CNP) is conjugated to another moiety, the moiety derived from the drug in the resulting product is referred to as a "drug moiety".

As used herein, the term "excipient" refers to a compound that is administered with a drug or drug conjugate, such as a buffer, an isotonicity adjuster, a preservative, a stabilizer, an anti-adsorbent, an oxidation protectant, or other auxiliary agent. However, in some cases, one excipient may have dual or triple functions. The term "excipient" may also refer to a diluent, adjuvant, or carrier with which a drug or drug conjugate is administered. Such pharmaceutically acceptable excipients may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to: peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical formulation is administered orally, water is a preferred excipient. Saline and aqueous dextrose are preferred excipients when administering pharmaceutical formulations intravenously or subcutaneously. In certain embodiments, saline solutions and aqueous dextrose and glycerol solutions are employed as liquid excipients for injectable solutions. Suitable pharmaceutically acceptable excipients include: starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The pharmaceutical preparation may also contain minor amounts of wetting or emulsifying agents, pH buffering agents, such as, for example, acetates, succinates, tris (Tris (hydroxymethyl) aminomethane), carbonates, phosphates, HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid), MES (2- (N-morpholinoethanesulfonic acid), or may contain detergents, such asPoloxamer, CHAPS,Or amino acids such as, for example, glycine, lysine or histidine. These pharmaceutical formulations may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The pharmaceutical formulation may be formulated as a suppository using conventional binders and excipients such as triglycerides. Oral formulations may include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such formulations will contain a therapeutically effective amount of the drug or drug portion, and a suitable amount of excipients, to provide the appropriate form of administration to the patient. The formulation should be suitable for the mode of administration.

As used herein, the term "formulation" or "pharmaceutical formulation" refers to a formulation containing one or more CNP conjugates and one or more excipients, as well as any product that results, directly or indirectly, from the combination, complexation, or aggregation of any two or more of the ingredients, or from the breakdown of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Thus, the pharmaceutical formulations of the present invention encompass any formulation or composition made by mixing one or more CNP conjugates with pharmaceutically acceptable excipients such as buffers and bulking agents.

As used herein, the term "free form" of a drug refers to an unmodified, pharmacologically fully active form of the drug, e.g., after release from a CNP conjugate or a pharmaceutically acceptable salt thereof.

As used herein, the term "functional group" means an atomic group that can react with other atomic groups. Functional groups include, but are not limited to, the following: carboxylic acid (- (c=o) OH), primary or secondary amine (-NH ₂, -NH-), maleimide, thiol (-SH), sulfonic acid (- (o=s=o) OH), carbonate, carbamate (-O (c=o) N <), hydroxyl (-OH), aldehyde (- (c=o) H), ketone (- (c=o) -), hydrazine (> N-N <), isocyanate, isothiocyanate, phosphoric acid (-O (p=o) OHOH), phosphonic acid (-O (p=o) OHH), haloacetyl, alkyl halide, acryl, aryl fluoride, hydroxylamine, disulfide, sulfonamide, sulfuric acid, vinyl sulfone, vinyl ketone, diazoane, ethylene oxide, and aziridine.

As used herein, the term "halogen" means fluorine, chlorine, bromine or iodine. It is generally preferred that the halogen is fluorine or chlorine.

As used herein, the term "interrupted" means that a moiety is inserted between two carbon atoms, or (if inserted at one end of the moiety) between a carbon or heteroatom and a hydrogen atom, in certain embodiments between a carbon and a hydrogen atom.

As used herein, the term "isotonic agent" refers to a compound that minimizes pain, irritation, and tissue damage caused by cellular injury due to osmotic pressure differences between the injection solution and plasma.

As used herein, the term "moiety" means a portion of a molecule lacking one or more atoms as compared to the corresponding reagent. For example, if a reagent of the formula "H-X-H" reacts with another reagent and becomes part of a reaction product, the corresponding partial reaction product has a "H-X-" or "-X-" structure, and each "-" represents a linkage with another moiety. Thus, the drug moiety (e.g., CNP moiety) is released from the conjugate in the form of a drug (e.g., CNP).

It is understood that if a sequence or chemical structure of an atomic group is provided that is linked to two moieties or interrupts one moiety, the sequence or chemical structure may be linked to both moieties in either direction, unless explicitly stated otherwise. For example, the moiety "-C (O) N (R ¹) -" may be attached to both moieties, or one moiety may be interrupted, as "-C (O) N (R ¹) -" or "-N (R ¹) C (O) -". Similarly, part of:

may be connected to both parts or may interrupt one of the following parts:

As a means of

When the CNP moiety comprises one or more acidic or basic groups, the unit dosage form also comprises its corresponding pharmaceutically or toxicologically acceptable salt, in particular its pharmaceutically usable salt. Thus, CNP moieties containing one or more acidic groups may be present and used as, for example, alkali metal, alkaline earth metal or ammonium salts. More precise examples of such salts include: sodium, potassium, calcium, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids, as well as other salts or amines known to those skilled in the art. The CNP moiety comprising one or more basic groups (i.e. protonatable groups) may be present and may be used in the form of its addition salts with inorganic or organic acids. Examples of suitable acids include: hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethyl acetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to those skilled in the art. Further methods of converting basic groups into cations, such as alkylation of amine groups, are known to those skilled in the art, yielding positively charged ammonium groups and counterions to the appropriate salts. If the CNP moiety contains both acidic and basic groups, the pharmaceutical formulation according to the invention comprises, in addition to the salt forms described above, also internal salts or betaines (zwitterionic). The corresponding salts can be obtained by conventional methods known to the person skilled in the art, for example by contacting these conjugates with organic or inorganic acids or bases in solvents or dispersants, or by anion exchange or cation exchange with other salts. The unit dosage forms of the present invention also include all salts of CNP conjugates, which are not directly suitable for use in medicine due to their low physiological compatibility, but may be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.

As used herein, the term "patient" refers to a subject, particularly a human subject, that may be treated or prevented in accordance with the present invention.

As used herein, the term "pharmaceutically acceptable" means a substance that does not cause injury when administered to a patient, and preferably means a substance approved by regulatory authorities, such as EMA (europe) and/or FDA (united states) and/or any other national regulatory authority for use in animals, preferably humans.

As used herein, the term "physiological condition" refers to an aqueous buffer having a pH of 7.4 and a temperature of 37 ℃.

As used herein, the term "polypeptide" refers to a chain of at least 2 and at most 50 amino acid monomer moieties linked by peptide (amide) bonds. For CNP drugs and CNP moieties only, sequences with more than 50 amino acids will be referred to as "polypeptides" for simplicity.

As used herein, the term "preservative" refers to a chemical substance that has an antimicrobial effect and prevents chemical degradation.

As used herein, the term "protein" refers to a chain of more than 50 amino acid monomer moieties linked by peptide bonds, wherein preferably the amino acid monomer moieties linked by peptide bonds are no more than 12,000, such as no more than 10,000 amino acid monomer moieties, no more than 8,000 amino acid monomer moieties, no more than 5,000 amino acid monomer moieties, or no more than 2,000 amino acid monomer moieties.

As used herein, the term "polymer" means a molecule comprising repeating structural units (i.e., monomers) linked by chemical bonds in a linear, cyclic, branched, crosslinked, or dendritic fashion, or a combination thereof, which may be of synthetic or biological origin, or a combination of both. It is understood that the polymer may also contain one or more other chemical groups and/or moieties, such as one or more functional groups. In certain embodiments, the soluble polymer has a molecular weight of at least 0.5kDa, e.g., a molecular weight of at least 1kDa, a molecular weight of at least 2kDa, a molecular weight of at least 3kDa, or a molecular weight of at least 5kDa. If the polymer is soluble, in certain embodiments, it has a molecular weight of at most 1000kDa, such as at most 750kDa, such as at most 500kDa, such as at most 300kDa, such as at most 200kDa, such as at most 100kDa.

It is understood that a protein or polypeptide is also a polymer with amino acids as repeating structural units, although the side chains of each amino acid may be different.

As used herein, the term "polymer" or "polymer moiety" means an agent or moiety comprising one or more polymers or polymer moieties. The polymeric reagent or moiety may also optionally comprise one or more other moieties, which in certain embodiments are selected from the group consisting of:

C _1-50 alkyl, C _2-50 alkenyl, C _2-50 alkynyl, C _3-10 cycloalkyl, 3-to 10-membered heterocyclyl, 8-to 11-membered heterobicycloyl, phenyl, naphthyl, indenyl, indanyl and tetrahydronaphthyl; and

A linkage selected from the group consisting of:

wherein the dotted line represents the remainder attached to the moiety or reagent, and

-R and-R ^a are independently from each other selected from the group consisting of: -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

Those skilled in the art understand that the polymerization products obtained by the polymerization are not all of the same molecular weight but exhibit a certain molecular weight distribution. Thus, as used herein, molecular weight range, molecular weight, range of number of monomers in the polymer, and number of monomers in the polymer refer to the number average molecular weight and number average molecular weight of the monomers, i.e., the arithmetic average of the molecular weights of the polymer or polymer portion and the arithmetic average of the number of monomers of the polymer or polymer portion.

Thus, in a polymer fraction comprising "x" monomer units, any integer given for "x" corresponds to the arithmetic mean of the monomers. Any integer range given for "x" provides the integer range over which the arithmetic mean of the monomers is located. The integer of "x" is given as "about x", meaning that the arithmetic average of the monomers is within the range of the integer x +/-10%, in certain embodiments within the range of the integer x +/-8%, in certain embodiments within the range of the integer x +/-5%, and in certain embodiments within the range of the integer x +/-2%.

As used herein, the term "PEG-based" in reference to a moiety or agent means that the moiety or agent comprises PEG. In certain embodiments, the PEG-based moiety or agent comprises at least 10% (w/w) PEG, such as at least 20% (w/w) PEG, such as at least 30% (w/w) PEG, such as at least 40% (w/w) PEG, such as at least 50% (w/w) PEG, such as at least 60% (w/w) PEG, such as at least 70% (w/w) PEG, such as at least 80% (w/w) PEG, such as at least 90% (w/w) PEG, such as at least 95% (w/w) PEG.

The remaining weight percent of the PEG-based moiety or reagent is selected from the following moieties and linkages:

A linkage selected from the group consisting of:

As used herein, the term "PEG-based, comprising at least X% PEG" in reference to a moiety or agent means that the moiety or agent comprises at least X% (w/w) ethylene glycol units (-CH ₂CH₂ O-), wherein the ethylene glycol units may be arranged in blocks, alternate, or may be randomly distributed within the moiety or agent, and in certain embodiments, all of the ethylene glycol units of the moiety or agent are present in one block; in certain embodiments, the remaining weight percent of the PEG-based moiety or reagent is a moiety selected from the group consisting of:

A linkage selected from the group consisting of:

As used herein, the term "hyaluronic acid-based, comprising at least X% hyaluronic acid" is used accordingly.

Those of ordinary skill in the art will also recognize that the conjugates of the present invention are prodrugs. As used herein, the term "prodrug" refers to a drug moiety (e.g., CNP moiety) that is reversibly covalently conjugated to a polymer moiety (e.g., Z) through a reversible linker moiety. The prodrug releases the reversibly covalently bound drug moiety in the form of the corresponding drug. In other words, a prodrug is a conjugate comprising a drug moiety (e.g., CNP moiety) covalently and reversibly conjugated to a polymer moiety via a reversible linker moiety, wherein the covalent and reversible conjugation of the polymer moiety to the reversible linker moiety is direct or through a spacer. Such prodrugs or conjugates release the previously conjugated drug moiety in the form of a free drug.

As used herein, the term "random coil" refers to a peptide or protein that in certain embodiments adopts/has/forms a conformation that substantially lacks defined secondary and tertiary structures, as determined by circular dichroism spectroscopy performed in an aqueous buffer at ambient temperature and ph 7.4. In certain embodiments, the ambient temperature is about 20 ℃ (i.e., 18 ℃ to 22 ℃), and in certain embodiments, the ambient temperature is 20 ℃.

As used herein, the term "reversible linkage" is a linkage cleavable under physiological conditions (aqueous buffer at ph7.4, 37 ℃) in the absence of an enzyme, with a half-life ranging from one hour to six months, such as one hour to four months, such as one hour to three months, one hour to two months, or one hour to one month. Thus, a stable ligation is one with a half-life of more than six months under physiological conditions (aqueous buffer, pH7.4, 37 ℃).

As used herein, the term "agent" means a compound that includes at least one functional group that is reactive with a functional group of another compound or drug. It is understood that a drug containing a functional group (e.g., primary or secondary amine or hydroxyl functionality) is also an agent.

As used herein, the term "reversible linker moiety" is a moiety that is covalently conjugated to a drug moiety (e.g., CNP moiety) and also covalently conjugated to a polymer moiety (e.g., -Z) by a reversible linkage, wherein the covalent conjugation to the polymer moiety is either direct or through a spacer moiety (e.g., -L ² -). In certain embodiments, the linkage between-Z and-L ² -is a stable linkage. Conjugates comprising a reversible linker moiety may be referred to as reversible conjugates.

As used herein, the term "spacer" or "spacer moiety" refers to a moiety that is suitable for joining two moieties. Suitable spacers may be selected from the group consisting of: c _1-50 alkyl, C _2-50 alkenyl or C _2-50 alkynyl, wherein C _1-50 alkyl, C _2-50 alkenyl or C _2-50 alkynyl may optionally be interrupted by one or more groups selected from the group consisting of: -NH-, -N (C _1-4 alkyl) -, -O-, -S-, -C (O) NH-, -C (O) N (C _1-4 alkyl) -, -O-C (O) -, -S (O) ₂ -, 4-7 membered heterocyclyl, phenyl and naphthyl.

As used herein, the term "substituted" means that one or more-H atoms of a molecule or moiety are replaced with a different atom or group of atoms, which is referred to as a "substituent".

In certain embodiments, the one or more substituents are selected from the group consisting of: halogen 、-CN、-COOR^x1、-OR^x1、-C(O)R^x1、-C(O)N(R^x1R^x1a)、-S(O)₂N(R^x1R^x1a)、-S(O)N(R^x1R^x1a)、-S(O)₂R^x1、-S(O)R^x1、-N(R^x1)S(O)₂N(R^x1aR^x1b)、-SR^x1、-N(R^x1R^x1a)、-NO₂、-OC(O)R^x1、-N(R^x1)C(O)R^x1a、-N(R^x1)S(O)₂R^x1a、-N(R^x1)S(O)R^x1a、-N(R^x1)C(O)OR^x1a、-N(R^x1)C(O)N(R^x1aR^x1b)、-OC(O)N(R^x1R^x1a)、-T⁰、C_1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl; wherein-T ⁰、C_1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally substituted by one or more identical or different-R ^x2 groups, And wherein C _1-50 alkyl, c _2-50 alkenyl and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of ：-T⁰-、-C(O)O-、-O-、-C(O)-、-C(O)N(R^x3)-、-S(O)₂N(R^x3)-、-S(O)N(R^x3)-、-S(O)₂-、-S(O)-、-N(R^x3)S(O)₂N(R^x3a)-、-S-、-N(R^x3)-、-OC(OR^x3)(R^x3a)-、-N(R^x3)C(O)N(R^x3a)- and-OC (O) N (R ^x3)-;-R^x1、-R^x1a、-R^x1b are each independently of the other selected from the group consisting of-H, -T ⁰、C_1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl; wherein-T ⁰、C_1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally substituted by one or more identical or different-R ^x2 groups, And wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl groups may be optionally interrupted by one or more groups selected from the group ：-T⁰-、-C(O)O-、-O-、-C(O)-、-C(O)N(R^x3)-、-S(O)₂N(R^x3)-、-S(O)N(R^x3)-;-S(O)₂-、-S(O)-、-N(R^x3)S(O)₂N(R^x3a)-、-S-、-N(R^x3)-、-OC(OR^x3)(R^x3a)-、-N(R^x3)C(O)N(R^x3a)- and-OC (O) N (R ^x3) -;

Each T ⁰ is independently selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclic group; wherein each T ⁰ is independently optionally substituted with one or more of the same or different-R ^x2;

each-R ^x2 is independently selected from the group consisting of: halogen, -CN, oxo (＝O)、-COOR^x4、-OR^x4、-C(O)R^x4、-C(O)N(R^x4R^x4a)、-S(O)₂N(R^x4R^x4a)、-S(O)N(R^x4R^x4a)、-S(O)₂R^x4、-S(O)R^x4、-N(R^x4)S(O)₂N(R^x4aR^x4b)、-SR^x4、-N(R^x4R^x4a)、-NO₂、-OC(O)R^x4、-N(R^x4)C(O)R^x4a、-N(R^x4)S(O)₂R^x4a、-N(R^x4)S(O)R^x4a、-N(R^x4)C(O)OR^x4a、-N(R^x4)C(O)N(R^x4aR^x4b)、-OC(O)N(R^x4R^x4a) and C _1-6 alkyl; wherein C _1-6 alkyl is optionally substituted with one or more of the same or different halogen;

-R ^x3、-R^x3a、-R^x4、-R^x4a、-R^x4b are each independently selected from the group consisting of: -H and C _1-6 alkyl; wherein the C _1-6 alkyl groups are optionally substituted with one or more of the same or different halogens.

In certain embodiments, one or more substituents are selected from the following groups, independently of each other: halogen 、-CN、-COOR^x1、-OR^x1、-C(O)R^x1、-C(O)N(R^x1R^x1a)、-S(O)₂N(R^x1R^x1a)、-S(O)N(R^x1R^x1a)、-S(O)₂R^x1、-S(O)R^x1、-N(R^x1)S(O)₂N(R^x1aR^x1b)、-SR^x1、-N(R^x1R^x1a)、-NO₂、-OC(O)R^x1、-N(R^x1)C(O)R^x1a、-N(R^x1)S(O)₂R^x1a、-N(R^x1)S(O)R^x1a、-N(R^x1)C(O)OR^x1a、-N(R^x1)C(O)N(R^x1aR^x1b)、-OC(O)N(R^x1R^x1a)、-T⁰、C_1-10 alkyl, C _2-10 alkenyl, and C _2-10 alkynyl; wherein-T ⁰、C_1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl are optionally substituted with one or more of the same or different-R ^x2, and wherein C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl are optionally interrupted ：-T⁰-、-C(O)O-、-O-、-C(O)-、-C(O)N(R^x3)-、-S(O)₂N(R^x3)-、-S(O)N(R^x3)-、-S(O)₂-、-S(O)-、-N(R^x3)S(O)₂N(R^x3a)-、-S-、-N(R^x3)-、-OC(OR^x3)(R^x3a)-、-N(R^x3)C(O)N(R^x3a)- and-OC (O) N (R ^x3) -, by one or more groups selected from the group consisting of;

each-R ^x1、-R^x1a、-R^x1b、-R^x3、-R^x3a is independently selected from the group consisting of: -H, halogen, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl;

each-R ^x2 is independently selected from the group consisting of: halogen, -CN, oxo (＝O)、-COOR^x4、-OR^x4、-C(O)R^x4、-C(O)N(R^x4R^x4a)、-S(O)₂N(R^x4R^x4a)、-S(O)N(R^x4R^x4a)、-S(O)₂R^x4、-S(O)R^x4、-N(R^x4)S(O)₂N(R^x4aR^x4b)、-SR^x4、-N(R^x4R^x4a)、-NO₂、-OC(O)R^x4、-N(R^x4)C(O)R^x4a、-N(R^x4)S(O)₂R^x4a、-N(R^x4)S(O)R^x4a、-N(R^x4)C(O)OR^x4a、-N(R^x4)C(O)N(R^x4aR^x4b)、-OC(O)N(R^x4R^x4a) and C _1-6 alkyl; wherein C _1-6 alkyl is optionally substituted with one or more of the same or different halogen; each-R ^x4、-R^x4a、-R^x4b is independently selected from the group consisting of: -H, halogen, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl.

In certain embodiments, one or more substituents are selected from the group consisting of, independently of each other: halogen 、-CN、-COOR^x1、-OR^x1、-C(O)R^x1、-C(O)N(R^x1R^x1a)、-S(O)₂N(R^x1R^x1a)、-S(O)N(R^x1R^x1a)、-S(O)₂R^x1、-S(O)R^x1、-N(R^x1)S(O)₂N(R^x1aR^x1b)、-SR^x1、-N(R^x1R^x1a)、-NO₂、-OC(O)R^x1、-N(R^x1)C(O)R^x1a、-N(R^x1)S(O)₂R^x1a、-N(R^x1)S(O)R^x1a、-N(R^x1)C(O)OR^x1a、-N(R^x1)C(O)N(R^x1aR^x1b)、-OC(O)N(R^x1R^x1a)、-T⁰、C_1-6 alkyl, C _2-6 alkenyl, and C _2-6 alkynyl; wherein-T ⁰、C_1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl may be optionally substituted with one or more of the same or different-R ^x2, wherein C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl may be optionally interrupted by one or more groups selected from ：-T⁰-、-C(O)O-、-O-、-C(O)-、-C(O)N(R^x3)-、-S(O)₂N(R^x3)-、-S(O)N(R^x3)-、-S(O)₂-、-S(O)-、-N(R^x3)S(O)₂N(R^x3a)-、-S-、-N(R^x3)-、-OC(OR^x3)(R^x3a)-、-N(R^x3)C(O)N(R^x3a and-OC (O) N (R ^x3) -;

each-R ^x1、-R^x1a、-R^x1b、-R^x2、-R^x3 and-R ^x3a is independently selected from the group consisting of: -H, halogen, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl;

Each T ⁰ is independently selected from: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclic group; wherein each T ⁰ is independently optionally substituted with one or more of the same or different-R ^x2.

In certain embodiments, up to 6-H atoms in the optionally substituted molecule are independently substituted with substituents, e.g., 5-H atoms are independently substituted with substituents, 4-H atoms are independently substituted with substituents, 3-H atoms are independently substituted with substituents, 2-H atoms are independently substituted with substituents, or 1-H atom is substituted with a substituent.

As used herein, the term "therapeutically effective amount" means an amount sufficient to cure, alleviate or partially inhibit the clinical manifestations of a given disease and its complications. The effective amount for each purpose will depend on the severity of the disease or injury and the weight and general state of the subject. It will be appreciated that determining the appropriate dosage can be accomplished using routine experimentation by constructing a matrix of values and testing different points in the matrix, all within the ordinary skill of a trained physician. Within the scope of the present invention, a therapeutically effective amount relates to a dose intended to achieve a therapeutic effect for a period of time, i.e. at least one day, such as two days, such as three days, such as four days, such as five days, such as six days, such as one week or such as two weeks.

As used herein, the term "traceless linker" means a reversible linker that releases the drug in free form after cleavage.

As used herein, the term "water-soluble" in reference to a polymer moiety means that when such polymer moiety is part of a CNP conjugate, at least 1g of CNP conjugate comprising such water-soluble polymer moiety can be dissolved in 1 liter of water at 20 ℃ to form a homogeneous solution.

In general, the term "comprising" also encompasses "consisting of … (consist of/consisting of)".

The unit dosage amount comprised by the unit dosage form of the invention depends on the actual weight of the patient.

In certain embodiments, the unit dosage ranges from about 12.3nmol CNP/kg to at least about 37nmol CNP/kg. In certain embodiments, the unit dosage ranges from 12.3 to 36.9nmol CNP/kg. In certain embodiments, the unit dosage range is at least 24.6nmol CNP/kg. In certain embodiments, the unit dosage range is about 24.6nmol CNP/kg. In certain embodiments, the unit dose range is 24.6nmol CNP/kg.

In certain embodiments, the unit dosage ranges from about 6 μg CNP/kg to at least about 100 μg gCNP/kg. In certain embodiments, the unit dosage ranges from about 6 μg CNP/kg to about 150 μg gCNP/kg.

In certain embodiments, the unit dosage range comprised in the unit dosage form of the invention is from 6 μ gCNP/kg to at least 100 μg CNP/kg. In certain embodiments, the unit dose ranges from 6 μ gCNP/kg to 150 μg CNP/kg.

It will be understood that "x" μg CNP/kg refers to "x" μg CNP per kg patient body weight, i.e., "x" μg of CNP moiety contained in the CNP conjugate. Similarly, it is understood that "y" nmol CNP/kg refers to "y" nmol CNP per kg patient body weight, i.e., "y" nmol CNP moiety contained in the CNP conjugate.

In certain embodiments, the patient is an adult. In certain embodiments, the patient is a pediatric patient. In certain embodiments, the patient is an infant.

In certain embodiments, the patient's weight ranges from about 2kg to about 80kg. In certain embodiments, the patient's weight ranges from about 4kg to about 60kg. In a certain embodiment, the patient's weight is about 5kg. In a certain embodiment, the patient has a weight of about 9kg. In a certain embodiment, the patient has a weight of about 10kg. In a certain embodiment, the patient has a weight of about 11kg. In a certain embodiment, the patient has a weight of about 12kg. In a certain embodiment, the patient has a weight of about 15kg. In a certain embodiment, the patient has a weight of about 20kg. In a certain embodiment, the patient has a weight of about 30kg. In a certain embodiment, the patient has a weight of about 40kg. In a certain embodiment, the patient has a weight of about 50kg. In a certain embodiment, the patient has a weight of about 60kg. In a certain embodiment, the patient has a weight of about 70kg. In a certain embodiment, the patient has a weight of about 80kg.

In certain embodiments, the unit dose range is 50-7000 μg CNP. In certain embodiments, the unit dose range is 100-5000 μg CNP. In certain embodiments, the unit dose range is 100-3000 μg CNP. In certain embodiments, the unit dose range is 100-2000 μg CNP. In certain embodiments, the unit dose range is 100-1000 μg CNP. In certain embodiments, the unit dose range is 150-750 μg CNP. In certain embodiments, the unit dose range is 150-500 μg CNP. In certain embodiments, the unit dose range is 150-350 μg CNP. In certain embodiments, the unit dose is about 700 μg CNP. In certain embodiments, the unit dose is about 600 μg CNP. In certain embodiments, the unit dose is about 500 μg CNP. In certain embodiments, the unit dose is about 400 μg CNP. In certain embodiments, the unit dose is about 300 μg CNP.

In certain embodiments, the unit dose is about 6 μg CNP/kg. In certain embodiments, the unit dose is about 20 μg CNP/kg. In certain embodiments, the unit dose is about 50 μ gCNP/kg. In certain embodiments, the unit dose is about 75 μg CNP/kg. In certain embodiments, the unit dose is about 100 μg CNP/kg. In certain embodiments, the unit dose is about 125 μg CNP/kg. In certain embodiments, the unit dose is about 150 μg CNP/kg.

In certain embodiments, the unit dose is 6 μg CNP/kg. In certain embodiments, the unit dose is 20 μg CNP/kg. In certain embodiments, the unit dose is 50 μg CNP/kg. In certain embodiments, the unit dose is 75 μg CNP/kg. In certain embodiments, the unit dose is 100 μg CNP/kg. In certain embodiments, the unit dose is 125 μg CNP/kg. In certain embodiments, the unit dose is 150 μg CNP/kg.

In certain embodiments, the unit dosage form is a liquid. In certain embodiments, the unit dosage form is a solid.

The solid unit dosage form is reconstituted prior to subcutaneous administration to a patient in need thereof. Reconstitution of a solid unit dosage form into a reconstituted formulation is accomplished by adding a predetermined amount of a reconstitution solution to the solid unit dosage form. Accordingly, another aspect of the present invention is a method of reconstituting a solid unit dosage form of the present invention, wherein the method comprises the steps of:

(a) The solid unit dosage form of the present invention is contacted with a reconstitution solution.

Reconstitution may be performed in a container providing a solid unit dosage form, such as in a vial; syringes, such as dual chamber syringes; ampoule bottle; cartridges, such as dual chamber cartridges; or the solid unit dosage form may be transferred to a different container and then reconstituted therein. In certain embodiments, the container in which reconstitution of the solid unit dosage form occurs is a vial. In certain embodiments, the container in which reconstitution of the solid unit dosage form occurs is a syringe. In certain embodiments, the container in which reconstitution of the solid unit dosage form occurs is a dual chamber syringe. In certain embodiments, the container in which reconstitution of the solid unit dosage form occurs is a cartridge. In certain embodiments, the container in which reconstitution of the solid unit dosage form occurs is a dual chamber cartridge.

In certain embodiments, the solid unit dosage forms of the present invention are provided in a first chamber of a dual chamber syringe and the reconstitution solution is provided in a second chamber of the dual chamber syringe.

The reconstitution solution is a sterile liquid (e.g., water or buffer) which may contain further additives (e.g., preservatives and/or antimicrobial agents).

In certain embodiments, the reconstituted solution comprises one or more preservatives, and/or antibacterial agents, and/or antioxidants.

In certain embodiments, the reconstituted solution comprises one or more preservatives.

The preservative may be selected from the group consisting of: m-cresol, benzoic acid, phenol, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, potassium sorbate, chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosal, sorbic acid, potassium sorbate, chlorocresol, benzalkonium chloride, 2-ethoxyethanol, chlorhexidine, chlorobutanol, phenylethyl alcohol, phenylmercuric acetate, and mixtures thereof.

In certain embodiments, the preservative is m-cresol. In certain embodiments, the preservative is benzyl alcohol. In certain embodiments, the preservative is benzoic acid. In certain embodiments, the preservative is phenol. In certain embodiments, the preservative is methylparaben. In certain embodiments, the preservative is p-hydroxy phenethyl ester. In certain embodiments, the preservative is propyl parahydroxybenzoate. In certain embodiments, the preservative is butyl parahydroxybenzoate. In certain embodiments, the preservative is potassium sorbate. In certain embodiments, the preservative is benzyl alcohol. In certain embodiments, the preservative is phenylmercuric nitrate. In certain embodiments, the preservative is thimerosal. In certain embodiments, the preservative is sorbic acid. In certain embodiments, the preservative is potassium sorbate. In certain embodiments, the preservative is chlorocresol. In certain embodiments, the preservative is benzalkonium chloride. In certain embodiments, the preservative is 2-ethoxyethanol. In certain embodiments, the preservative is chlorhexidine. In certain embodiments, the preservative is chlorobutanol. In certain embodiments, the preservative is phenethyl alcohol. In certain embodiments, the preservative is phenylmercuric acetate.

In certain embodiments, the concentration of preservative ranges from 1 to 10mg/ml. In certain embodiments, the concentration of preservative ranges from 1.5 to 3.5mg/ml. In certain embodiments, the concentration of preservative ranges from 2 to 3mg/ml.

The antioxidant may be selected from the group consisting of: methionine, butylhydroxytoluene, butylhydroxyanisole, tocopherol, propyl gallate, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), polyethylenimine, vitamin E, and mixtures thereof. In certain embodiments, the preservative is methionine. In certain embodiments, the preservative is butyl hydroxy toluene. In certain embodiments, the preservative is butyl hydroxy anisole. In certain embodiments, the preservative is tocopherol. In certain embodiments, the preservative is propyl gallate. In certain embodiments, the preservative is ethylenediamine tetraacetic acid. In certain embodiments, the preservative is polyethylenimine. In certain embodiments, the preservative is vitamin E.

The term "methionine" as defined herein is intended to encompass D-methionine and L-methionine and mixtures thereof. In certain embodiments, the term "methionine" refers to L-methionine. In certain embodiments, the term "methionine" refers to D-methionine. In certain embodiments, the term "methionine" refers to D-methionine or L-methionine in a mixture. In certain embodiments, the term "methionine" refers to L-methionine hydrochloride.

As defined herein, the term "EDTA" is intended to encompass all EDTA forms known in the art, such as EDTA salts, including EDTA metal salts, such as EDTA disodium salt, EDTA dipotassium salt, EDTA calcium salt, EDTA magnesium salt, or mixtures thereof. In certain embodiments, EDTA refers to EDTA disodium salt. In certain embodiments, the term "EDTA" refers to EDTA di-calcium salt. In certain embodiments, the term "EDTA" refers to anhydrous EDTA.

In certain embodiments, the molar ratio of antioxidant to CNP moiety is from about 0.1:1 to about 100:1. In certain embodiments, the molar ratio of antioxidant to CNP moiety is from about 0.1:1 to about 70:1. In certain embodiments, the molar ratio of antioxidant to CNP moiety is from about 0.1:1 to about 15:1. In certain embodiments, the molar ratio of antioxidant to CNP moiety is from about 1:1 to about 10:1. In certain embodiments, the molar ratio of antioxidant to CNP moiety is from about 3:1 to about 7:1.

In certain embodiments, the reconstituted solution does not contain an antimicrobial agent. In certain embodiments, the reconstitution solution comprises one or more excipients.

In certain embodiments, the reconstitution solution is sterile water. In certain embodiments, the reconstitution solution is sterile water containing 0.7-1.1% benzyl alcohol. In certain embodiments, the reconstitution solution is sterile water containing 0.9% benzyl alcohol.

In certain embodiments, the reconstitution solution comprises a pH adjusting agent.

As used herein, the term "pH adjuster" refers to a compound that is used to adjust the pH of a reconstituted solution.

In certain embodiments, the pH adjuster may be an acid or an acidic salt thereof. The acid may be selected from: acetic acid, citric acid, succinic acid, hydrochloric acid, phosphoric acid, carbonic acid, nitric acid, and mixtures thereof.

In certain embodiments, the pH adjuster may be a base or an alkaline salt thereof. The base may be selected from the group consisting of: tris (hydroxymethyl) aminomethane), sodium hydroxide, potassium hydroxide, lysine, and mixtures thereof.

In certain embodiments, the volume of the reconstitution solution ranges from about 0.1ml to about 4ml. In certain embodiments, the volume of the reconstitution solution is about 1ml, such as about 2ml, such as about 3ml, or such as about 4ml.

In certain embodiments, the volume of reconstitution solution is about 0.79ml. In certain embodiments, the volume of reconstitution solution is 0.79ml. In certain embodiments, the volume of the reconstitution solution is about 1ml. In certain embodiments, the volume of reconstitution solution is 1ml. In certain embodiments, the volume of the reconstitution solution is about 1.1ml. In certain embodiments, the volume of the reconstitution solution is 1.1ml. In certain embodiments, the volume of the reconstitution solution is about 1.25ml. In certain embodiments, the volume of the reconstitution solution is 1.25ml.

It is understood that the volume of a unit dose or injection is based on the actual weight of the patient and the concentration of the formulated solution. In certain embodiments, the concentration of CNP in the reconstituted solution does not exceed 7mg/ml. In certain embodiments, the concentration of CNP in the reconstituted solution is not less than 0.5mg/ml. In certain embodiments, the concentration of CNP in the reconstituted solution is 0.75mg/ml. In certain embodiments, the concentration of CNP in the reconstituted solution is 1mg/ml. In certain embodiments, the concentration of CNP in the reconstituted solution is 2.2mg/ml. In certain embodiments, the concentration of CNP in the reconstituted solution is 3.6mg/ml. In certain embodiments, the concentration of CNP in the reconstituted solution is 4.6mg/ml. In certain embodiments, the concentration of CNP in the reconstituted solution is 5mg/ml. In certain embodiments, the concentration of CNP in the reconstituted solution is 5.5mg/ml.

After reconstitution, the unit dose volume is no more than 4ml. In certain embodiments, the unit dose ranges in volume from about 0.01ml to about 1.1ml. In certain embodiments, the unit dose volume ranges from 0.01 to 0.75ml. In certain embodiments, the unit dose volume ranges from 0.01 to 0.50ml.

In certain embodiments, the unit dose volume is about 0.03ml. In certain embodiments, the unit dose volume is about 0.05ml. In certain embodiments, the unit dose volume is about 0.1ml. In certain embodiments, the unit dose volume is about 0.2ml. In certain embodiments, the unit dose volume is about 0.25ml. In certain embodiments, the unit dose volume is about 0.3ml. In certain embodiments, the unit dose volume is about 0.35ml. In certain embodiments, the unit dose volume is about 0.4ml. In certain embodiments, the unit dose volume is about 0.5ml. In certain embodiments, the unit dose volume is about 0.6ml. In certain embodiments, the unit dose volume is about 0.75ml. In certain embodiments, the unit dose volume is about 1ml.

In certain embodiments, the patient is an infant and the unit dose volume ranges from about 10 to 100 μl. In certain embodiments, the patient is an infant and the unit dose volume ranges from about 10 to 50 μl. In certain embodiments, the patient is an infant and the unit dose volume ranges from about 10 to 30 μl.

In certain embodiments, the patient is an infant and the unit dose is about 10 μl in volume. In certain embodiments, the patient is an infant and the unit dose volume is about 15 μl. In certain embodiments, the patient is an infant and the unit dose volume is about 20 μl.

In certain embodiments, the patient is an infant and the unit dose is 10 μl in volume. In certain embodiments, the patient is an infant and the unit dose is 15 μl in volume. In certain embodiments, the patient is an infant and the unit dose is 20 μl in volume.

In certain embodiments, the patient is an infant, the unit dose is 20 μg CNP/kg and the unit dose has a volume of about 10 μl. In certain embodiments, the patient is an infant, the unit dose is 20 μ gCNP/kg and the unit dose volume is about 15 μl. In certain embodiments, the patient is an infant, the unit dose is 20 μg CNP/kg and the unit dose has a volume of about 20 μl.

In certain embodiments, the unit dose is 6 μg CNP/kg and the volume of the unit dose is 0.06ml. In certain embodiments, the unit dose is 20 μg CNP/kg and the volume of the unit dose is 0.3ml. In certain embodiments, the unit dose is 50 μg CNP/kg and the volume of the unit dose is 0.2ml. In certain embodiments, the unit dose is 75 μg CNP/kg and the volume of the unit dose is 0.4ml. In certain embodiments, the unit dose is 100 μg CNP/kg and the volume of the unit dose is 0.5ml. In certain embodiments, the unit dose is 150 μg CNP/kg and the volume of the unit dose is 0.5ml.

In certain embodiments, the pH of the liquid unit dosage form is from about pH4 to about pH6. In certain embodiments, the pH of the liquid unit dosage form is from about pH4.5 to about pH5.5. In certain embodiments, the pH of the liquid unit dosage form is about 5. In certain embodiments, the pH of the liquid unit dosage form is 5.

In certain embodiments, the unit dosage form of the present invention further comprises a buffer, an isotonic agent, and a pH adjusting agent.

In certain embodiments, the concentration of buffer in the unit dosage form ranges from 1.3 to 57.6mM. In certain embodiments, the buffer is present in the unit dosage form at a concentration ranging from 1.7 to 33mM. In certain embodiments, the concentration of buffer in the unit dosage form ranges from 5.1 to 20.3mM. In certain embodiments, the buffer is present in the unit dosage form at a concentration of about 10mM.

Exemplary buffers may be selected from the group consisting of: succinic acid, citric acid, lactic acid, acetic acid, glutamic acid, fumaric acid, aspartic acid, glutaric acid, phosphoric acid, histidine, gluconic acid, tartaric acid, malic acid, and mixtures thereof. It will be clear to those skilled in the art that the corresponding conjugate base or salt of the buffer may also be included, such as succinate, citrate, lactate, acetate, glutamate, fumarate, aspartate, glutarate, phosphate, gluconate, tartrate, malate, and mixtures thereof.

In certain embodiments, the buffer is succinic acid. In certain embodiments, the buffer is citric acid. In certain embodiments, the buffer is lactic acid. In certain embodiments, the buffer is acetic acid. In certain embodiments, the buffer is glutamic acid. In certain embodiments, the buffer is fumaric acid. In certain embodiments, the buffer is aspartic acid. In certain embodiments, the buffer is glutaric acid. In certain embodiments, the buffer is phosphoric acid. In certain embodiments, the buffer is histidine. In certain embodiments, the buffer is gluconic acid. In certain embodiments, the buffer is tartaric acid. In certain embodiments, the buffer is malic acid.

The isotonic agent may be selected from the group consisting of: trehalose, mannitol, sucrose, raffinose, gelatin, lactose, dibasic calcium phosphate, sorbitol, xylitol, glycine, histidine, hydroxyethyl starch, glucose, dextran,Propylene glycol and mixtures thereof.

In certain embodiments, the isotonic agent may be selected from the group consisting of: trehalose, mannitol, sucrose, raffinose, gelatin, lactose, dibasic calcium phosphate, sorbitol, xylitol, glycine, histidine, hydroxyethyl starch, glucose, dextran, propylene glycol and mixtures thereof.

In certain embodiments, the isotonic agent is selected from the group consisting of: trehalose, sucrose and glycine. In certain embodiments, the isotonic agent is a non-reducing sugar, such as trehalose or sucrose.

In certain embodiments, the isotonic agent is trehalose.

As defined herein, the term "trehalose" is intended to encompass all salts and hydrated states of trehalose, such as anhydrous trehalose or trehalose dihydrate. In certain embodiments, the term "trehalose" refers to anhydrous trehalose. In certain embodiments, the term "trehalose" refers to trehalose dihydrate.

In certain embodiments, the unit dosage form comprises succinic acid and trehalose.

In certain embodiments, the unit dosage form comprises:

CNP conjugate 0.9-82.1mg/ml

Succinic acid 1.3-57.6mM

Trehalose dihydrate 67-111.6mg/ml,

And the pH value ranges from pH4.0 to pH6.0.

In certain embodiments, the unit dosage form comprises:

CNP conjugate 19.8-73.6mg/ml

Succinic acid 1.7-50mM

Trehalose dihydrate 63-100mg/ml,

And the pH value ranges from pH4.0 to pH6.0.

In certain embodiments, the unit dosage form comprises:

CNP conjugate 27.5-50.5mg/ml

Succinic acid 5.1-20.3mM

Trehalose dihydrate 67-95mg/ml,

And the pH value ranges from pH4.0 to pH6.0.

In certain embodiments, the unit dosage form comprises about 8.2mg/ml CNP conjugate, about 10mM succinic acid, about 89mg/ml trehalose dihydrate, and optionally Tris and/or hydrochloric acid, and has a pH of about 5.

In certain embodiments, the unit dosage form comprises 8.2mg/ml CNP conjugate, 10mM succinic acid, 89mg/ml trehalose dihydrate and optionally Tris and/or hydrochloric acid, and has a pH of 5.

In certain embodiments, the unit dosage form comprises about 11mg/ml CNP conjugate, about 10mM succinic acid, about 88.5mg/ml trehalose dihydrate and optionally Tris and/or hydrochloric acid, and has a pH of about 5.

In certain embodiments, the unit dosage form comprises 11mg/ml CNP conjugate, 10mM succinic acid, 88.5mg/ml trehalose dihydrate and optionally Tris and/or hydrochloric acid, and has a pH of 5.

In certain embodiments, the unit dosage form comprises about 24.2mg/ml CNP conjugate, about 10mM succinic acid, about 85mg/ml trehalose dihydrate, and optionally Tris and/or hydrochloric acid, and has a pH of about 5.

In certain embodiments, the unit dosage form comprises 24.2mg/ml CNP conjugate, 10mM succinic acid, 85mg/ml trehalose dihydrate and optionally Tris and/or hydrochloric acid, and has a pH of 5.

In certain embodiments, the unit dosage form comprises about 39.6mg/ml CNP conjugate, about 10mM succinic acid, about 80mg/ml trehalose dihydrate, and optionally Tris and/or hydrochloric acid, and has a pH of about 5.

In certain embodiments, the unit dosage form comprises 39.6mg/ml CNP conjugate, 10mM succinic acid, 80mg/ml trehalose dihydrate and optionally Tris and/or hydrochloric acid, and has a pH of 5.

In certain embodiments, the unit dosage form comprises about 50.5mg/ml CNP conjugate, about 10mM succinic acid, about 77mg/ml trehalose dihydrate, and optionally Tris and/or hydrochloric acid, and has a pH of about 5.

In certain embodiments, the unit dosage form comprises 50.5mg/ml CNP conjugate, 10mM succinic acid, 77mg/ml trehalose dihydrate and optionally Tris and/or hydrochloric acid, and has a pH of 5.

In certain embodiments, the unit dosage form comprises about 54.9mg/ml CNP conjugate, about 10mM succinic acid, about 75mg/ml trehalose dihydrate, and optionally Tris and/or hydrochloric acid, and has a pH of about 5.

In certain embodiments, the unit dosage form comprises 54.9mg/ml CNP conjugate, 10mM succinic acid, 75mg/ml trehalose dihydrate, and optionally Tris and/or hydrochloric acid, and has a pH of about 5.

In certain embodiments, the unit dosage form comprises about 60.4mg/ml CNP conjugate, about 10mM succinic acid, about 73mg/ml trehalose dihydrate, and optionally Tris and/or hydrochloric acid, and has a pH of about 5.

In certain embodiments, the unit dosage form comprises 60.4mg/ml CNP conjugate, 10mM succinic acid, 73mg/ml trehalose dihydrate and optionally Tris and/or hydrochloric acid, and has a pH of 5.

In certain embodiments, the unit dosage form comprises, based on the total weight of the solid unit dosage form:

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): about 8.2% (w/w) CNP conjugate, about 1.2% (w/w) succinic acid, about 89.1% (w/w) trehalose dihydrate, and about 1.5% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): 8.2% (w/w) CNP conjugate, 1.2% (w/w) succinic acid, 89.1% (w/w) trehalose dihydrate and 1.5% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): about 10.7% (w/w) CNP conjugate, about 1.2% (w/w) succinic acid, about 86.8% (w/w) trehalose dihydrate, and about 1.3% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): 10.7% (w/w) CNP conjugate, 1.2% (w/w) succinic acid, 86.8% (w/w) trehalose dihydrate and 1.3% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): about 21.6% (w/w) CNP conjugate, about 1.1% (w/w) succinic acid, about 76.1% (w/w) trehalose dihydrate, and about 1.2% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): 21.6% (w/w) CNP conjugate, 1.1% (w/w) succinic acid, 76.1% (w/w) trehalose dihydrate and 1.2% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): about 32.4% (w/w) CNP conjugate, about 1.0% (w/w) succinic acid, about 65.4% (w/w) trehalose dihydrate, and about 1.2% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): 32.4% (w/w) CNP conjugate, 1.0% (w/w) succinic acid, 65.4% (w/w) trehalose dihydrate and 1.2% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): about 38.9% (w/w) CNP conjugate, about 0.9% (w/w) succinic acid, about 59.2% (w/w) trehalose dihydrate, and about 1% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): 38.9% (w/w) CNP conjugate, 0.9% (w/w) succinic acid, 59.2% (w/w) trehalose dihydrate and 1% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): about 41.5% (w/w) CNP conjugate, about 0.9% (w/w) succinic acid, about 56.6% (w/w) trehalose dihydrate, and about 1% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): 41.5% (w/w) CNP conjugate, 0.9% (w/w) succinic acid, 56.6% (w/w) trehalose dihydrate and 1% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): about 44.4% (w/w) CNP conjugate, about 0.9% (w/w) succinic acid, about 53.7% (w/w) trehalose dihydrate, and about 1% (w/w) Tris.

In certain embodiments, the unit dosage form comprises (based on the total weight of the solid unit dosage form): 44.4% (w/w) CNP conjugate, 0.9% (w/w) succinic acid, 53.7% (w/w) trehalose dihydrate and 1% (w/w) Tris.

The applicant has surprisingly found that when the unit dosage form of the invention is administered to a patient in need thereof, the incidence of hypotension is less than 10%, preferably less than 8%, most preferably less than 5%, even more preferably less than 3%. In certain embodiments, the incidence of hypotension is less than 1% after administration of the unit dosage form of the invention to a patient in need thereof. In certain embodiments, no hypotension occurs.

Furthermore, surprisingly, it was found that no therapeutic presence of anti-CNP antibodies was detected when treated with the conjugates of the invention. In certain embodiments, no anti-CNP binding antibodies are detected after repeated weekly exposure to the conjugates of the invention for 1-9 months. In certain embodiments, no anti-CNP binding antibodies were detected after 52 weeks of repeated weekly exposure to the conjugates of the invention.

Furthermore, it has surprisingly been found that administering 100 μg CNP/kg weekly to a pediatric patient in need of CNP treatment, aged 2-10, such as 2-5, or such as 5-10, produces a similar response measured in terms of annual growth rate.

In certain embodiments, the CNP moiety of the CNP conjugate has the sequence of SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25 or SEQ ID NO: 30. In certain embodiments, the CNP moiety has the sequence of SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24 or SEQ ID NO. 25. In certain embodiments, the CNP moiety has the sequence of SEQ ID NO. 20. In certain embodiments, the CNP moiety has the sequence of SEQ ID NO. 21. In certain embodiments, the CNP moiety has the sequence of SEQ ID NO. 22. In certain embodiments, the CNP moiety has the sequence of SEQ ID NO. 23. In certain embodiments, the CNP moiety has the sequence of SEQ ID NO. 24. In certain embodiments, the CNP moiety has the sequence of SEQ ID NO. 25.

In certain embodiments, the CNP conjugate has the following formula (Ia) or (Ib):

Wherein the method comprises the steps of

-D is a CNP moiety;

-L ¹ -is a reversible linker moiety;

-L ² -is a single chemical bond or a spacer moiety;

-Z is a polymeric moiety;

x is an integer selected from the group consisting of: 1.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16; and

Y is an integer selected from the group consisting of: 1.2, 3, 4 and 5.

-D in formula (Ia) or (Ib) is covalently and reversibly conjugated to-L ¹ -.

In certain embodiments, x in formula (Ia) is an integer selected from the group consisting of: 1.2, 3, 4, 6 and 8. In certain embodiments, x in formula (Ia) is an integer selected from the group consisting of: 1.2, 4 and 6. In certain embodiments, x in formula (Ia) is an integer selected from the group consisting of: 1. 4 and 6, and in certain embodiments, x in formula (Ia) is 1.

In certain embodiments, y in formula (Ib) is an integer selected from the group consisting of: 2. 3,4 and 5. In certain embodiments, y in formula (Ib) is an integer selected from the group consisting of: 2. 3 and 4. In certain embodiments, y in formula (Ib) is an integer selected from the group consisting of: 2 and 3. In certain embodiments, y in formula (Ib) is an integer selected from the group consisting of: 1. 2 and 3. In certain embodiments, y of formula (Ib) is 1. In certain embodiments, y of formula (Ib) is 2.

In certain embodiments, the CNP conjugate has formula (Ia), and x=1.

In certain embodiments, the-D of formula (Ia) or (Ib) has the sequence of SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25 or SEQ ID NO: 30. In certain embodiments, -D of formula (Ia) or (Ib) has the sequence of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO: 25.

In certain embodiments, -D of formula (Ia) or (Ib) has the sequence of SEQ ID NO: 20. In certain embodiments, -D of formula (Ia) or (Ib) has the sequence of SEQ ID NO: 21. In certain embodiments, -D of formula (Ia) or (Ib) has the sequence of SEQ ID NO: 22. In certain embodiments, -D of formula (Ia) or (Ib) has the sequence of SEQ ID NO: 23. In certain embodiments, -D of formula (Ia) or (Ib) has the sequence of SEQ ID NO: 24. In certain embodiments, -D of formula (Ia) or (Ib) has the sequence of SEQ ID NO: 25.

The-L ¹ -moiety in formula (Ia) or (Ib) is conjugated to any one of the following: -the functional group of the amino acid residue side chain of D, -the N-terminal amine functional group or C-terminal carboxyl functional group of D, or-the nitrogen atom in the main chain polypeptide chain of D. The attachment to the N-terminal or C-terminal end, respectively, can be carried out directly or indirectly via the corresponding amine or carboxyl functional group, wherein the spacer moiety is first conjugated to the amine or carboxyl functional group of the spacer moiety-L ¹ -conjugate.

The-L ¹ -moiety in formula (Ia) or (Ib) is a reversible linker, whereby the drug (i.e.D-H) is released in free form, i.e.L ¹ -is a traceless linker. Suitable reversible linkers are known to those skilled in the art, as for example the reversible linker moieties disclosed in WO2005/099768A2, WO2006/136586A2, WO2011/089216Al and WO2013/024053Al (which are incorporated herein by reference).

In certain embodiments, -L ¹ -is a reversible linker as described in WO 2011/012720 Al, WO2011/089214Al, WO2011/089215Al, WO2013/024052Al and WO2013/160340Al (which are incorporated herein by reference).

The moiety-L ¹ -may be attached to-D by any type of attachment, provided that it is reversible. In certain embodiments, -L ¹ -is attached to-D by a linkage selected from the group consisting of: amides, esters, carbamates, acetals, aminals, imines, oximes, hydrazones, disulfides and acylguanidines. In certain embodiments, -L ¹ -is attached to-D by a linkage selected from the group consisting of: amides, esters, carbamates and acylguanidines. It will be appreciated that these linkages may not be reversible in themselves, but that the inclusion of adjacent groups in-L ¹ -may render the linkages reversible.

In certain embodiments, the moiety-L ¹ -is attached to-D through an amide bond.

Part-L ¹ -is disclosed in WO2009/095479A 2. Thus, in certain embodiments, the moiety-L ¹ -has the following formula (II):

wherein the dashed line represents the nitrogen attached to-D (which is a CNP moiety) by the formation of an amide bond;

-X-is -C(R⁴R^4a)-;-N(R⁴)-;-O-;-C(R⁴R^4a)-C(R⁵R^5a)-;-C(R⁵R^5a)-C(R⁴R^4a)-;-C(R⁴R^4a)-N(R⁶)-;-N(R⁶)-C(R⁴R^4a)-;-C(R⁴R^4a)-O-;-O-C(R⁴R^4a)-; or-C (R ⁷R^7a) -;

x ¹ is C; or S (O);

-X ² -is-C (R ⁸R^8a) -; or-C (R ⁸R^8a)-C(R⁹R^9a) -;

X ³ is =o; =s; or = N-CN;

-R¹、-R^1a、-R²、-R^2a、-R⁴、-R^4a、-R⁵、-R^5a、-R⁶、-R⁸、-R^8a、-R⁹、-R^9a Independently selected from the group consisting of: -H and C _1-6 alkyl;

-R ³、-R^3a is independently selected from the group consisting of: -H and C _1-6 alkyl, provided that if one or both of-R ³、-R^3a are not-H, they are attached to the N to which they are attached through an sp ³ -hybridized carbon atom;

-R ⁷ is-N (R ¹⁰R^10a); or-NR ¹⁰-(C＝O)-R¹¹;

-R ^7a、-R¹⁰、-R^10a、-R¹¹ is independently from each other-H or C _1-6 alkyl;

Optionally one or more of the ,-R^1a/-R^4a、-R^1a/-R^5a、-R^1a/-R^7a、-R^4a/-R^5a、-R^8a/-R^9a pairs form a chemical bond;

optionally one or more of the ,-R¹/-R^1a、-R²/-R^2a、-R⁴/-R^4a、-R⁵/-R^5a、-R⁸/-R^8a、-R⁹/-R^9a pairs are linked together with the atom to which they are attached to form a C _3-10 cycloalkyl group; or a 3-to 10-membered heterocyclyl group;

optionally one or more of the pairs ,-R¹/-R⁴、-R¹/-R⁵、-R¹/-R⁶、-R¹/-R^7a、-R⁴/-R⁵、-R⁴/-R⁶、-R⁸/-R⁹ and-R ²/-R³ are linked together with the atoms to which they are attached to form ring a;

optionally, -R ³/-R^3a together with the nitrogen atom to which they are attached form a 3-10 membered heterocyclic ring;

a is selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclic group; and

Wherein-L ¹ -is substituted by-L ² -Z, and wherein-L ¹ -is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (II) is not substituted by-L ² -Z or a substituent;

Wherein the method comprises the steps of

-L ² -is a single chemical bond or a spacer; and

-Z is a water soluble polymer moiety;

In certain embodiments, -L ¹ -in formula (II) is substituted with a moiety, -L ² -Z. In certain embodiments, -L ¹ -in formula (II) is not further substituted.

It will be appreciated that if-R ³/-R^3a in formula (II) is taken together with the nitrogen atom to which they are attached to form a 3-10 membered heterocyclic ring, only such 3-10 membered heterocyclic ring is possible to form, wherein the atom directly attached to the nitrogen is an sp ³ -hybridized carbon atom. In other words, the 3-10 membered heterocyclic ring formed by-R ³/-R^3a together with the nitrogen atom to which they are attached has the structure:

Wherein the method comprises the steps of

The dashed line represents the remainder attached to-L ¹ -;

the ring contains 3 to 10 atoms, which contains at least one nitrogen; and

R ^# and R ^## represent sp ³ -hybridized carbon atoms.

It is also understood that 3-10 membered heterocycles may be further substituted.

Exemplary embodiments of suitable 3-10 membered heterocycles formed by-R ³/-R^3a of formula (II) together with the nitrogen atom to which they are attached are as follows:

Wherein the method comprises the steps of

The dotted line indicates the attachment to the remainder of the molecule; and

-R is selected from the group consisting of: -H and C _1-6 alkyl.

-L ¹ -in formula (II) may optionally be further substituted. In general, any substituent may be used as long as the cleavage principle is not affected, i.e., the hydrogen marked with an asterisk in formula (II) is not substituted, and the following moiety of formula (II)

The nitrogen of (c) is still part of a primary, secondary or tertiary amine, i.e. -R ³ and-R ^3a are independently of each other-H or are linked to-N <, via an sp ³ -hybridised carbon atom.

In certain embodiments, -R ¹ or-R ^1a in formula (II) is substituted with-L ² -Z. In certain embodiments, -R ² or-R ^2a in formula (II) is substituted with-L ² -Z. In certain embodiments, -R ³ or-R ^3a in formula (II) is substituted with-L ² -Z. In certain embodiments, -R ⁴ in formula (II) is substituted with-L ² -Z. In certain embodiments, -R ⁵ or-R ^5a in formula (II) is substituted with-L ² -Z. In certain embodiments, -R ⁶ in formula (II) is substituted with-L ² -Z. in certain embodiments, -R ⁷ or-R ^7a in formula (II) is substituted with-L ² -Z. In certain embodiments, -R ⁸ or-R ^8a in formula (II) is substituted with-L ² -Z. In certain embodiments, -R ⁹ or-R ^9a in formula (II) is substituted with-L ² -Z.

In certain embodiments, -R ⁴ of formula (II) is substituted with-L ² -Z.

In some embodiments of the present invention, in some embodiments, -X-in formula (II) is-C (R ⁴R^4a) -or-N (R ⁴) -. In certain embodiments, the-X-in formula (II) is-C (R ⁴R^4a) -.

In certain embodiments, X ¹ of formula (II) is C.

In certain embodiments, = X ³ of formula (II) is =o.

In certain embodiments, -X ² -in formula (II) is-C (R ⁸R^8a) -.

In certain embodiments, -R ⁸ and-R ^8a of formula (II) are independently selected from the group consisting of: -H, methyl and ethyl. In certain embodiments, at least one of-R ⁸ and-R ^8a of formula (II) is-H. In certain embodiments, -R ⁸ and-R ^8a of formula (II) are both-H.

In certain embodiments, -R ¹ and-R ^1a of formula (II) are independently selected from the group consisting of: -H, methyl and ethyl. In certain embodiments, at least one of-R ¹ and-R ^1a of formula (II) is-H. In certain embodiments, -R ¹ and-R ^1a of formula (II) are both-H.

In certain embodiments, -R ² and-R ^2a of formula (II) are independently selected from the group consisting of: -H, methyl and ethyl. In certain embodiments, at least one of-R ² and-R ^2a of formula (II) is-H. In certain embodiments, -R ² and-R ^2a of formula (II) are both H.

In certain embodiments, -R ³ and-R ^3a of formula (II) are independently selected from the group consisting of: -H, methyl, ethyl, propyl and butyl. In certain embodiments, at least one of-R ³ and-R ^3a in formula (II) is methyl. In certain embodiments, both-R ³ and-R ^3a in formula (II) are-H. In certain embodiments, -R ³ and-R ^3a in formula (II) are both methyl. In certain embodiments, -R ³ of formula (II) is-H, and-R ^3a of formula (II) is methyl.

In certain embodiments, -R ⁴ and-R ^4a of formula (II) are independently selected from the group consisting of: -H, methyl and ethyl. In certain embodiments, at least one of-R ⁴ and-R ^4a in formula (II) is-H. In certain embodiments, both-R ⁴ and-R ^4a in formula (II) are-H.

In certain embodiments, moiety-L ¹ -has formula (IIa):

-R ¹、-R^1a、-R²、-R^2a、-R³、-R^3a、-R⁴、-R^4a and-X ² -are used as defined in formula (II); and wherein-L ¹ -is substituted by-L ² -Z, and wherein-L ¹ -is optionally further substituted, provided that the asterisked hydrogen in formula (IIa) is not substituted by-L ² -Z or a substituent.

In certain embodiments, -L ¹ -of formula (IIa) is substituted with a moiety, -L ² -Z. In certain embodiments, the-L ¹ -moiety of formula (IIa) is not further substituted.

In certain embodiments, -R ¹ and-R ^1a of formula (IIa) are independently selected from the group consisting of: -H, methyl and ethyl. In certain embodiments, at least one of-R ¹ and-R ^1a of formula (IIa) is-H. In certain embodiments, -R ¹ and-R ^1a of formula (IIa) are both-H.

In certain embodiments, -R ⁴ and-R ^4a of formula (IIa) are independently selected from: -H, methyl and ethyl. In certain embodiments, at least one of-R ⁴ and-R ^4a of formula (IIa) is-H. In certain embodiments, -R ⁴ and-R ^4a of formula (IIa) are both-H.

In certain embodiments, -X ² -of formula (IIa) is-C (R ⁸R^8a) -.

In certain embodiments, -R ⁸ and-R ^8a of formula (IIa) are independently selected from the group consisting of: -H, methyl and ethyl. In certain embodiments, at least one of-R ⁸ and-R ^8a of formula (IIa) is-H. In certain embodiments, -R ⁸ and-R ^8a of formula (IIa) are both-H.

In certain embodiments, -R ² and-R ^2a of formula (IIa) are independently selected from the group consisting of: -H, methyl and ethyl. In certain embodiments, at least one of-R ² and-R ^2a of formula (IIa) is-H. In certain embodiments, -R ² and-R ^2a of formula (IIa) are both H.

In certain embodiments, -R ³ and-R ^3a of formula (IIa) are independently selected from the group consisting of: -H, methyl, ethyl, propyl and butyl. In certain embodiments, at least one of-R ³ and-R ^3a of formula (IIa) is methyl. In certain embodiments, -R ³ and-R ^3a of formula (IIa) are both-H. In certain embodiments, -R ³ and-R ^3a of formula (IIa) are both methyl. In certain embodiments, -R ³ of formula (IIa) is-H, and-R ^3a of formula (IIa) is methyl.

In certain embodiments, moiety-L ¹ -has formula (IIb):

-R ²、-R^2a、-R³、-R^3a and-X ² -use as defined in formula (II); and

Wherein-L ¹ -is substituted by-L ² -Z, and wherein-L ¹ -is optionally further substituted, provided that the asterisked hydrogen in formula (IIb) is not substituted by-L ² -Z or a substituent.

In certain embodiments, -L ¹ -in formula (IIb) is substituted with a moiety, -L ² -Z. In certain embodiments, the moiety-L ¹ -in formula (IIb) is not further substituted.

In certain embodiments, -X ² -in formula (IIb) is-C (R ⁸R^8a) -.

In certain embodiments, -R ⁸ and-R ^8a of formula (IIb) are independently selected from the group consisting of: -H, methyl and ethyl. In certain embodiments, at least one of-R ⁸ and-R ^8a in formula (IIb) is-H. In certain embodiments, both-R ⁸ and-R ^8a in formula (IIb) are-H.

In certain embodiments, -R ² and-R ^2a of formula (IIb) are independently selected from the group consisting of: -H, methyl and ethyl. In certain embodiments, at least one of-R ² and-R ^2a in formula (IIb) is-H. In certain embodiments, -R ² and-R ^2a in formula (IIb) are both H.

In certain embodiments, -R ³ and-R ^3a of formula (IIb) are independently selected from: -H, methyl, ethyl, propyl and butyl. In certain embodiments, at least one of-R ³ and-R ^3a in formula (IIb) is methyl. In certain embodiments, both-R ³ and-R ^3a in formula (IIb) are-H. In certain embodiments, -R ³ and-R ^3a in formula (IIb) are both methyl. In certain embodiments, -R ³ in formula (IIb) is-H, and-R ^3a in formula (IIb) is methyl.

In certain embodiments, moiety-L ¹ -has formula (IIb'):

Wherein the method comprises the steps of

The dashed line represents the nitrogen attached to-D (which is the CNP moiety) by the formation of an amide bond;

the dotted line marked with an asterisk indicates the connection to-L ² -;

-R ²、-R^2a、-R³、-R^3a and-X ² -use as defined in formula (II); and is also provided with

Wherein-L ¹ -is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIb') is not substituted with a substituent.

In certain embodiments, the-L ¹ -moiety in formula (IIb') is not further substituted.

In certain embodiments, -X ² -of formula (IIb') is-C (R ⁸R^8a) -.

In certain embodiments, -R ⁸ and-R ^8a in formula (IIb') are independently selected from the group consisting of: -H, methyl and ethyl. In certain embodiments, at least one of-R ⁸ and-R ^8a in formula (IIb') is-H. In certain embodiments, both-R ⁸ and-R ^8a in formula (IIb') are-H.

In certain embodiments, -R ² and-R ^2a of formula (IIb') are independently selected from the group consisting of: -H, methyl and ethyl. In certain embodiments, at least one of-R ² and-R ^2a in formula (IIb') is-H. In certain embodiments, -R ² and-R ^2a in formula (IIb') are both H.

In certain embodiments, -R ³ and-R ^3a of formula (IIb') are independently selected from: -H, methyl, ethyl, propyl and butyl. In certain embodiments, at least one of-R ³ and-R ^3a in formula (IIb') is methyl. In certain embodiments, both-R ³ and-R ^3a in formula (IIb') are-H. In certain embodiments, -R ³ and-R ^3a in formula (IIb') are both methyl. In certain embodiments, -R ³ in formula (IIb ') is-H, and-R ^3a in formula (IIb') is methyl.

In certain embodiments, moiety-L ¹ -has the following formula (IIc):

wherein the dashed line represents the nitrogen attached to-D (which is a CNP moiety) by the formation of an amide bond; and

Wherein-L ¹ -is substituted by-L ² -Z, and wherein-L ¹ -is optionally further substituted, provided that the asterisked hydrogen in formula (IIc) is not substituted by-L ² -Z or a substituent.

In certain embodiments, -L ¹ -of formula (IIc) is substituted with a moiety, -L ² -Z. In certain embodiments, the-L ¹ -moiety of formula (IIc) is not further substituted.

In certain embodiments, moiety-L ¹ -is selected from the group consisting of: formulas (IIc-i), (IIc-ii), (IIc-iii), (IIc-iv) and (IIc-v):

Wherein the method comprises the steps of

The unlabeled dashed line represents the nitrogen attached to-D (which is the CNP moiety) by the formation of an amide bond; and

The dotted line marked with an asterisk indicates the connection to-L ² -Z; and

-L ¹ -is optionally further substituted, provided that the asterisk-marked hydrogen in formulae (IIc-i), (IIc-ii), (IIc-iii), (IIc-iv) and (IIc-v) is not substituted.

In certain embodiments, the-L ¹ -moiety in formulas (IIc-i), (IIc-ii), (IIc-iii), (IIc-iv), and (IIc-v) is not further substituted.

In certain embodiments, moiety-L ¹ -has the following formula (IIc-ii):

Wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the attachment to-L ² -Z.

In certain embodiments, -L ¹ -of formula (IIc-ii) is substituted with a moiety, -L ² -Z.

In certain embodiments, the optional further substituents of-L ¹ -of formula (II), (IIa), (Uh'), (IIc-a), (IIc-b), (IIc-i), (IIc-II), (IIc-iii), (IIc-iv), (IIc-v) are as described above.

Another part-L ¹ -is disclosed in WO2016/020373 Al. Thus, in certain embodiments, moiety-L ¹ -has the following formula (III):

Wherein the method comprises the steps of

The dashed lines represent primary or secondary amine, or hydroxyl groups, respectively, linked to-D (which is a CNP moiety) by amide or ester formation;

-R ¹、-R^1a、-R²、-R^2a、-R³ and-R ^3a are independently from each other selected from the following group ：-H、-C(R⁸R^8aR^8b)、-C(＝O)R⁸、-C≡N、-C(＝NR⁸)R^8a、-CR⁸(＝CR^8aR^8b)、-C≡CR⁸ and-T;

-R ⁴、-R⁵ and-R ^5a are independently of each other selected from the group: -H, -C (R ⁹R^9aR^9b) and-T;

a1 and a2 are independently 0 or 1;

-R⁶、-R^6a、-R⁷、-R^7a、-R⁸、-R^8a、-R^8b、-R⁹、-R^9a and-R ^9b are each independently selected from the group consisting of: -H, halogen 、-CN、-COOR¹⁰、-OR¹⁰、-C(O)R¹⁰、-C(O)N(R¹⁰R^10a)、-S(O)₂N(R¹⁰R^10a)、-S(O)N(R¹⁰R^10a)、-S(O)₂R¹⁰、-S(O)R¹⁰、-N(R¹⁰)S(O)₂N(R^10aR^10b)、-SR¹⁰、-N(R¹⁰R^10a)、-NO₂、-OC(O)R¹⁰、-N(R¹⁰)C(O)R^10a、-N(R¹⁰)S(O)₂R^10a、-N(R¹⁰)S(O)R^10a、-N(R¹⁰)C(O)OR^10a、-N(R¹⁰)C(O)N(R^10aR^10b)、-OC(O)N(R¹⁰R^10a)、-T、C_1-20 alkyl, C _2-20 alkenyl and C _2-20 alkynyl; wherein-T, C _1-20 alkyl, C _2-20 alkenyl and C _2-20 alkynyl may be optionally substituted with one or more of the same or different-R ¹¹, and wherein C _1-20 alkyl, C _2-20 alkenyl and C _2-20 alkynyl may be optionally interrupted by one or more groups selected from ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R¹²)-、-S(O)₂N(R¹²)-、-S(O)N(R¹²)-、-S(O)₂-、-S(O)-、-N(R¹²)S(O)₂N(R^12a)-、-S-、-N(R¹²)-、-OC(OR¹²)(R^12a)-、-N(R¹²)C(O)N(R^12a)- and-OC (O) N (R ¹²) -;

-R ¹⁰、-R^10a、-R^10b are each independently selected from the group consisting of: -H, -T, C _1-20 alkyl, C _2-20 alkenyl and C _2-20 alkynyl; wherein-T, C _1-20 alkyl, C _2-20 alkenyl and C _2-20 alkynyl may be optionally substituted with one or more of the same or different-R ¹¹, wherein C _1-20 alkyl, C _2-20 alkenyl and C _2-20 alkynyl may be optionally interrupted by one or more groups selected from ：-T-、-C(O)O-、-O-、-C(O)-、C(O)N(R¹²)-、-S(O)₂N(R¹²)-、-S(O)N(R¹²)-、-S(O)₂-、-S(O)-、-N(R¹²)S(O)₂N(R^12a)-、-S-、-N(R¹²)-、-OC(OR¹²)(R^12a)-、-N(R¹²)C(O)N(R^12a)- and-OC (O) N (R ¹²) -;

Each T is independently selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclic group; wherein each T is independently optionally substituted with one or more of the same or different-R ¹¹;

each-R ¹¹ is independently selected from: halogen, -CN, oxo (＝O)、-COOR¹³、-OR¹³、-C(O)R¹³、-C(O)N(R¹³R^13a)、-S(O)₂N(R¹³R^13a)、-S(O)N(R¹³R^13a)、-S(O)₂R¹³、-S(O)R¹³、-N(R¹³)S(O)₂N(R^13aR^13b)、-SR¹³、-N(R¹³R^13a)、-NO₂、-OC(O)R¹³、-N(R¹³)C(O)R^13a、-N(R¹³)S(O)₂R^13a、-N(R¹³)S(O)R^13a、-N(R¹³)C(O)OR^13a、-N(R¹³)C(O)N(R^13aR^13b)、-OC(O)N(R¹³R^13a) and C _1-6 alkyl; wherein C _1-6 alkyl is optionally substituted with one or more of the same or different halogen;

-R ¹²、-R^12a、-R¹³、-R^13a、-R^13b are each independently selected from the group consisting of: -H and C _1-6 alkyl; wherein C _1-6 alkyl is optionally substituted with one or more of the same or different halogen;

Optionally one or more of the ,-R¹/-R^1a、-R²/-R^2a、-R³/-R^3a、-R⁶/-R^6a、-R⁷/-R^7a pairs are linked together with the atom to which they are attached to form a C _3-10 cycloalkyl or 3-10 membered heterocyclyl;

Optionally, one or more of the following pairs are joined together with the atoms to which they are attached to form a ring A：-R¹/-R²、-R¹/-R³、-R¹/-R⁴、-R¹/-R⁵、-R¹/-R⁶、-R¹/-R⁷、-R²/-R³、-R²/-R⁴、-R²/-R⁵、-R²/-R⁶、-R²/-R⁷、-R³/-R⁴、-R³/-R⁵、-R³/-R⁶、-R³/-R⁷、-R⁴/-R⁵、-R⁴/-R⁶、-R⁴/-R⁷、-R⁵/-R⁶、-R⁵/-R⁷ and-R ⁶/-R⁷;

A is selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclic group;

wherein-L ¹ -is substituted with-L ² -Z, and wherein-L ¹ -is optionally further substituted;

Wherein the method comprises the steps of

-L ² -is a single chemical bond or a spacer; and

-Z is a water soluble polymer moiety.

In certain embodiments, the optional further substituents of-L ¹ -of formula (III) are as described above. In certain embodiments, -L ¹ -in formula (III) is substituted with a moiety, -L ² -Z. In certain embodiments, -L ¹ -in formula (III) is not further substituted.

Other embodiments of-L ¹ -are disclosed in EP1536334B1, W02009/009712A1, W02008/034122A1, WO2009/143412A2, WO2011/082368A2 and US8618124B2, which are incorporated herein by reference in their entirety.

Other embodiments of-L ¹ -are disclosed in US8946405B2 and US8754190B2, which are incorporated herein by reference in their entirety. Thus, moiety-L ¹ -has the following formula (IV):

Wherein the method comprises the steps of

The dashed line indicates the attachment to-D (which is the CNP moiety), and wherein the attachment is through a functional group of-D selected from the group consisting of: -OH, -SH, and-NH ₂;

m is 0 or l;

At least one or both of R ¹ and R ² are, independently of each other, selected from the group consisting of: -CN, -NO ₂, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -C (O) R ³、-S(O)R³、-S(O)₂R³ and-SR ⁴,

-One and only one of R ¹ and R ² is selected from the group consisting of: -H, optionally substituted alkyl, optionally substituted aralkyl and optionally substituted heteroaralkyl;

-R ³ is selected from the group consisting of: -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heteroaralkyl, -OR ⁹, and-N (R ⁹)₂;

-R ⁴ is selected from the group consisting of: optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heteroaralkyl;

each-R ⁵ is independently selected from the group consisting of: -H, optionally substituted alkyl, optionally substituted alkenyl alkyl, optionally substituted alkynyl alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl and optionally substituted heteroaralkyl;

-R ⁹ is selected from the group consisting of: -H and optionally substituted alkyl;

-Y-is absent, and-X-is-O-or-S-; or alternatively

-Y-is-N (Q) CH ₂ -, and-X-is-O-;

Q is selected from the group consisting of: optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heteroaralkyl;

Optionally, -R ¹ and-R ² may be linked to form a 3-8 membered ring; and

Optionally, -R ⁹ together with the nitrogen to which they are attached form a heterocycle;

Wherein the method comprises the steps of

-L ² -is a single chemical bond or a spacer; and

-Z is a water soluble polymer moiety.

In certain embodiments, the optional further substituents of-L ¹ -of formula (IV) are as described above. In certain embodiments, -L ¹ -of formula (IV) is substituted with a moiety, -L ² -Z. In certain embodiments, -L ¹ -of formula (IV) is not further substituted.

Only in the context of formula (IV), the terms used have the following meanings:

as used herein, the term "alkyl" includes straight, branched or cyclic saturated hydrocarbon groups containing from 1 to 8 carbon atoms, or in some embodiments, from 1 to 6 or from 1 to 4 carbon atoms.

The term "alkoxy" includes oxygen-bonded alkyl groups including methoxy, ethoxy, isopropoxy, cyclopropyloxy, cyclobutoxy, and the like.

The term "alkenyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon double bond.

The term "alkynyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon triple bond.

The term "aryl" includes aromatic hydrocarbon groups of 6 to 18 carbons, and in certain embodiments 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. The term "heteroaryl" includes aromatic rings containing 3 to 15 carbon atoms and containing at least one N, O or S atom; in certain embodiments, aromatic rings containing 3 to 7 carbon atoms and at least one N, O or S atom include groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, indenyl, and the like.

In certain embodiments, the alkenyl, alkynyl, aryl, or heteroaryl moiety may be coupled to the remainder of the molecule through an alkylene linkage. In these cases, the substituents will be referred to as alkenyl alkyl, alkynyl alkyl, arylalkyl or heteroarylalkyl, meaning that the alkylene moiety is located between the alkenyl, alkynyl, aryl or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl or heteroaryl moiety is coupled.

The term "halogen" includes bromine, fluorine, chlorine and iodine.

The term "heterocycle" refers to a 4-8 membered aromatic or non-aromatic ring containing 3-7 carbon atoms and at least one N, O or S atom. Examples are piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine and tetrahydrofuranyl, and the exemplary groups provided above for the term "heteroaryl".

When the ring system is optionally substituted, suitable substituents are selected from the group consisting of: alkyl, alkenyl, alkynyl or another ring, each substituent optionally being further substituted. Optional substituents on any group (including those described above) include halogen, nitro, cyano, -OR, -SR, -NR ₂、-OCOR、-NRCOR、-COOR、-CONR₂、-SOR、-SO₂R、-SONR₂, and-SO ₂NR₂, where each R is independently alkyl, alkenyl, alkynyl, aryl, OR heteroaryl, OR two R groups together with the atoms to which they are attached form a ring.

Other embodiments of-L ¹ -are disclosed in WO2013/036857A1, which is incorporated herein by reference in its entirety. Thus, in certain embodiments, moiety-L ¹ -has the following formula (V):

Wherein the method comprises the steps of

The dashed line represents the attachment to-D (which is the CNP moiety), and wherein the attachment is through the amine functionality of-D;

-R ¹ is selected from the group consisting of: optionally substituted C ₁-C₆ straight, branched or cyclic alkyl; optionally substituted aryl; optionally substituted heteroaryl; an alkoxy group; and-NR ⁵ ₂;

-R ² is selected from the group consisting of: -H; optionally substituted C ₁-C₆ alkyl; optionally substituted aryl; and optionally substituted heteroaryl;

-R ³ is selected from the group consisting of: -H; optionally substituted C ₁-C₆ alkyl; optionally substituted aryl; and optionally substituted heteroaryl;

-R ⁴ is selected from the group consisting of: -H; optionally substituted C ₁-C₆ alkyl; optionally substituted aryl; and optionally substituted heteroaryl;

each-R ⁵ is independently selected from the group consisting of: -H; optionally substituted C ₁-C₆ alkyl; optionally substituted aryl; and optionally substituted heteroaryl; or when two-R ⁵ are taken together, may be cycloalkyl or cycloheteroalkyl;

Wherein the method comprises the steps of

-L ² -is a single chemical bond or a spacer; and

-Z is a water-soluble polymer.

In certain embodiments, the optional further substituents of-L ¹ -of formula (V) are as described above.

In certain embodiments, -L ¹ -of formula (V) is substituted with a moiety, -L ² -Z.

In certain embodiments, -L ¹ -of formula (V) is not further substituted.

Only in the context of formula (V), the terms used have the following meanings:

"alkyl", "alkenyl" and "alkynyl" include straight, branched or cyclic hydrocarbon groups of 1 to 8 carbons, or 1 to 6 carbons, or 1 to 4 carbons, wherein alkyl is a saturated hydrocarbon, alkenyl contains one or more carbon-carbon double bonds, and alkynyl contains one or more carbon-carbon triple bonds. Unless otherwise indicated, these contain 1-6C.

"Aryl" includes aromatic hydrocarbon groups of 6 to 18 carbon atoms, and in certain embodiments 6 to 10 carbon atoms, including groups such as phenyl, naphthyl, and anthracene. "heteroaryl" includes aromatic rings containing 3 to 15 carbon atoms and containing at least one N, O or S atom, in certain embodiments 3 to 7 carbon atoms and containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, indenyl, and the like.

The term "substituted" means that the alkyl, alkenyl, alkynyl, aryl, or heteroaryl group contains one or more substituents in place of one or more hydrogen atoms. Substituents may generally be selected from: halogen, including F, cl, br and I; lower alkyl groups including straight, branched and cyclic alkyl groups; lower haloalkyl including fluoroalkyl, chloroalkyl, bromoalkyl, and iodoalkyl; OH; lower alkoxy including straight, branched and cyclic alkoxy; SH; lower alkylthio, including straight, branched and cyclic alkylthio; amino, alkylamino, dialkylamino, silyl, including alkylsilyl, alkoxysilyl and arylsilyl; a nitro group; cyano group; a carbonyl group; carboxylic acids, carboxylic acid esters, carboxylic acid amides, aminocarbonyl groups; an aminoacyl group; a carbamate; urea; a thiocarbamate; thiourea; a ketone; sulfone; sulfonamide; aryl groups including phenyl, naphthyl, and anthracenyl; heteroaryl, comprising: 5-membered heteroaryl groups including pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole and tetrazole, 6-membered heteroaryl groups including pyridine, pyrimidine and pyrazine, and fused heteroaryl groups including benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole and benzisothiazole.

Another embodiment of-L ¹ -is disclosed in WO2022/115563A1, which is incorporated herein by reference in its entirety. Thus, in certain embodiments, -L ¹ -has the following formula (Va):

Wherein the dotted line marked with an asterisk indicates the attachment to-L ² -Z and the non-marked dotted line indicates the attachment to-D.

In certain embodiments, -L ¹ -has formula (Va), the dotted line marked with an asterisk represents a linkage to-L ² -Z, and the unlabeled dotted line represents a linkage to-D, wherein-D is the CNP moiety of the amino acid sequence:

SEQ ID NO:97(CNP-38N6Q,N14Q):

LQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC，

wherein the cysteines at positions 22 and 38 are linked by a disulfide bridge; and

Wherein the linkage to-L ¹ -occurs at the N-terminus or ring of the peptide.

Another embodiment of-L ¹ -is disclosed in US7585837B2, which is incorporated herein by reference in its entirety. Thus, in certain embodiments, moiety-L ¹ -has the following formula (VI):

Wherein the method comprises the steps of

R ¹ and R ² are independently selected from the group consisting of: hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkylaryl, aralkyl, halogen, nitro, -SO ₃H、-SO₂NHR⁵, amino, ammonium, carboxyl, PO ₃H₂, and OPO ₃H₂;

R ³、R⁴ and R ⁵ are independently selected from the group consisting of: hydrogen, alkyl and aryl;

Wherein the method comprises the steps of

-L ² -is a single chemical bond or a spacer;

-Z is a water soluble polymer moiety.

Suitable substituents of formula (VI) are alkyl (e.g. C _1-6 alkyl), alkenyl (e.g. C _2-6 alkenyl), alkynyl (e.g. C _2-6 alkynyl), aryl (e.g. phenyl), heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl (e.g. an aromatic 4-7 membered heterocycle) or halogen moiety.

In certain embodiments, -L ¹ -of formula (VI) is substituted with a moiety, -L ² -Z. The optional other substituents of-L ¹ -of formula (VI) are as described above in certain embodiments.

In certain embodiments, -L ¹ -of formula (VI) is not further substituted.

Only in the context of formula (VI), the terms used have the following meanings:

The terms "alkyl", "alkoxy", "alkoxyalkyl", "aryl", "alkylaryl" and "aralkyl" refer to alkyl radicals having from 1 to 8 carbon atoms (in some embodiments, from 1 to 4 carbon atoms), such as methyl, ethyl, propyl, isopropyl and butyl, and aryl radicals having from 6 to 10 carbon atoms, such as phenyl and naphthyl. The term "halogen" includes bromine, fluorine, chlorine and iodine.

Another embodiment of-L ¹ -is disclosed in WO2002/089789A1, which is incorporated herein by reference in its entirety. Thus, moiety-L ¹ -has the following formula (VII):

Wherein the method comprises the steps of

L ₁ is a bifunctional linking group,

Y ₁ and Y ₂ are independently O, S or NR ⁷;

R ²、R³、R⁴、R⁵、R⁶ and R ⁷ are independently selected from the group consisting of: hydrogen, C _1-6 alkyl, C _3-12 branched alkyl, C _3-8 cycloalkyl, C _1-6 substituted alkyl, C _3-8 substituted cycloalkyl, aryl, substituted aryl, aralkyl, C _1-6 heteroalkyl, substituted C _1-6 heteroalkyl, C _1-6 alkoxy, phenoxy, and C _1-6 heteroalkoxy;

ar is a moiety which when included in formula (VII) forms a polysubstituted aromatic hydrocarbon or polysubstituted heterocyclyl;

X is a chemical bond or a moiety actively transported into the target cell, a hydrophobic moiety, or a combination thereof, y is 0 or 1;

Wherein the method comprises the steps of

-L ² -is a single chemical bond or a spacer; and

-Z is a water soluble polymer moiety.

In certain embodiments, -L ¹ -of formula (VII) is substituted by a moiety, -L ² -Z. In certain embodiments, the optional further substituents of-L ¹ -of formula (VII) are as described above.

In certain embodiments, -L ¹ -of formula (VII) is not further substituted.

Only in the context of formula (VII), the terms used have the following meanings:

The term "alkyl" is understood to include, for example, straight, branched, substituted C _1-12 alkyl groups, including alkoxy, C _3-8 cycloalkyl or substituted cycloalkyl groups, and the like.

The term "substituted" is understood to include the addition or substitution of one or more atoms contained in a functional group or compound with one or more different atoms.

Substituted alkyl groups include carboxyalkyl, aminoalkyl, dialkylamino, hydroxyalkyl, and mercaptoalkyl groups; substituted cycloalkyl groups include moieties such as 4-chlorocyclohexyl; aryl includes moieties such as naphthyl; substituted aryl groups include moieties such as 3-bromo-phenyl; aralkyl includes moieties such as tolyl; heteroalkyl groups include moieties such as ethyl thiophene; substituted heteroalkyl groups include moieties such as 3-methoxythiophene; alkoxy includes moieties such as methoxy; and phenoxy includes moieties such as 3-nitrophenoxy. Halogen is understood to include fluorine, chlorine, iodine and bromine.

In certain embodiments, -L ¹ -comprises a substructure of formula (VIII):

Wherein the method comprises the steps of

The dotted line marked with an asterisk represents the nitrogen (which is the CNP moiety) attached to-D by the formation of an amide bond;

the unlabeled dashed line represents the remainder attached to-L ¹ -; and

Wherein the method comprises the steps of

-L ² -is a single chemical bond or a spacer; and

-Z is a water soluble polymer moiety.

In certain embodiments, -L ¹ -of formula (VIII) is substituted with a moiety, -L ² -Z. The optional other substituents of-L ¹ -of formula (VIII) are as described above.

In certain embodiments, -L ¹ -of formula (VIII) is not further substituted.

In certain embodiments, -L ¹ -comprises a substructure of the following formula (IX):

Wherein the method comprises the steps of

The dotted line marked with an asterisk represents the nitrogen linked to-D (which is the CNP moiety) by the formation of a urethane linkage;

The unlabeled dashed line represents the remainder attached to-L ¹ -;

Wherein the method comprises the steps of

-L ² -is a single chemical bond or a spacer; and

-Z is a water soluble polymer moiety.

The optional other substituents of-L ¹ -of formula (IX) are as described above. In certain embodiments, -L ¹ -of formula (IX) is substituted with a moiety, -L ² -Z. In certain embodiments, -L ¹ -of formula (IX) is not further substituted.

The moiety-D may be attached to-L ¹ -through any of the functional groups of D-H and to-L ¹ -through the amine functional group of D-H. If CNP has the sequence of SEQ ID NO:24, this may be an N-terminal amine function or an amine function provided by a lysine side chain (i.e., by the lysines at positions 9, 11, 15, 16, 20 and 26).

The-L ¹ -ring attached to the CNP moiety significantly reduces the affinity of the CNP conjugate for NPR-B compared to the N-terminal or CNP acyclic moiety, which in turn reduces the risk of developing cardiovascular side effects (such as hypotension).

Thus, in certain embodiments, -L ¹ -is conjugated to the side chain of an amino acid residue of the-D loop moiety or the backbone of the-D loop moiety. In certain embodiments, -L ¹ -is covalently and reversibly conjugated to the side chain of the amino acid residue of the-D loop moiety. if-D is a CNP moiety having the sequence of SEQ ID NO:24, then in certain embodiments, -L ¹ -is conjugated to the amine functionality provided by lysine at position 26 of the corresponding drug D-H.

The-L ² -moiety is a chemical bond or a spacer moiety. In certain embodiments, -L ² -is a bond. In certain embodiments, -L ² -is a spacer moiety.

The moiety-L ² -may be attached to-L ¹ -by replacing any existing-H unless explicitly excluded.

When-L ² -is not a single bond, -L ² -is selected from the group ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R^y1)-、-S(O)₂N(R^y1)-、-S(O)N(R^y1)-、-S(O)₂-、-S(O)-、-N(R^y1)S(O)₂N(R^y1a)-、-S-、-N(R^y1)-、-OC(OR^y1)(R^y1a)-、-N(R^y1)C(O)N(R^y1a)-、-OC(O)N(R^y1)-、C_1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl; wherein-T-, C _1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl may be optionally substituted with one or more of the same or different-R ^y2, and wherein C _1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl may be optionally interrupted ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R^y3)-、-S(O)₂N(R^y3)-、-S(O)N(R^y3)-、-S(O)₂-、-S(O)-、-N(R^y3)S(O)₂N(R^y3a)-、-S-、-N(R^y3)-、-OC(OR^y3)(R^y3a)-、-N(R^y3)C(O)N(R^y3a)- and-OC (O) N (R ^y3) -, by one or more groups selected from the group consisting of;

-R ^y1 and-R ^y1a are independently of each other selected from the group: -H, -T, C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl; wherein-T, C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl may be optionally substituted with one or more of the same or different-R ^y2, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl may be optionally interrupted by one or more groups selected from ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R^y4)-、-S(O)₂N(R^y4)-、-S(O)N(R^y4)-、-S(O)₂-、-S(O)-、-N(R^y4)S(O)₂N(R^y4a)-、-S-、-N(R^y4)-、-OC(OR^y4)(R^y4a)-、-N(R^y4)C(O)N(R^y4a)- and-OC (O) N (R ^y4) -;

Each T is independently selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclic, 8-30 membered carbopolycyclic and 8-30 membered heteropolycyclic; wherein each T is independently optionally substituted with one or more of the same or different-R ^y2;

each-R ^y2 is independently selected from the group consisting of: halogen, -CN, oxo (＝O)、-COOR^y5、-OR^y5、-C(O)R^y5、-C(O)N(R^y5R^y5a)、-S(O)₂N(R^y5R^y5a)、-S(O)N(R^y5R^y5a)、-S(O)₂R^y5、-S(O)R^y5、-N(R^y5)S(O)₂N(R^y5aR^y5b)、-SR^y5、-N(R^y5R^y5a)、-NO₂、-OC(O)R^y5、-N(R^y5)C(O)R^y5a、-N(R^y5)S(O)₂R^y5a、-N(R^y5)S(O)R^y5a、-N(R^y5)C(O)OR^y5a、-N(R^y5)C(O)N(R^y5aR^y5b)、-OC(O)N(R^y5R^y5a) and C _1-6 alkyl; wherein C _1-6 alkyl is optionally substituted with one or more of the same or different halogen; and each of-R ^y3、-R^y3a、-R^y4、-R^y4a、-R^y5、-R^y5a and-R ^y5b is independently selected from the group consisting of: -H and C _1-6 alkyl, wherein C _1-6 alkyl is optionally substituted with one or more identical or different halogens.

When-L ² -is not a single bond, -L ² -is selected from the group ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R^y1)-、-S(O)₂N(R^y1)-、-S(O)N(R^y1)-、-S(O)₂-、-S(O)-、-N(R^y1)S(O)₂N(R^y1a)-、-S-、-N(R^y1)-、-OC(OR^y1)(R^y1a)-、-N(R^y1)C(O)N(R^y1a)-、-OC(O)N(R^y1)-、C_1-20 alkyl, C _2-20 alkenyl, and C _2-20 alkynyl; wherein-T-, C _1-20 alkyl, C _2-20 alkenyl, C _2-20 alkynyl may be optionally substituted with one or more of the same or different-R ^y2, wherein C _1-20 alkyl, C _2-20 alkenyl and C _2-20 alkynyl may be optionally interrupted by one or more groups selected from ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R^y3)-、-S(O)₂N(R^y3)-、-S(O)N(R^y3)-、-S(O)₂-、-S(O)-、-N(R^y3)S(O)₂N(R^y3a)-、-S-、-N(R^y3)-、-OC(OR^y3)(R^y3a)-、-N(R^y3)C(O)N(R^y3a)- and-OC (O) N (R ^y3) -;

-R ^y1 and-R ^y1a are independently of each other selected from the group: -H, -T, C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl; wherein-T, C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl are optionally substituted with one or more of the same or different-R ^y2, and wherein C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl are optionally interrupted by one or more groups selected from ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R^y4)-、-S(O)₂N(R^y4)-、-S(O)N(R^y4)-、-S(O)₂-、-S(O)-、-N(R^y4)S(O)₂N(R^y4a)-、-S-、-N(R^y4)-、-OC(OR^y4)(R^y4a)-、-N(R^y4)C(O)N(R^y4a)- and-OC (O) N (R ^y4) -;

Each T is independently selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclic, 8-30 membered carbopolycyclic, and 8-30 membered heteropolycyclic; wherein each T is independently optionally substituted with one or more of the same or different-R ^y2;

-R ^y2 is selected from the group consisting of: halogen, -CN, oxo (＝O)、-COOR^y5、-OR^y5、-C(O)R^y5、-C(O)N(R^y5R^y5a)、-S(O)₂N(R^y5R^y5a)、-S(O)N(R^y5R^y5a)、-S(O)₂R^y5、-S(O)R^y5、-N(R^y5)S(O)₂N(R^y5aR^y5b)、-SR^y5、-N(R^y5R^y5a)、-NO₂、-OC(O)R^y5、-N(R^y5)C(O)R^y5a、-N(R^y5)S(O)₂R^y5a、-N(R^y5)S(O)R^y5a、-N(R^y5)C(O)OR^y5a、-N(R^y5)C(O)N(R^y5aR^y5b)、-OC(O)N(R^y5R^y5a) and C _1-6 alkyl; wherein C _1-6 alkyl is optionally substituted with one or more of the same or different halogen; and

Each-R ^y3、-R^y3a、-R^y4、-R^y4a、-R^y5、-R^y5a and-R ^y5b is independently selected from the group consisting of: -H and C _1-6 alkyl; wherein the C _1-6 alkyl groups are optionally substituted with one or more of the same or different halogens.

When-L ² -is not a single bond, -L ² -is selected from the group ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R^y1)-、-S(O)₂N(R^y1)-、-S(O)N(R^y1)-、-S(O)₂-、-S(O)-、-N(R^y1)S(O)₂N(R^y1a)-、-S-、-N(R^y1)-、-OC(OR^y1)(R^y1a)-、-N(R^y1)C(O)N(R^y1a)-、-OC(O)N(R^y1)-、C_1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl; wherein-T-, C _1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl are optionally substituted with one or more of the same or different-R ^y2, and wherein C _1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl are optionally interrupted by ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R^y3)-、-S(O)₂N(R^y3)-、-S(O)N(R^y3)-、-S(O)₂-、-S(O)-、-N(R^y3)S(O)₂N(R^y3a)-、-S-、-N(R^y3)-、-OC(OR^y3)(R^y3a)-、-N(R^y3)C(O)N(R^y3a)- and-OC (O) N (R ^y3) -, with one or more groups selected from the group consisting of;

-R ^y1 and-R ^y1a are independently selected from the group consisting of: -H, -T, C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl;

Each T is independently selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclic, 8-30 membered carbopolycyclic and 8-30 membered heteropolycyclic;

each-R ^y2 is independently selected from the group consisting of: halogen and C _1-6 alkyl; and

In certain embodiments, -L ² -is a C _1-20 alkyl chain, optionally interrupted by one or more groups independently selected from the group consisting of: -O-, -T-and-C (O) N (R ^y1) -; and the C _1-20 alkyl chain is optionally substituted with one or more groups independently selected from the group consisting of: -OH, -T and-C (O) N (R ^y6R^y6a); wherein-R ^y1、-R^y6、-R^y6a is independently selected from the group consisting of: h and C _1-4 alkyl, and wherein T is selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclic, 8-30 membered carbopolycyclic and 8-30 membered heteropolycyclic.

In certain embodiments, -L ² -has a molecular weight in the range of 14-750g/mol.

In certain embodiments, -L ² -has a chain length of 1 to 20 atoms.

As used herein, with respect to the moiety-L ² -, the term "chain length" refers to the number of atoms of-L ² -present in the shortest connection between-L ¹ -and-Z.

In certain embodiments, -L ² -has the following formula (i):

Wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the connection to-L ¹ -;

the unlabeled dashed line represents a link to-Z;

-R ¹ is selected from the group consisting of: -H, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl;

n is selected from the group consisting of: 0.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18;

Wherein the moiety of formula (i) is optionally further substituted.

In certain embodiments, -R ¹ of formula (i) is selected from the group consisting of: -H, methyl, ethyl, propyl and butyl. In certain embodiments, -R ¹ of formula (i) is selected from the group consisting of: -H, methyl, ethyl and propyl. In certain embodiments, -R ¹ of formula (i) is selected from the group consisting of: from-H and methyl. In certain embodiments, -R ¹ of formula (i) is methyl.

In certain embodiments, n in formula (i) is selected from the group consisting of: 0.1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In certain embodiments, n in formula (i) is selected from the group consisting of: 0.1, 2, 3, 4 and 5. In certain embodiments, n in formula (i) is selected from: 0.1, 2 and 3. In certain embodiments, n in formula (i) is selected from: 0 and 1. In certain embodiments, n in formula (i) is 0.

In certain embodiments, -L ² -is a moiety selected from the group consisting of:

Wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the connection to-L ¹ -;

The unlabeled dashed line indicates the attachment to-Z, and

Wherein parts (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), (xi), (xii), (xiii), (xiv), (xv), (xvi) and (xvi) are optionally further substituted.

In certain embodiments, -L ² -is selected from the group consisting of:

Wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the connection to-L ¹ -; and

The unlabeled dashed line indicates the attachment to-Z.

In certain embodiments, -L ² -is selected from the group consisting of:

Wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the connection to-L ¹ -; and

The unlabeled dashed line indicates the attachment to-Z.

In certain embodiments, -L ² -has the formula (xvi):

Wherein the method comprises the steps of

The dotted line marked with an asterisk indicates the connection to-L ¹ -; and

The unlabeled dashed line indicates the attachment to-Z.

In certain embodiments, moiety-L ¹-L² -is selected from the group consisting of:

Wherein the method comprises the steps of

The dashed line marked with an asterisk indicates the attachment to-Z.

In certain embodiments, moiety-L ¹-L² -has the following formula (IId-ii):

Wherein the method comprises the steps of

The dashed line marked with an asterisk indicates the attachment to-Z.

In certain embodiments, moiety-L ¹-L² -has the formula (IId-ii'):

Wherein the method comprises the steps of

The dashed line marked with an asterisk indicates the attachment to-Z.

Wherein the method comprises the steps of

The dashed line marked with an asterisk indicates the attachment to-Z.

In certain embodiments, -Z of formula (Ia) or (Ib) has a molecular weight of from 5 to 200 kDa. In certain embodiments, -Z of formula (Ia) or (Ib) has a molecular weight of 8-100 kDa. In certain embodiments, -Z of formula (Ia) or (Ib) has a molecular weight of from 10 to 80 kDa. In certain embodiments, -Z of formula (Ia) or (Ib) has a molecular weight of from 12 to 60 kDa. In certain embodiments, -Z of formula (Ia) or (Ib) has a molecular weight of 15-40 kDa. In certain embodiments, the-Z of formula (Ia) or (Ib) has a molecular weight of about 20 kDa. In certain embodiments, the-Z of formula (Ia) or (Ib) has a molecular weight of about 40 kDa.

The polymer moiety-Z of formula (Ia) or (Ib) comprises a polymer. In certain embodiments, -Z in formula (Ia) or (Ib) comprises a polymer selected from the group consisting of: 2-methacryloyloxyethyl phosphorylcholine, poly (acrylic acid), poly (acrylic acid ester), poly (acrylamide), poly (alkoxy) polymer, poly (amide), poly (amidoamine), poly (amino acid), poly (anhydride), poly (asparagine), poly (butyric acid), poly (glycolic acid), poly (butylene terephthalate, poly (caprolactone), poly (carbonate), poly (cyanoacrylate), poly (dimethylacrylamide), poly (ester), poly (ethylene glycol), poly (ethylene oxide), poly (ethyl phosphate), poly (ethyl oxazoline), poly (glycolic acid), poly (hydroxyethyl acrylate), poly (hydroxyethyl oxazoline), poly (hydroxy methacrylate), poly (hydroxypropyl methacrylamide), poly (hydroxypropyl methacrylate), poly (iminocarbonate), poly (lactic acid-co-polymer), poly (methacrylamide), poly (methyl acrylate), poly (methyl oxazoline), poly (organophosphazene), poly (orthoester), poly (oxazoline), poly (polypropyleneglycol), poly (urethane), amino acid ester, polyvinyl alcohol, poly (vinylamine), poly (vinylmethyl ether), poly (vinylpyrrolidone), silicone, cellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannans, pectins, rhamnogalacturonan, starches, hydroxyalkyl starches, hydroxyethyl starch and other carbohydrate based polymers, xylans, and copolymers thereof.

In certain embodiments, -Z of formula (Ia) or (Ib) comprises a protein. Preferred proteins are selected from the group consisting of: the carboxy-terminal peptide of chorionic gonadotrophin, as described in US2012/0035101Al, which is incorporated herein by reference; albumin; XTEN sequences, as described in WO2011123813 A2, incorporated herein by reference; proline/alanine random coil sequence as described in WO2011/144756Al, which is incorporated herein by reference; proline/alanine/serine random coil sequences as described in WO2008/155134Al and WO 2013/024949 Al, which are incorporated herein by reference; and Fc fusion proteins.

In certain embodiments, -Z of formula (Ia) or (Ib) is a poly-sarcosine. In certain embodiments, -Z of formula (Ia) or (Ib) comprises poly (N-methylglycine). In certain embodiments, -Z of formula (Ia) or (Ib) comprises a random coil protein moiety. In certain embodiments, -Z of formula (Ia) or (Ib) comprises a random coil protein moiety. In certain embodiments, -Z of formula (Ia) or (Ib) comprises two random coil protein moieties. In certain embodiments, -Z of formula (Ia) or (Ib) comprises three random coil protein moieties. In certain embodiments, -Z of formula (Ia) or (Ib) comprises four random coil protein moieties. In certain embodiments, -Z of formula (Ia) or (Ib) comprises five random coil protein moieties. In certain embodiments, -Z of formula (Ia) or (Ib) comprises six random coil protein moieties. In certain embodiments, -Z of formula (Ia) or (Ib) comprises seven random coil protein moieties. In certain embodiments, -Z of formula (Ia) or (Ib) comprises eight random coil protein moieties.

In certain embodiments, such random coil protein moieties comprise at least 25 amino acid residues and up to 2000 amino acids. In certain embodiments, such random coil protein moieties comprise at least 30 amino acid residues and up to 1500 amino acid residues. In certain embodiments, such random coil protein moieties comprise at least 50 amino acid residues and up to 500 amino acid residues.

In certain embodiments, -Z of formula (Ia) or (Ib) comprises a fatty acid derivative. In certain embodiments, -Z in formula (Ia) or (Ib) is a fatty acid derivative. In certain embodiments, -Z in formula (Ia) is a fatty acid derivative, and x is 1.

In certain embodiments, -Z of formula (Ia) or (Ib) is a fatty acid derivative as disclosed in WO2006/097537A2 (which is incorporated herein by reference).

In certain embodiments, -Z of formula (Ia) or (Ib) includes fatty acid derivatives as disclosed in WO2021/055497Al (which is incorporated herein by reference). Thus, in certain embodiments, —z in formula (Ia) or (Ib) has the following structure (w):

Wherein the dotted line represents-L ² -or-L ¹ -attached to formula (Ia) or (Ib).

In certain embodiments, -Z has formula (w), and-L ¹ -has formula (V).

In certain embodiments, -Z-L ²-L¹ -has the following formula (w-a):

wherein the dashed line represents-D attached to formula (Ia) or (Ib).

In certain embodiments, the CNP has a sequence selected from the group consisting of:

PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO:98)；

PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO:30)；

PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 99); and

PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO:100)。

In certain embodiments, the CNP has a sequence selected from the group consisting of: SEQ ID NO. 98, SEQ ID NO. 30, SEQ ID NO. 99 and SEQ ID NO. 100, -Z has the formula (w), and-L ¹ -is a reversible linker moiety. In certain embodiments, the CNP has a sequence selected from the group consisting of: SEQ ID NO. 98, SEQ ID NO. 30, SEQ ID NO. 99 and SEQ ID NO. 100, -Z has the formula (w), and-L ¹ -has the formula (V). In certain embodiments, the CNP has a sequence selected from the group consisting of: SEQ ID NO. 98, SEQ ID NO. 30, SEQ ID NO. 99 and SEQ ID NO. 100, -Z-L ²-L¹ -has the formula (w-a). the-L ¹ -may be linked to the CNP via a lysine other than lysine within the ring structure, or may be linked to the N-terminus.

In certain embodiments, the CNPs of SEQ ID NO. 98, SEQ ID NO. 30, SEQ ID NO. 99 and SEQ ID NO. 100 further comprise an acetyl group, such as an acetyl group located at the N-terminus of the peptide. In certain embodiments, the CNPs of SEQ ID NO. 98, SEQ ID NO. 30, SEQ ID NO. 99 and SEQ ID NO. 100 further comprise a-OH or-NH ₂ group at the C-terminus. In certain embodiments, CNP and-L ¹ -of SEQ ID NO:98, SEQ ID NO:30, SEQ ID NO:99 and SEQ ID NO:100 are attached to residues of the CNP loop portion or to sites outside the CNP portion.

In certain embodiments, -L ¹ -is attached to a lysine residue, such as the lysine residue in bold in SEQ ID NO:98, SEQ ID NO:30, SEQ ID NO:99, and SEQ ID NO: 100:

PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO:98)；

PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO:30)；

PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 99); and

PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO:100)。

In certain embodiments, the CNP is selected from the group consisting of:

Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO:101)；

Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH₂(SEQ ID NO:102)；

Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO:103)；

Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH ₂ (SEQ ID NO: 104); and

Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH₂(SEQ ID NO:105)。

In certain embodiments, -Z in formula (Ia) or (Ib) is a hyaluronic acid-based polymer.

In certain embodiments, -Z of formula (Ia) or (Ib) is a polymer moiety as disclosed in WO2013/024047Al (which is incorporated herein by reference).

In certain embodiments, -Z of formula (Ia) or (Ib) is a polymer moiety as disclosed in WO2013/024048Al (which is incorporated herein by reference).

In certain embodiments, -Z in formula (Ia) or (Ib) is a PEG-based polymer. In certain embodiments, -Z is a branched or multi-arm PEG-based polymer.

In certain embodiments, -Z of formula (Ia) or (Ib) is a branched polymer. In certain embodiments, -Z in formula (Ia) or (Ib) is a branched polymer having one, two, three, four, five, or six branching points. In certain embodiments, -Z of formula (Ia) or (Ib) is a branched polymer having one, two, or three branching points. In certain embodiments, -Z of formula (Ia) or (Ib) is a branched polymer having one branching point. In certain embodiments, -Z of formula (Ia) or (Ib) is a branched polymer having two branching points. In certain embodiments, -Z of formula (Ia) or (Ib) is a branched polymer having three branching points. In certain embodiments, the branching point is selected from the group consisting of: -N <, -CH < and > C <.

In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) is PEG-based.

In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) has a molecular weight of 5-500kDa and includes 5kDa and 500kDa. In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) has a molecular weight of 10-250kDa and includes 10kDa and 250kDa. In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) has a molecular weight of 12-100kDa and includes 12kDa and 100kDa. In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) has a molecular weight of 15-80kDa and includes 15kDa and 80kDa. In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) has a molecular weight of 10-80kDa and includes 10kDa and 80kDa. In certain embodiments, the molecular weight is about 10kDa. In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) has a molecular weight of about 20kDa. In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) has a molecular weight of about 30kDa. In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) has a molecular weight of about 40kDa. In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) has a molecular weight of about 50kDa. In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) has a molecular weight of about 60kDa. In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) has a molecular weight of about 70kDa. In certain embodiments, such branched moiety-Z of formula (Ia) or (Ib) has a molecular weight of about 80kDa. In certain embodiments, such branching moiety-Z of formula (Ia) or (Ib) has a molecular weight of about 40kDa.

In certain embodiments, -Z comprises the following moieties:

in certain embodiments, -Z comprises an amide bond.

In certain embodiments, -Z of formula (Ia) or (Ib) comprises a moiety of formula (a) below:

Wherein the method comprises the steps of

The dashed line represents the remainder attached to-L ² -or-Z;

BP ^a is a branching point selected from the group consisting of: -N <, -CR < and > C <;

-R is selected from the group consisting of: -H and C _1-6 alkyl;

If BP ^a is-N < or-CR <, then a is 0; and if BP ^a is > C <, a is 1;

-S ^a-、-S^a'-、-S^a" -and-S ^a"' -are independently of each other a chemical bond or are selected from the group: c _1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl; wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl may be optionally substituted with one or more of the same or different-R ¹, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl may be optionally interrupted by one or more groups selected from ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R²)-、S(O)₂N(R²)-、-S(O)N(R²)-、-S(O)₂-、-S(O)-、-N(R²)S(O)₂N(R^2a)-、-S-、-N(R²)-、-OC(OR²)(R^2a)-、-N(R²)C(O)N(R^2a)- and-OC (O) N (R ²) -;

each-T-is independently selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclic, 8-30 membered carbopolycyclic and 8-30 membered heteropolycyclic; wherein each-T-is independently optionally substituted with one or more of the same or different-R ¹;

each-R ¹ is independently selected from the group consisting of: halogen, -CN, oxo (＝O)、-COOR³、-OR³、-C(O)R³、-C(O)N(R³R^3a)、-S(O)₂N(R³R^3a)、-S(O)N(R³R^3a)、-S(O)₂R³、-S(O)R³、-N(R³)S(O)₂N(R^3aR^3b)、-SR³、-N(R³R^3a)、-NO₂、-OC(O)R³、-N(R³)C(O)R^3a、-N(R³)S(O)₂R^3a、-N(R³)S(O)R^3a、-N(R³)C(O)OR^3a、-N(R³)C(O)N(R^3aR^3b)、-OC(O)N(R³R^3a) and C _1-6 alkyl; wherein C _1-6 alkyl is optionally substituted with one or more of the same or different halogen;

-R ²、-R^2a、-R³、-R^3a and-R ^3b are each independently selected from the group consisting of: -H and C _1-6 alkyl, wherein C _1-6 alkyl is optionally substituted with one or more of the same or different halogens; and

-P ^a'、-P^a" and-P ^a"' are independently polymeric moieties.

Optionally, the moiety of formula (a) is substituted with one or more substituents.

In certain embodiments, BP ^a of formula (a) is-N <. In certain embodiments, BP ^a of formula (a) is-CR <. In certain embodiments, -R is-H.

Thus, in certain embodiments, a in formula (a) is 0.

In certain embodiments, BP ^a of formula (a) is > C <.

In certain embodiments, -S ^a -in formula (a) is a bond.

In certain embodiments, -S ^a -of formula (a) is selected from the group consisting of: c _1-10 alkyl, C _2-10 alkenyl, and C _2-10 alkynyl, wherein C _1-10 alkyl, C _2-10 alkenyl, and C _2-10 alkynyl are optionally interrupted by one or more chemical groups selected from ：-C(O)O-、-O-、-C(O)-、-C(O)N(R⁴)-、-S(O)₂N(R⁴)-、-S(O)N(R⁴)-、-S(O)₂-、-S(O)-、-N(R⁴)S(O)₂N(R^4a)-、-S-、-N(R⁴)-、-OC(OR⁴)(R^4a)-、-N(R⁴)C(O)N(R^4a)- and-OC (O) N (R ⁴) -; wherein-R ⁴ and-R ^4a are independently selected from the group consisting of: -H, methyl, ethyl, propyl and butyl. In certain embodiments, -S ^a -in formula (a) is selected from: methyl, ethyl, propyl, butyl, optionally interrupted by one or more chemical groups selected from the group consisting of: -O-, -C (O) -and-C (O) N (R ⁴) -.

In certain embodiments, -S ^a' -in formula (a) is a bond.

In certain embodiments, -S ^a' -of formula (a) is selected from the group consisting of: c _1-10 alkyl, C _2-10 alkenyl, and C _2-10 alkynyl, wherein C _1-10 alkyl, C _2-10 alkenyl, and C _2-10 alkynyl are optionally interrupted by one or more chemical groups selected from ：-C(O)O-、-O-、-C(O)-、-C(O)N(R⁴)-、-S(O)₂N(R⁴)-、-S(O)N(R⁴)-、-S(O)₂-、-S(O)-、-N(R⁴)S(O)₂N(R^4a)-、-S-、-N(R⁴)-、-OC(OR⁴)(R^4a)-、-N(R⁴)C(O)N(R^4a)- and-OC (O) N (R ⁴) -; wherein-R ⁴ and-R ^4a are independently selected from the group consisting of: -H, methyl, ethyl, propyl and butyl. In certain embodiments, -S ^a' -in formula (a) is selected from the group consisting of: methyl, ethyl, propyl, butyl, optionally interrupted by one or more chemical groups selected from the group consisting of: -O-, -C (O) -and-C (O) N (R ⁴) -.

In certain embodiments, -S ^a" -in formula (a) is a bond.

In certain embodiments, -S ^a" -of formula (a) is selected from the group consisting of: c _1-10 alkyl, C _2-10 alkenyl, and C _2-10 alkynyl, wherein C _1-10 alkyl, C _2-10 alkenyl, and C _2-10 alkynyl are optionally interrupted by one or more chemical groups selected from ：-C(O)O-、-O-、-C(O)-、-C(O)N(R⁴)-、-S(O)₂N(R⁴)-、-S(O)N(R⁴)-、-S(O)₂-、-S(O)-、-N(R⁴)S(O)₂N(R^4a)-、-S-、-N(R⁴)-、-OC(OR⁴)(R^4a)-、-N(R⁴)C(O)N(R^4a)- and-OC (O) N (R ⁴) -; wherein-R ⁴ and-R ^4a are independently selected from the group consisting of: -H, methyl, ethyl, propyl and butyl. In certain embodiments, -S ^a" -in formula (a) is selected from the group consisting of: methyl, ethyl, propyl, butyl, optionally interrupted by one or more chemical groups selected from the group consisting of: -O-, -C (O) -and-C (O) N (R ⁴) -.

In certain embodiments, -S ^a"' -in formula (a) is a bond.

In certain embodiments, -S ^a"' -of formula (a) is selected from the group consisting of: c _1-10 alkyl, C _2-10 alkenyl, and C _2-10 alkynyl, wherein C _1-10 alkyl, C _2-10 alkenyl, and C _2-10 alkynyl are optionally interrupted by one or more chemical groups selected from ：-C(O)O-、-O-、-C(O)-、-C(O)N(R⁴)-、-S(O)₂N(R⁴)-、-S(O)N(R⁴)-、-S(O)₂-、-S(O)-、-N(R⁴)S(O)₂N(R^4a)-、-S-、-N(R⁴)-、-OC(OR⁴)(R^4a)-、-N(R⁴)C(O)N(R^4a)- and-OC (O) N (R ⁴) -; wherein-R ⁴ and-R ^4a are independently selected from the group consisting of: -H, methyl, ethyl, propyl and butyl. In certain embodiments, -S ^a"' -in formula (a) is selected from the group consisting of: methyl, ethyl, propyl, butyl, optionally interrupted by one or more chemical groups selected from the group consisting of: -O-, -C (O) -and-C (O) N (R ⁴) -.

In certain embodiments, -P ^a'、-P^a" and-P ^a"' of formula (a) independently comprise a polymer selected from the group consisting of: 2-methacryloyloxyethyl phosphorylcholine, poly (acrylic acid), poly (acrylic acid ester), poly (acrylamide), poly (alkoxy) polymer, poly (amide), poly (amidoamine), poly (amino acid), poly (anhydride), poly (asparagine), poly (butyric acid), poly (glycolic acid), poly (butylene terephthalate, poly (caprolactone), poly (carbonate), poly (cyanoacrylate), poly (dimethylacrylamide), poly (ester), poly (ethylene glycol), poly (ethylene oxide), poly (ethyl phosphate), poly (ethyl oxazoline), poly (glycolic acid), poly (hydroxyethyl acrylate), poly (hydroxyethyl oxazoline), poly (hydroxy methacrylate), poly (hydroxypropyl methacrylamide), poly (hydroxypropyl methacrylate), poly (iminocarbonate), poly (lactic acid-co-polymer), poly (methacrylamide), poly (methyl acrylate), poly (methyl oxazoline), poly (organophosphazene), poly (orthoester), poly (oxazoline), poly (polypropyleneglycol), poly (urethane), amino acid ester, polyvinyl alcohol, poly (vinylamine), poly (vinylmethyl ether), poly (vinylpyrrolidone), silicone, cellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannans, pectins, rhamnogalacturonan, starches, hydroxyalkyl starches, hydroxyethyl starches and polymers based on other carbohydrates, xylans, and copolymers thereof

In certain embodiments, -P ^a'、-P^a" and-P ^a"' of formula (a) independently have a molecular weight of 5-50kDa and include 5kDa and 50kDa, in certain embodiments have a molecular weight of 5-40kDa and include 5kDa and 40kDa, in certain embodiments have a molecular weight of 7.5-35kDa and include 7.5kDa and 35kDa, in certain embodiments have a molecular weight of 10-30kDa and include 10kDa and 30 kDa.

In certain embodiments, the molecular weight of-P ^a'、-P^a" and-P ^a"' in formula (a) is about 5kDa. In certain embodiments, the molecular weight of-P ^a'、-P^a" and-P ^a"' in formula (a) is about 7.5kDa. In certain embodiments, the molecular weight of-P ^a'、-P^a" and-P ^a"' in formula (a) is about 10kDa. In certain embodiments, the molecular weight of-P ^a'、-P^a" and-P ^a"' in formula (a) is about 12.5kDa. In certain embodiments, the molecular weight of-P ^a'、-P^a" and-P ^a"' in formula (a) is about 15kDa. In certain embodiments, the molecular weight of-P ^a'、-P^a" and-P ^a"' in formula (a) is about 20kDa.

In certain embodiments, -P ^a'、-P^a" and-P ^a"' of formula (a) independently comprise a PEG-based moiety. In certain embodiments, -P ^a'、-P^a" and-P ^a"' of formula (a) independently comprise a PEG-based moiety comprising at least 20% PEG, in certain embodiments at least 30% PEG, in certain embodiments at least 40% PEG, in certain embodiments at least 50% PEG, in certain embodiments at least 60% PEG, in certain embodiments at least 70% PEG, in certain embodiments at least 80% PEG, in certain embodiments at least 90% PEG.

In certain embodiments, -P ^a'、-P^a" and-P ^a"' of formula (a) independently comprise a protein moiety; in certain embodiments, the random coil protein moiety; and in certain embodiments, a random coil protein moiety selected from the group consisting of: PA, PAS, PAG, PG and XTEN sections.

In certain embodiments, -P ^a'、-P^a" and-P ^a"' in formula (a) are PA moieties. In certain embodiments, -P ^a'、-P^a" and-P ^a"' of formula (a) are PAS moieties. In certain embodiments, -P ^a'、-P^a" and-P ^a"' of formula (a) are PAG moieties. In certain embodiments, -P ^a'、-P^a" and-P ^a"' of formula (a) are PG moieties. In certain embodiments, -P ^a'、-P^a" and-P ^a"' of formula (a) are XTEN moieties.

In certain embodiments, -Z comprises a moiety of formula (a). In certain embodiments, -Z comprises two moieties of formula (a). In another embodiment, -Z comprises three moieties of formula (a). In certain embodiments, -Z comprises four moieties of formula (a). In certain embodiments, -Z comprises five moieties of formula (a). In certain embodiments, -Z comprises six moieties of formula (a).

In certain embodiments, -Z comprises the following moiety of formula (b):

Wherein the method comprises the steps of

The dashed line represents the remainder attached to-L ² -, or attached to-Z;

b1 is selected from the group consisting of: 0.1, 2, 3, 4, 5, 6, 7 and 8;

b2 is selected from the group consisting of: 1. 2,3, 4, 5, 6, 7 and 8;

b3 is an integer from 150 to 1000 and includes 150 and 1000; in certain embodiments, the range is 150-500 and includes 150 and 1000; and in certain embodiments, ranges from 200 to 460 and includes 200 and 460; and

B4 is an integer from 150 to 1000 and includes 150 and 1000; in certain embodiments, the range is from 150 to 500 and includes integers of 150 and 500; and in certain embodiments ranges from 200 to 460 and includes 200 and 460.

Optionally, the moiety of formula (b) is substituted with one or more substituents.

In certain embodiments, b3 and b4 in formula (b) are the same integer. In certain embodiments, b3 and b4 in formula (b) are each integers from 200 to 250, and in certain embodiments, b3 and b4 in formula (b) are about 225. In certain embodiments, b3 and b4 in formula (b) are each integers from 400 to 500, and in certain embodiments, b3 and b4 in formula (b) are about 450.

In certain embodiments, b1 in formula (b) is selected from the group consisting of: 0.1, 2, 3 and 4. In certain embodiments, b1 in formula (b) is selected from the group consisting of: 1.2 and 3. In certain embodiments, b1 of formula (b) is 2.

In certain embodiments, b2 in formula (b) is selected from the group consisting of: 1. 2, 3,4 and 5. In certain embodiments, b2 in formula (b) is selected from the group consisting of: 2.3 and 4. In certain embodiments, b2 in formula (b) is 3.

In certain embodiments, b1 of formula (b) is 2, b2 of formula (b) is 3, and both b3 and b4 are about 450. In certain embodiments, b1 of formula (b) is 2, b2 of formula (b) is 3, and both b3 and b4 are about 225.

In certain embodiments, -Z comprises a moiety of formula (b). In certain embodiments, -Z comprises two moieties of formula (b). In certain embodiments, -Z comprises three moieties of formula (b). In certain embodiments, -Z comprises four moieties of formula (b). In certain embodiments, -Z comprises five moieties of formula (b). In certain embodiments, -Z comprises six moieties of formula (b).

In certain embodiments, -Z comprises a moiety of formula (c):

Wherein the method comprises the steps of

The dashed line represents the remainder attached to-L ² -, or attached to-Z;

c1 and c2 are independently integers from 150 to 500 and include 150 and 500; in certain embodiments, it is 200-460, and includes 200 and 460.

Optionally, the moiety of formula (c) is substituted with one or more substituents.

In certain embodiments, c1 and c2 in formula (c) are both the same integer.

In certain embodiments, c1 and c2 in formula (c) are 200-250, and include 200 and 250; and in certain embodiments, it is about 225. In certain embodiments, c1 and c2 in formula (c) are 400-500, and include 400 and 500; and in certain embodiments, c1 and c2 in formula (c) are about 450.

In certain embodiments, moiety-Z is a branched PEG-based polymer comprising at least 10% PEG, having one branching point and two PEG-based polymer arms, having a molecular weight of about 40kDa. Thus, each of the two PEG-based polymer arms has a molecular weight of about 20kDa. In certain embodiments, the branching point is-CH <.

In certain embodiments, -Z comprises a moiety of formula (c). In certain embodiments, -Z comprises two moieties of formula (c). In certain embodiments, -Z comprises three moieties of formula (c). In certain embodiments, -Z comprises four moieties of formula (c). In certain embodiments, -Z comprises five moieties of formula (c). In certain embodiments, -Z comprises six moieties of formula (c).

In certain embodiments, moiety-Z has the following formula (d):

Wherein the method comprises the steps of

The dashed line indicates the connection to-L ² -;

-Z ^b -selected from the group consisting of: c _1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl; wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl may be optionally substituted with one or more of the same or different-R ¹, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl may be optionally interrupted by one or more groups selected from ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R²)-、-S(O)₂N(R²)-、-S(O)N(R²)-、-S(O)₂-、-S(O)-、-N(R²)S(O)₂N(R^2a)-、-S-、-N(R²)-、-OC(OR²)(R^2a)-、-N(R²)C(O)N(R^2a)- and-OC (O) N (R ²) -;

-R ²、-R^2a、-R³、-R^3a and-R ^3b are each independently selected from the group consisting of: -H and C _1-6 alkyl, wherein C _1-6 alkyl is optionally substituted with one or more of the same or different halogens;

And

-Z ^a is

Wherein the method comprises the steps of

Use of BP ^a、-S^a-、-S^a'-、-S^a"-、-S^a"'-、-P^a'、-P^a"、-P^a"' and a as defined for formula (a).

Optionally, the moiety of formula (d) is substituted with one or more substituents.

In certain embodiments, BP ^a、-S^a-、-S^a'-、-S^a"-、-S^a"'-、-P^a'、-P^a" and-P ^a"' of formula (d) are as defined above for formula (a).

In certain embodiments, -Z ^a of formula (d) is formula (b). In certain embodiments, b1, b2, b3, and b4 are as described in formula (b).

In certain embodiments, the-Z moiety in formula (Ia) or (Ib) has the following formula (e):

Wherein the method comprises the steps of

The dashed line indicates the connection to-L ² -;

e is selected from the group consisting of: 1.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15; and

-Z ^a is

Wherein b1, b2, b3 and b4 are as defined in formula (b).

Optionally, the moiety of formula (e) is substituted with one or more substituents.

In certain embodiments, b1, b2, b3 and b4 for formula (e) are as defined above for formula (b).

In certain embodiments, e in formula (e) is 1. In certain embodiments, e in formula (e) is 2. In certain embodiments, e in formula (e) is 3. In certain embodiments, e in formula (e) is 4. In certain embodiments, e in formula (e) is 5. In certain embodiments, e in formula (e) is 6. In certain embodiments, e in formula (e) is 7. In certain embodiments, e in formula (e) is 8. In certain embodiments, e in formula (e) is 9. In certain embodiments, e in formula (e) is 10. In certain embodiments, e in formula (e) is 11. In certain embodiments, e in formula (e) is 12. In certain embodiments, e in formula (e) is 13. In certain embodiments, e in formula (e) is 14. In certain embodiments, e in formula (e) is 15.

In certain embodiments, e in formula (e) is selected from the group consisting of: 2.3, 4, 5, 6, 7, 8 and 9. In certain embodiments, e in formula (e) is selected from the group consisting of: 3.4, 5 and 6. In certain embodiments, e in formula (e) is 5.

In certain embodiments, e in formula (e) is 5, b1 in formula (e) is 2, b2 in formula (e) is 3, and both b3 and b4 in formula (e) are about 450.

In certain embodiments, the moiety-Z in formula (Ia) or (Ib) has the following formula (e-i) or (e-i'):

Wherein the method comprises the steps of

The dashed line indicates the attachment to-L ² -,

E is selected from the group consisting of: 1.2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15;

-Z ^a is

Wherein the method comprises the steps of

B1, b2, b3 and b4 are as defined for formula (b).

In certain embodiments, b1, b2, b3 and b4 in formulas (e-i) and (e-i') are as defined above for formula (b). In certain embodiments, e in formulas (e-i) and (e-i') is as described for formula (e). In certain embodiments, b1 of formulas (e-i) and (e-i ') is 2, b2 of formulas (e-i) and (e-i ') is 3, and b3 and b4 of formulas (e-i) and (e-i ') are both about 450.

In certain embodiments, the-Z in formula (Ia) or (Ib) has formula (e-i).

In certain embodiments, moiety-Z is a branched PEG-based polymer comprising at least 10% PEG, has three branching points and four PEG-based polymer arms, and has a molecular weight of about 40 kDa. Thus, each of the four PEG-based polymer arms has a molecular weight of about 10 kDa. In certain embodiments, each of the three branch points is-CH <.

In certain embodiments, moiety-Z has the following formula (f):

Wherein the method comprises the steps of

The dashed line indicates the connection to-L ² -;

BP ^f is a branching point selected from the group consisting of: -N <, -CR < and > C <;

-R is selected from the group consisting of: -H and C _1-6 alkyl;

if BP ^f is-N < or-CR <, then f is 0; and if BP ^f is > C <, then f is 1;

-S ^f-、-S^f'-、-S^f" -and-S ^f"' -are independently a chemical bond, or are independently selected from the group consisting of: c _1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl; wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl may be optionally substituted with one or more of the same or different-R ¹, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl may be optionally interrupted by one or more groups selected from ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R²)-、-S(O)₂N(R²)-、-S(O)N(R²)-、-S(O)₂-、-S(O)-、-N(R²)S(O)₂N(R^2a)-、-S-、-N(R²)-、-OC(OR²)(R^2a)-、-N(R²)C(O)N(R^2a)- and-OC (O) N (R ²) -;

Each R ¹ is independently selected from the group consisting of: halogen, -CN, oxo (＝O)、-COOR³、-OR³、-C(O)R³、-C(O)N(R³R^3a)、-S(O)₂N(R³R^3a)、-S(O)N(R³R^3a)、-S(O)₂R³、-S(O)R³、-N(R³)S(O)₂N(R^3aR^3b)、-SR³、-N(R³R^3a)、-NO₂、-OC(O)R³、-N(R³)C(O)R^3a、-N(R³)S(O)₂R^3a、-N(R³)S(O)R^3a、-N(R³)C(O)OR^3a、-N(R³)C(O)N(R^3aR^3b)、-OC(O)N(R³R^3a) and C _1-6 alkyl; wherein C _1-6 alkyl is optionally substituted with one or more of the same or different halogen;

And

-Z ^a'、-Z^a" and Z ^a"' are independently

Wherein the method comprises the steps of

Optionally, the moiety of formula (f) is substituted with one or more substituents.

In certain embodiments, BP ^a、-S^a-、-S^a'-、-S^a"-、-S^a"'-、-P^a'、-P^a" and-P ^a"' of formula (f) are as defined above for formula (a).

In certain embodiments, BP ^f of formula (f) is-CR <, and r is 0. In certain embodiments, -R is-H.

In certain embodiments, -S ^f -of formula (f) is a bond.

In certain embodiments, -Z ^a'、-Z^a" and-Z ^a"' in formula (f) have the same structure. In certain embodiments, -Z ^a'、-Z^a" and-Z ^a"' in formula (f) are formula (b).

In certain embodiments, b1, b2, b3, and b4 are as described in formula (b).

In certain embodiments, -S ^f -in formula (f) is a bond, BP ^a in formula (f) is-CR <, wherein-R is-H. In certain embodiments, -S ^f -in formula (f) is a bond, -BP ^a in formula (f) is-CR <, wherein-R is-H, and-Z ^a'、-Z^a" and-Z ^a"' in formula (f) are formula (b).

In certain embodiments, -Z has the following formula (g):

Wherein the method comprises the steps of

The dashed line indicates the connection to-L ² -;

-S ^g-、-S^g' -and-S ^g" -are independently selected from the group consisting of: c _1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl; wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally substituted with one or more of the same or different-R ¹, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally interrupted by one or more groups selected from ：-T-、-C(O)O-、-O-、-C(O)-、-C(O)N(R²)-、-S(O)₂N(R²)-、-S(O)N(R²)-、-S(O)₂-、-S(O)-、-N(R²)S(O)₂N(R^2a)-、-S-、-N(R²)-、-OC(OR²)(R^2a)-、-N(R²)C(O)N(R^2a)- and-OC (O) N (R ²) -;

-Z ^a and-Z ^a are independently

Wherein the method comprises the steps of

BP^a、-S^a-、-S^a'-、-S^a"-、-S^a"^'-、-P^a'、-P^a"、-P^a"^' And a is as defined for formula (a).

Optionally, a moiety of formula (g) is substituted with one or more substituents.

In certain embodiments, BP ^a、-S^a-、-S^a'-、-S^a"-、-S^a"^'-、-P^a'、-P^a "and-P ^a"^' of formula (g) are as defined above for formula (a).

In certain embodiments, -S ^g -of formula (g) is selected from the group consisting of: c _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl, which are optionally substituted by one or more identical or different-R ¹ groups,

Wherein the method comprises the steps of

-R ¹ is selected from the group consisting of: halogen, oxo (＝O)、-COOR³、-OR³、-C(O)R³、-C(O)N(R³R^3a)、-S(O)₂N(R³R^3a)、-S(O)N(R³R^3a)、-S(O)₂R³、-S(O)R³、-N(R³)S(O)₂N(R^3aR^3b)、-SR³、-N(R³R^3a)、-NO₂、-OC(O)R³、-N(R³)C(O)R^3a、-N(R³)S(O)₂R^3a、-N(R³)S(O)R^3a、-N(R³)C(O)OR^3a、-N(R³)C(O)N(R^3aR^3b)、-OC(O)N(R³R^3a) and C _1-6 alkyl; wherein C _1-6 alkyl is optionally substituted with one or more of the same or different halogen; and-R ³、-R^3a and-R ^3b are independently selected from: -H, methyl, ethyl, propyl and butyl.

In certain embodiments, -S ^g -of formula (g) is selected from: c _1-6 alkyl.

In certain embodiments, -S ^g' -in formula (g) is selected from the group consisting of: c _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl, which are optionally substituted by one or more identical or different-R ¹ groups,

Wherein the method comprises the steps of

-R ¹ is selected from the group consisting of: halogen, oxo (＝O)、-COOR³、-OR³、-C(O)R³、-C(O)N(R³R^3a)、-S(O)₂N(R³R^3a)、-S(O)N(R³R^3a)、-S(O)₂R³、-S(O)R³、-N(R³)S(O)₂N(R^3aR^3b)、-SR³、-N(R³R^3a)、-NO₂、-OC(O)R³、-N(R³)C(O)R^3a、-N(R³)S(O)₂R^3a、-N(R³)S(O)R^3a、-N(R³)C(O)OR^3a、-N(R³)C(O)N(R^3aR^3b)、-OC(O)N(R³R^3a) and C _1-6 alkyl; wherein C _1-6 alkyl is optionally substituted with one or more of the same or different halogen; and

-R ³、-R^3a and-R ^3b are independently selected from: -H, methyl, ethyl, propyl and butyl.

In certain embodiments, -S ^g' -in formula (g) is C _1-6 alkyl.

In certain embodiments, -S ^g" -of formula (g) is selected from the group consisting of: c _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl, optionally substituted with one or more identical or different-R ¹ groups, wherein

In certain embodiments, -S ^g" -in formula (g) is C _1-6 alkyl.

In certain embodiments, -Z ^a and-Z ^a' in formula (g) have the same structure. In certain embodiments, -Z ^a and-Z ^a' in formula (g) have formula (b).

In certain embodiments, BP ^a、-S^a-、-S^a'-、-S^a"-、-S^a"'-、-P^a'、-P^a" and-P ^a"' in formula (g-i) are as defined above for formula (a).

In certain embodiments, -S ^g-、-S^g' -and-S ^g" -in formula (g-i) are as defined for formula (g).

In certain embodiments, -Z ^a and-Z ^a' of formula (g-i) have the same structure. In certain embodiments, -Z ^a and-Z ^a' of formula (g-i) have formula (b). In certain embodiments, pairs b1, b2, b3 and b4 are as described for formula (b).

In certain embodiments, -Z has the following formula (h):

Wherein the method comprises the steps of

The dashed line indicates the connection to-L ² -; and

Each-Z ^c is a moiety

Wherein the method comprises the steps of

Each c1 is independently an integer from about 200 to 250.

Optionally, the moiety of formula (h) is substituted with one or more substituents.

In certain embodiments, two c1 of formula (h) are the same. In certain embodiments, both c 1's of formula (h) are about 225.

In certain embodiments, moiety-Z has the following formula (h-i):

Wherein the method comprises the steps of

The dashed line indicates the connection to-L ² -; and

Each-Z ^c is a moiety

Each c1 is independently an integer from 200 to 250.

Optionally, the moiety of formula (h-i) is substituted with one or more substituents.

In certain embodiments, two c1 of formula (h-i) are the same. In certain embodiments, both c 1's of formula (h-i) are about 225.

In certain embodiments, the CNP conjugate has the following formula (IIf):

Wherein the method comprises the steps of

The dashed line marked with an asterisk indicates a connection to-Z having the following structure,

Wherein each-Z ^a is

Wherein each c1 is independently an integer from 200 to 250-.

In certain embodiments, each c1 in formula (IIf) is about 225.

In certain embodiments, -D of formula (IIf) is a CNP moiety, i.e., the conjugate of formula (IIf) is a CNP conjugate. In certain embodiments, -D of formula (IIf) is a CNP moiety having the sequence of SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO: 30. In certain embodiments, formula (IIf) -D is a CNP moiety having the sequence of SEQ ID NO: 24. In certain embodiments, formula (IIf) -D is a CNP moiety having the sequence of SEQ ID NO: 20. In certain embodiments, formula (IIf) -D is a CNP moiety having the sequence of SEQ ID NO. 21. In certain embodiments, formula (IIf) -D is a CNP moiety having the sequence of SEQ ID NO. 22. In certain embodiments, formula (IIf) -D is a CNP moiety having the sequence of SEQ ID NO. 23. In certain embodiments, formula (IIf) -D is a CNP moiety having the sequence of SEQ ID NO: 30.

In certain embodiments, -D of formula (IIf) is a CNP moiety linked to-L ¹ -through the nitrogen of the N-terminal amine function of the CNP.

In certain embodiments, -D of formula (IIf) is a CNP moiety that is linked to-L ¹ -through the nitrogen provided by the amine functionality of the lysine side chain of the CNP moiety.

In certain embodiments, if the CNP moiety is SEQ ID NO:24, then the lysine side chain is not part of the loop formed by the disulfide bridge between the cysteine residues at positions 22 and 38.

Thus, in certain embodiments, if the CNP has the sequence of SEQ ID NO:24, the CNP moiety is linked to-L ¹ -in the CNP conjugate of formula (IIf) via an amine functionality provided by the lysine side chain at position 9.

In certain embodiments, if the CNP has the sequence of SEQ ID NO:24, the CNP moiety is linked to-L ¹ -in the CNP conjugate of formula (IIf) via an amine functional group provided by a lysine side chain at position 11.

In certain embodiments, if the CNP has the sequence of SEQ ID NO:24, the CNP moiety is linked to-L ¹ -in the CNP conjugate of formula (IIf) via an amine functional group provided by a lysine side chain at position 15.

In certain embodiments, if the CNP has the sequence of SEQ ID NO:24, the CNP moiety is linked to-L ¹ -in the CNP conjugate of formula (IIf) via an amine functional group provided by a lysine side chain at position 16.

In certain embodiments, if the CNP has the sequence of SEQ ID NO:24, the CNP moiety is linked to-L ¹ -in the CNP conjugate of formula (IIf) via an amine functional group provided by a lysine side chain at position 20.

In certain embodiments, if the CNP moiety is SEQ ID NO:24, then the lysine side chain is part of a loop formed by a disulfide bridge between cysteine residues at positions 22 and 38.

Thus, in certain embodiments, if the CNP has the sequence of SEQ ID NO:24, the CNP moiety is linked to-L ¹ -in the CNP conjugate of formula (IIf) via an amine functional group provided by a lysine side chain at position 26.

In certain embodiments, the CNP conjugate has formula (IIf) wherein c1 is about 225, -D is a CNP moiety having the sequence of SEQ ID NO:20, and is linked to-L ¹ -by an amine functionality provided by a lysine side chain at position 30.

In certain embodiments, the CNP conjugate has formula (IIf) wherein c1 is about 225, -D is a CNP moiety having the sequence of SEQ ID NO:21, and is linked to-L ¹ -by an amine functionality provided by a lysine side chain at position 29.

In certain embodiments, the CNP conjugate has formula (IIf) wherein c1 is about 225, -D is a CNP moiety having the sequence of SEQ ID NO:22, and is linked to-L ¹ -by an amine functionality provided by a lysine side chain at position 28.

In certain embodiments, the CNP conjugate has formula (IIf) wherein c1 is about 225, -D is a CNP moiety having the sequence of SEQ ID NO:23, and is linked to-L ¹ -by an amine functionality provided by a lysine side chain at position 27.

In certain embodiments, the CNP conjugate has formula (IIf) wherein c1 is about 225, -D is a CNP moiety having the sequence of SEQ ID NO:30, and is linked to-L ¹ -by an amine functionality provided by a lysine side chain at position 27.

It will be appreciated that the positions of the above cysteines and lysines vary depending on the length of the CNP moiety, and that one skilled in the art would have no difficulty identifying the corresponding cysteines and lysines in longer or shorter versions of the CNP moiety, and also understand that some lysines may not be present in shorter CNP moieties, for example. It is further understood that more lysine residues may be present in the non-loop-forming portion and/or the loop-forming portion of the CNP moiety, for example, due to site-directed mutagenesis.

In certain embodiments, the CNP conjugate has formula (IIf) wherein c1 is about 225, -D is a CNP moiety having the sequence of SEQ ID NO:24, and is linked to-L ¹ -by an amine functionality provided by a lysine side chain at position 26.

In certain embodiments, the CNP conjugate has the formula (IIf'):

Wherein the method comprises the steps of

The unlabeled dashed line represents the nitrogen provided by the lysine side chain at position 26 forming an amide linkage to the CNP moiety of SEQ ID NO. 24; and

Wherein the method comprises the steps of

Each-Z ^a is

Wherein the method comprises the steps of

Each c1 is independently an integer from 200 to 250.

In certain embodiments, each c1 of formula (IIf') is about 225.

In certain embodiments, the unit dosage form of the invention comprises a CNP conjugate of formula (IIf):

Wherein the method comprises the steps of

Wherein each-Z ^a is

Wherein each c1 is independently an integer from 200 to 250, and

Wherein the unit dose is about 50 μg CNP/kg.

Wherein the method comprises the steps of

The unlabeled dashed line represents the nitrogen provided by the lysine side chain at position 26 forming an amide linkage to the CNP moiety of SEQ ID NO. 24;

Wherein the method comprises the steps of

Each-Z ^a is

Wherein the method comprises the steps of

Each c1 is independently an integer from 200 to 250, and

Wherein the unit dose is about 75 μg CNP/kg.

Wherein the method comprises the steps of

Each-Z ^c is

Wherein the method comprises the steps of

Each c1 is independently an integer from 200 to 250, and

Wherein the unit dose is about 100 μg CNP/kg.

In certain embodiments, the unit dosage form of the invention comprises a CNP conjugate of formula (IIf') or a pharmaceutically acceptable salt thereof:

Wherein the method comprises the steps of

Each-Z ^c is

Wherein the method comprises the steps of

Each c1 is independently an integer from 200 to 250, and

Wherein the unit dose is 50 μg CNP/kg.

Wherein the method comprises the steps of

Each-Z ^a is

Wherein the method comprises the steps of

Each c1 is independently an integer from 200 to 250, and

Wherein the unit dose is 75 μg CNP/kg.

In certain embodiments, the unit dosage forms of the present invention comprise a CNP conjugate having the following formula (IIf):

Wherein the method comprises the steps of

Each-Z ^a is

Wherein the method comprises the steps of

Each c1 is independently an integer from 200 to 250, and

Wherein the unit dose is 100 μg CNP/kg.

Another aspect of the invention is a method of treating, reducing the risk of, or delaying a disease treatable with CNP in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of about 6 μg CNP/kg to at least about 150 μg CNP/kg.

In certain embodiments, the invention is a method of treating, reducing the risk of, or delaying a disease treatable with CNP in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of about 6 μg CNP/kg to at least about 100 μg CNP/kg.

In certain embodiments, the invention relates to unit dosage forms for the treatment of diseases treatable with CNP.

In certain embodiments, the disease treatable with CNP is selected from the group consisting of: bone related disorders (e.g., skeletal dysplasia), cancer, autoimmune diseases, fibrotic diseases, inflammatory diseases, central nervous system diseases (e.g., neurodegenerative diseases), infectious diseases, pulmonary diseases, cardiovascular diseases, metabolic diseases, and ophthalmic diseases.

In certain embodiments, the disease treatable with CNP is selected from the group consisting of: chondrogenesis imperfecta, hypochondral, short stature, dwarfism, osteochondral dysplasia, lethal dysplasia, osteogenesis imperfecta, chondral dysplasia, punctate chondral dysplasia, homozygous chondral dysplasia, curved limb dysplasia, congenital lethal hypophosphatemia, perinatal lethal osteogenesis imperfecta, short rib multi-finger syndrome, acroroot punctate chondral dysplasia, jansen (Jansen) metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, osteogenesis imperfecta, bone malformation dysplasia, congenital short femur, Langer (Langer) in-limb osteodysplasia, nivegan (NIEVERGELT) in-limb osteodysplasia, luo Binnuo (Robinow) syndrome, lychnhate (Reinhardt) syndrome, acroosteodysplasia, peripheral osteodysplasia, kenist (Kniest) dysplasia, fibrochondral hyperplasia, robert (Roberts) syndrome, acrodysplasia, small limb deformity, moroo (Morquio) syndrome, kenist (Kniest) syndrome, post-acquired dystrophy, metaphyseal dysplasia, Neurofibromatosis, leggews (Legius) syndrome, LEOPARD syndrome, noonan (Noonan) syndrome, hereditary gum fibromatosis, neurofibromatosis type 1, leggews (Legius) syndrome, heart face skin syndrome, costello syndrome, SHOX deficiency, idiopathic short stature, growth hormone deficiency, osteoarthritis, collarbone dysgenesis, craniosynostosis (e.g., mu Enke (Muenke) syndrome, kruezone (Crouzon) syndrome, apertson syndrome, jack-Weiss (Jackson-Weiss) syndrome), pafeiffer's (Pfeiffer) syndrome or Kluyzon (Crouzon) cutaneous skeletal syndrome), digital deformity, short finger, flexor finger, multi-finger, parallel finger, segmental dysplasia, endogenous chondrioma, fibrodysplasia, hereditary multiple exotoses, hypophosphatemic rickets, jacfe-Li Jin Stent (Jaffe-Lichtenstein) syndrome, marfan's syndrome, mcCune-Albumt's syndrome, sclerosteosis, fragile bone sclerosis, hemorrhagic shock, hypertension, vascular restenosis, arteriosclerosis, Acute decompensated heart failure, congestive heart failure, cardiac edema, renal edema, hepatic edema, acute renal insufficiency, chronic renal insufficiency, glaucoma, elevated intraocular pressure, multiple myeloma, myeloproliferative syndromes, leukemia, plasma cell leukemia, lymphoma, glioblastoma, prostate cancer, bladder cancer, breast cancer, growth retardation, cranial deformities, orthodontics defects, cervical marrow pressure, spinal stenosis, hydrocephalus, hearing loss due to chronic otitis, cardiovascular diseases, neurological diseases, and obesity.

In certain embodiments, the disease treatable with CNP is selected from: chondrogenesis imperfecta (e.g., homozygous chondrogenesis imperfecta), chondrodysplasia, short stature, dwarfism, osteochondral dysplasia, lethal dysplasia, osteogenesis imperfecta, chondral chondrogenesis imperfecta, punctate chondrogenesis imperfecta, curved limb dysplasia, congenital lethal hypophosphatemia, perinatal lethal osteogenesis imperfecta, short rib multi-finger syndrome, acroroot punctate chondrogenesis imperfecta, jansen (Jansen) metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, osteogenesis imperfecta, bone malformation dysplasia, congenital short femur, Langer (Langer) type in-limb bone dysplasia, nivegate (NIEVERGELT) type in-limb bone dysplasia, luo Binnuo (Robinow) syndrome, lychnhate (Reinhardt) syndrome, acroosteogenesis, peripheral osteogenesis, kenist (Kniest) dysplasia, fibrochondral hyperplasia, robert (Roberts) syndrome, acrogenesis, lower limb deformity, morkey (Morquio) syndrome, kenist (Kniest) syndrome, post-acquired nutritional dysplasia, metaphyseal dysplasia, neurofibromatosis, Legenus (Legius) syndrome, LEOPARD syndrome, knonan (Noonan) syndrome, hereditary gum fibromatosis, type 1 neurofibromatosis, legenus (Legius) syndrome, cardiac skin syndrome, costello (Costello) syndrome, SHOX deficiency, idiopathic short stature, growth hormone deficiency, osteoarthritis, collarbone dysgenesis, craniosynostosis (e.g., mu Enke (Muenke) syndrome, kluzon (Crouzon) syndrome, apert) syndrome, jackson-Weiss syndrome, pafeiffer's (Pfeiffer) syndrome or Kluyzon (Crouzon) cutaneous skeletal syndrome), digital deformity, short finger, flexor finger, multi-finger, parallel finger, segmental dysplasia, endogenous chondrioma, fibrodysplasia, hereditary multiple exotoses, hypophosphatemic rickets, jacfe-Li Jin Stent (Jaffe-Lichtenstein) syndrome, marfan's syndrome, mcCune-Albumt's syndrome, sclerosteosis, fragile bone sclerosis, hemorrhagic shock, hypertension, vascular restenosis, arteriosclerosis, Acute decompensated heart failure, congestive heart failure, cardiac edema, renal edema, hepatic edema, acute renal insufficiency, chronic renal insufficiency, glaucoma, elevated intraocular pressure, multiple myeloma, myeloproliferative syndromes, leukemia, plasma cell leukemia, lymphoma, glioblastoma, prostate cancer, bladder cancer, breast cancer, growth retardation, cranial deformities, orthodontics defects, cervical marrow compression, spinal stenosis, hydrocephalus, hearing loss due to chronic otitis, obesity, diseases involving aberrant RAS-mitogen-activated protein kinase signaling, pulmonary hypertension, vascular lesions, endothelial dysfunction, cirrhosis, liver cirrhosis, liver ascites, liver fibrosis, liver and kidney syndrome, asthma, pulmonary fibrosis, chronic kidney disease, heart and kidney syndrome, dyspnea and lysosomal storage diseases (e.g. mucopolysaccharidosis).

In certain embodiments, the disease treatable with CNP is one or more cardiovascular diseases selected from the group consisting of: arrhythmia (e.g., cardiac or sinus arrhythmia); atrial fibrillation; atrial flutter; bradycardia; brugada syndrome; a complex sign of premature heart beat; heart concussion; heart block; long QT syndrome; shrink in parallel; pre-excitation syndrome; tachycardia; ventricular fibrillation; ventricular flutter; diseases of the cardiac conduction system; low cardiac output; the heart enlarges; dilated cardiomyopathy; hypertrophy (e.g., left ventricular hypertrophy or right ventricular hypertrophy); cardiomyopathy (e.g., alcoholic, dilated, hypertrophic, restrictive, diabetic, or Chagas (Chagas) type cardiomyopathy); arrhythmogenic right ventricular dysplasia; endocardial stretch fiber hyperplasia; endocardial myocardial fibrosis; type IIb glycogen storage disease; kans-Seer (Kearns-Sayr) syndrome; myocardial reperfusion injury; myocarditis; myoglycanases; endocarditis (e.g., bacterial or non-infectious endocarditis); sudden cardiac arrest; sudden cardiac death; sudden cardiac arrest outside the hospital; heart kidney syndrome; paroxysmal dyspnea; cardiac oedema, heart failure (e.g., diastolic or systolic heart failure); heart valve disease; aortic insufficiency; aortic valve stenosis; heart valve prolapse; mitral insufficiency; mitral valve stenosis; pulmonary artery occlusion; pulmonary insufficiency; stenosis of the pulmonary valve; tricuspid valve closure; tricuspid insufficiency; tricuspid valve stenosis; myocardial ischemia; acute coronary syndrome; angina pectoris; coronary heart disease; kuni si (Kounis) syndrome; myocardial infarction; pulmonary heart disease; ventricular dysfunction (e.g., left ventricular or right ventricular dysfunction); ventricular outflow tract obstruction; aortic valve stenosis and pulmonary valve stenosis; hypertension; atherosclerosis; restenosis; critical limb ischemia; peripheral arterial disease; ischemia (e.g., ischemia-reperfusion injury or ischemic injury); abnormal fluid accumulation in the heart and myocardial edema.

In certain embodiments, the disease treatable with CNP is selected from the group consisting of: ischemic heart disease (e.g., myocardial infarction), congestive heart failure, arrhythmia, and atherosclerosis.

In certain embodiments, the disease treatable with CNP is one or more central nervous system diseases selected from the group of: cerebral ischemia (e.g., ischemic hypoxia); cerebral infarction; transient ischemic attacks; vertebral basal artery insufficiency; cerebrovascular disease; stroke; intracranial hemorrhage; corneal neovascularization; cornea transplantation; graft versus host disease; graft rejection; glaucoma (e.g., closed angle, neovascular, open angle or low tension glaucoma); ischemic optic neuropathy; central serous chorioretinopathy; retinopathy (e.g., diabetic or hypertensive retinopathy); retinal degeneration; macular degeneration; geographic atrophy; macular edema; stergard disease; vitelliform macular dystrophy; wet macular degeneration; retinal cleavage; retinal detachment; retinal perforations; retinal hemorrhage; retinal neovascularization; retinal vein occlusion; retinal artery occlusion; retinopathy of prematurity and proliferative vitreoretinopathy.

In certain embodiments, the disease treatable with CNP is selected from the group consisting of: hypothalamic esterase, hypochondral dysplasia, mu Enke (Muenke) syndrome, hypertension, osteogenesis imperfecta and achondroplasia.

In certain embodiments, the disease treatable with CNP is selected from the group consisting of: achondroplasia, hypochondral development, short stature, noonan (Noonan) syndrome or SHOX deficiency.

In certain embodiments, the disease treatable with CNP is hypophosphatasia. In certain embodiments, the disease treatable with CNP is hypochondrogenesis. In certain embodiments, the disease treatable with CNP is Mu Enke (Muenke) syndrome. In certain embodiments, the disease treatable with CNP is hypertension. In certain embodiments, the disease treatable with CNP is osteogenesis imperfecta. In certain embodiments, the disease treatable with CNP is achondroplasia.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia (e.g., achondroplasia), comprising at least 24nmol CNP/kg unit dose of CNP conjugate or pharmaceutically acceptable salt thereof, and administered to a human patient, wherein the administration results in an annual growth rate (AGV) of about 5.4 cm/year, such as about 5.42 cm/year.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia, such as achondroplasia, comprising about 24nmol CNP/kg unit dose of CNP conjugate or pharmaceutically acceptable salt thereof, and administered to a human patient, wherein the administration results in an annual growth rate (AGV) of 5.42 cm/year.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia (e.g., achondroplasia), comprising a unit dose of about 100 μg CNP/kg of CNP conjugate or pharmaceutically acceptable salt thereof, and administered to a human patient, wherein the administration results in an annual growth rate (AGV) of about 5.4 cm/year, such as about 5.42 cm/year.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia, such as achondroplasia, comprising a CNP conjugate or pharmaceutically acceptable salt thereof, in a unit dose of 100 μg CNP/kg, and administered to a human patient, wherein the administration results in an annual growth rate (AGV) of 5.42 cm/year.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia, such as achondroplasia, comprising a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof, in a unit dose of 100 μg CNP/kg, and administered to a human patient aged 2-10 years, wherein the administration results in an annual growth rate (AGV) of 5.42 cm/year.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia, such as achondroplasia, comprising a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof, in a unit dose of 100 μg CNP/kg, and administered to a human patient aged 2-5 years, wherein the administration results in an annual growth rate (AGV) of about 5.95 cm/year.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia, such as achondroplasia, comprising a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof, in a unit dose of 100 μg CNP/kg, and administered to a human patient aged 2-5 years, wherein the administration results in an annual growth rate (AGV) of 5.95 cm/year.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia (e.g., achondroplasia), comprising a CNP conjugate or pharmaceutically acceptable salt thereof in a unit dosage range of 12.3-36.9nmol CNP/kg, wherein the unit dosage form is administered to a human patient suffering from open epiphysis and the treatment reduces the incidence of achondroplasia related adverse events in the human patient.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia (such as achondroplasia), comprising a CNP conjugate or pharmaceutically acceptable salt thereof at a unit dose of 24.6nmol CNP/kg, wherein the unit dosage form is administered to a human patient suffering from open epiphysis and the treatment reduces the incidence of achondroplasia related adverse events in the human patient.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia (such as achondroplasia), comprising a unit dose of 100 μg CNP/kg of CNP conjugate or a pharmaceutically acceptable salt thereof, wherein the unit dosage form is administered to a human patient having an open epiphysis and the treatment reduces the incidence of an adverse event associated with achondroplasia in the human patient.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia, such as achondroplasia, comprising a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof in a unit dose of 100 μg CNP/kg, wherein the unit dosage form is administered to a human patient suffering from open epiphysis and the treatment reduces the incidence of achondroplasia related adverse events in the human patient.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia (e.g., achondroplasia), comprising a CNP conjugate or pharmaceutically acceptable salt thereof at a unit dose of 24.6nmol CNP/kg, wherein the unit dosage form is administered once weekly via subcutaneous injection to a human patient having an open epiphysis, and wherein each administration is associated with a frequency of injection site reaction of less than 3%, such as less than 2%, such as less than 1%, or the absence of injection site reaction.

In certain embodiments, the invention relates to a unit dosage form for treating skeletal dysplasia (e.g., achondroplasia), comprising a unit dose of 100 μg CNP/kg of a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof, wherein the unit dosage form is administered once weekly via subcutaneous injection to a human patient having an open epiphysis, and wherein each administration is associated with a frequency of less than 3% of injection site reactions, such as less than 2%, such as less than 1%, or the absence of injection site reactions.

Another aspect of the invention is a method of treating achondroplasia in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of about 6 μg CNP/kg to at least about 150 μg CNP/kg.

Optionally, the unit dose is from about 50 μg CNP/kg to about 150 μg CNP/kg, from about 75 μg gCNP/kg to about 125 μg CNP/kg, from about 90 μg CNP/kg to about 110 μg CNP/kg, or about 100 μg CNP/kg, or 100 μg CNP/kg. Such doses are preferably administered once a week. Such doses are preferably administered once a week by subcutaneous injection. Preferred compounds in such methods are compounds of formula (IIf) or (IIf'), the dosages of which refer to the mass of the CNP1-38 moiety of the compound. Thus, a preferred regimen is to administer the compound of formula (IIf) or (IIf ') subcutaneously once per week in a unit dose of about 100 μg CNP/kg CNP1-38 fraction of the compound of formula (IIf) or (IIf'). Dosages of other compounds can be adjusted to achieve equimolar delivery of the CNP moiety of the compound of interest, just as the CNP moiety of the compound of formula (IIf) or (IIf'), with a tolerance of +/-20% or +/-10%. The dosage regimen of the other compound or compound of formula (IIf) or (IIf ') can be adjusted to deliver +/-20% or +/-10% (based on moles of CNP moieties) of the same area under the curve as the compound of formula (IIf) or (IIf') administered subcutaneously once per week. The same regimen can be used to treat other diseases that can be treated with the CNPs disclosed herein.

In certain embodiments, the invention is a method of treating achondroplasia in a human patient, the method comprising the step of administering the CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of 6 μg CNP/kg, 20 μg CNP/kg, 50 μg CNP/kg, 75 μg gCNP/kg, 100 μg CNP/kg, or 125 μg CNP/kg.

In certain embodiments, the invention is a method of treating achondroplasia in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of 50-100 μg CNP/kg.

In certain embodiments, the invention is a method of treating achondroplasia in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of 50 μg CNP/kg.

In certain embodiments, the invention is a method of treating achondroplasia in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 100 μg CNP/kg.

In certain embodiments, the invention is a method of treating achondroplasia in a human patient, comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 50-100 μg CNP/kg, wherein the CNP conjugate is a compound of formula (IIf'):

Wherein the method comprises the steps of

Each-Z ^c is

And wherein

Each c1 is independently an integer from 200 to 250.

In certain embodiments, the invention is a method of treating achondroplasia in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 50 μg CNP/kg, wherein the CNP conjugate is a compound of formula (IIf'):

Wherein the method comprises the steps of

Each-Z ^c is

And wherein

Each c1 is independently an integer from 200 to 250.

In certain embodiments, the invention is a method of treating achondroplasia in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 100 μg CNP/kg, wherein the CNP conjugate is a compound of formula (IIf'):

Wherein the method comprises the steps of

Each-Z ^c is

And wherein

Each c1 is independently an integer from 200 to 250.

In certain embodiments, the invention is a method of treating achondroplasia in a human patient, comprising the step of administering a unit dose comprising a unit dose of 24.6nmol CNP/kg, wherein the unit dose comprises a CNP conjugate of formula (IIf) or (IIf'), or a pharmaceutically acceptable salt thereof, wherein the patient has an age in the range of 2-10 years, and the administration results in an annual growth rate of about 5.42 cm/year.

In certain embodiments, the invention is a method of treating achondroplasia in a human patient, the method comprising the step of administering a unit dose comprising a unit dose of 100 μg CNP/kg, wherein the unit dose comprises a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof, wherein the patient has an age in the range of 2-10 years and the administration results in an annual growth rate of about 5.42 cm/year.

In certain embodiments, the invention is a method of treating achondroplasia in a human patient, the method comprising the step of administering a unit dose comprising a unit dose of 100 μg CNP/kg, wherein the unit dose comprises a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof, wherein the patient has an age in the range of 2-10 years and the administration results in an annual growth rate of 5.42 cm/year.

In certain embodiments, the invention is a method of treating achondroplasia in a human patient, the method comprising the step of administering a unit dose comprising a unit dose of 100 μg CNP/kg, wherein the unit dose comprises a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof, wherein the patient has an age in the range of 2-5 years and the administration results in an annual growth rate of about 5.95 cm/year.

In certain embodiments, the invention is a method of treating achondroplasia in a human patient, the method comprising the step of administering a unit dose comprising a unit dose of 100 μg CNP/kg, wherein the unit dose comprises a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof, wherein the patient has an age in the range of 2-5 years and the administration results in an annual growth rate of 5.95 cm/year.

The unit dose may be administered one or more times. For multiple administrations, the interval is preferably weekly, but may be twice weekly, once every two weeks, once monthly, etc. In certain embodiments, one unit dose is administered via subcutaneous injection once a week. In certain embodiments, one unit dose is administered via once-a-month subcutaneous injection. In certain embodiments, for a patient group weighing 55kg or more, one unit dose may be split and administered via two simultaneous or sequential injections.

The CNP conjugate, or a pharmaceutically acceptable salt thereof, may be administered for at least six months, one year, five years, ten years, up to the patient's age 18 years old, up to the patient's epiphyseal closure, or indefinitely. In certain embodiments, the CNP conjugate or pharmaceutically acceptable salt thereof may be administered until the patient is over 18 years old. In certain embodiments, the CNP conjugate or pharmaceutically acceptable salt thereof may be administered until the epiphyseal closure of the patient.

Treatment may begin prenatally, at birth, or at the time of diagnosis of a defect or risk associated with CNP.

If a unit dose of a particular drug, such as a reversible CNP conjugate of formula (IIf) or (IIf '), is determined, the unit dose can be used as a guide for other reversible CNP conjugates such that the unit dose of the other reversible conjugates is the same as the unit dose of CNP of formula (IIf) or (IIf'), calculated as moles of CNP. This guidance is particularly useful when the release half-life of the CNP moiety released by the other conjugate is within plus or minus 20% of the release half-life of the CNP conjugate of formula (IIf) or (IIf'). Thus, for example, if a unit dose of a reversible conjugate of CNP-38 is determined, an otherwise similar reversible conjugate of CNP-53 can be administered by multiplying the dose of CNP-38 by the molecular weight ratio of CNP-53/CNP-38.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of about 6 μg CNP/kg to at least about 150 μg CNP/kg.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of 6 μg CNP/kg, 20 μg CNP/kg, 50 μg CNP/kg, 75 μg CNP/kg, 100 μg CNP/kg, or 125 μg CNP/kg.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of 50-100 μg CNP/kg.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of 50 μg CNP/kg.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 100 μg CNP/kg.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of about 12.3nmol CNP/kg to about 36.9nmol CNP/kg for at least 52 weeks, the administration resulting in an annual growth rate (AGV) of about 5.4 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of at least 24nmol CNP/kg for at least 52 weeks, the administration resulting in an annual growth rate (AGV) of about 5.4 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of about 24.6nmol CNP/kg for at least 52 weeks, the administration resulting in an annual growth rate (AGV) of about 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 24.6nmol CNP/kg for at least 52 weeks, the administration resulting in an annual growth rate (AGV) of about 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 24.6nmol CNP/kg for at least 52 weeks until the epiphyseal closure of the patient, the patient being between 2 and 10 years old, the administration resulting in an annual growth rate (AGV) of 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate or a pharmaceutically acceptable salt thereof at a unit dose of 24.6nmol CNP/kg for at least 52 weeks until the epiphyseal closure of the patient, the patient being aged 2-5 years old, the administration resulting in an annual growth rate (AGV) of about 5.95 cm/year, such as 5.95 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of 24.6nmol CNP/kg for at least 104 weeks, the administration resulting in an annual growth rate (AGV) of about 5.42 cm/year.

Annual growth rate may be determined by measuring the patient's height growth for at least 52 weeks (e.g., at least 104 weeks or 156 weeks), or by measuring a portion of a year (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months) and then extrapolating the value to an entire year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the steps of:

(a) Administering the CNP conjugate or pharmaceutically acceptable salt thereof to a human patient at a unit dose of 24.6nmol CNP/kg for at least 52 weeks;

(b) Absolute height was measured at time points of 0 month, 6 months, 12 months, respectively;

(c) AGVs were calculated for periods of 0-6 months and for periods of 6-12 months.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of at least 100 μg CNP/kg for at least 52 weeks, the administration resulting in an annual growth rate (AGV) of about 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of about 100 μg CNP/kg for at least 52 weeks, the administration resulting in an annual growth rate (AGV) of about 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 100 μg CNP/kg for at least 52 weeks, the administration resulting in an annual growth rate (AGV) of 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 100 μg CNP/kg for at least 52 weeks until the epiphyseal closure of the patient, the patient being between 2 and 10 years old, the administration resulting in an annual growth rate (AGV) of 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate or a pharmaceutically acceptable salt thereof at a unit dose of 100 μg CNP/kg for at least 52 weeks until the epiphyseal closure of the patient, the patient being aged 2-5 years, the administration resulting in an annual growth rate (AGV) of about 5.95 cm/year, such as 5.95 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 100 μg CNP/kg for at least 104 weeks, the administration resulting in an annual growth rate (AGV) of 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose in the range of 12.3-36.9nmol CNP/kg for at least 52 weeks, wherein the CNP conjugate unit dosage form is administered once weekly to a human patient suffering from open epiphysis via subcutaneous injection, and wherein each administration is associated with a frequency of less than 3% of injection site reactions, such as less than 2%, such as less than 1%, or the absence of injection site reactions.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate or a pharmaceutically acceptable salt thereof at a unit dose of 24.6nmol CNP/kg for at least 52 weeks, wherein the CNP conjugate or unit dosage form is administered once weekly via subcutaneous injection to a human patient having an open epiphyseal, and wherein each administration is associated with a frequency of less than 3% of injection site reactions, such as less than 2%, such as less than 1%, or the absence of injection site reactions.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate or a pharmaceutically acceptable salt thereof at a unit dose of 100 μg CNP/kg for at least 52 weeks, wherein the CNP conjugate or unit dosage form is administered once weekly via subcutaneous injection to a human patient having an open epiphysis, and wherein each administration is associated with a frequency of less than 3% of injection site reactions, such as less than 2%, such as less than 1%, or the absence of injection site reactions.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof at a unit dose of 100 μg CNP/kg for at least 52 weeks, wherein the CNP conjugate or unit dosage form is administered once weekly to a human patient suffering from open epiphyseal via subcutaneous injection and the frequency of injection site reactions per administration is less than 3%, such as less than 2%, such as less than 1%, or no injection site reactions are present.

In certain embodiments, the frequency of injection site reactions is less than 3%. In certain embodiments, the frequency of injection site reactions is less than 2%. In certain embodiments, the frequency of injection site reactions is less than 1%. In certain embodiments, there is no injection site reaction.

In certain embodiments, the invention is a method of increasing the growth rate of a achondroplasia human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose in the range of 12.3-36.9nmol CNP/kg for at least 52 weeks, wherein the CNP conjugate or unit dosage form is administered to a human patient suffering from an open epiphysis, and the treatment reduces the incidence of achondroplasia related adverse events in the human patient.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient with achondroplasia, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 24.6nmol CNP/kg for at least 52 weeks, wherein the CNP conjugate or unit dosage form is administered to a human patient with open epiphysis, and the treatment reduces the incidence of achondroplasia-related adverse events in the human patient.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient suffering from achondroplasia, comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof at a unit dose of 100 μg CNP/kg for at least 52 weeks, wherein the CNP conjugate or unit dose form is administered to a human patient suffering from open epiphysis and the treatment reduces the incidence of achondroplasia related adverse events in the human patient.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising administering a CNP conjugate or a pharmaceutically acceptable salt of formula (IIf) or (IIf') in a unit dose of 50-100 μg CNP/kg.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof in a unit dose of 50 μg CNP/kg.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof at a unit dose of 100 μg CNP/kg.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof in a unit dose of at least 100 μg CNP/kg for at least 52 weeks, the administration resulting in an annual growth rate (AGV) of about 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof at a unit dose of about 100 μg CNP/kg for at least 52 weeks, the administration resulting in an annual growth rate (AGV) of about 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof at a unit dose of 100 μg CNP/kg for at least 52 weeks, the administration resulting in an annual growth rate (AGV) of 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, comprising the step of administering a CNP conjugate or a pharmaceutically acceptable salt of formula (IIf) or (IIf') at a unit dose of 100 μg CNP/kg for at least 52 weeks until the epiphyseal closure of the patient, who is between 2 and 10 years old, resulting in an annual growth rate (AGV) of about 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient, comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof at a unit dose of 100 μg CNP/kg for at least 52 weeks until the epiphyseal closure of the patient, who is 2-5 years old, resulting in an annual growth rate (AGV) of about 5.95 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof at a unit dose of 100 μg CNP/kg for at least 104 weeks, the administration resulting in an annual growth rate (AGV) of at least about 5.42 cm/year.

In certain embodiments, the invention is a method of increasing the growth rate of a human patient comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof at a unit dose of 100 μg CNP/kg for at least 104 weeks, the administration resulting in an annual growth rate (AGV) of about 5.42 cm/year.

In certain embodiments, the invention is a method of increasing long bone growth in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of about 6 μg CNP/kg to at least about 150 μg CNP/kg.

In certain embodiments, the invention is a method of increasing long bone growth in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of 6 μg CNP/kg, 20 μg CNP/kg, 50 μg CNP/kg, 75 μg CNP/kg, 100 μg CNP/kg, or 125 μg CNP/kg.

In certain embodiments, the invention is a method of increasing long bone growth in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of 50-100 μg CNP/kg.

In certain embodiments, the invention is a method of increasing long bone growth in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, in a unit dose of 50 μg CNP/kg.

In certain embodiments, the invention is a method of increasing long bone growth in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 100 μg CNP/kg.

In certain embodiments, the invention is a method of increasing long bone growth in a human patient comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof in a unit dose of 50-100 μg CNP/kg.

In certain embodiments, the invention is a method of increasing long bone growth in a human patient comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof in a unit dose of 50 μg CNP/kg.

In certain embodiments, the invention is a method of increasing long bone growth in a human patient comprising the step of administering a CNP conjugate of formula (IIf) or (IIf') or a pharmaceutically acceptable salt thereof at a unit dose of 100 μg CNP/kg.

In certain embodiments, administration of the unit dose is performed by subcutaneous injection using a syringe, needle, pen-type injector, or auto-injector. In certain embodiments, administration of the unit dose is by subcutaneous injection using a syringe. In certain embodiments, administration of the unit dose is performed by subcutaneous injection using a pen injector. In certain embodiments, administration of the unit dose is by subcutaneous injection using an auto-injector.

The present methods are useful for treating or effectively preventing a patient suffering from or at risk of a disease (e.g., genetic risk) treatable with CNP. The present methods can also be used to treat a population of patients suffering from or at risk of such diseases. Such a population may include at least 10, 100, or 1000 patients, or perhaps all patients on behalf of a particular institution.

Although clinical trials may be used to determine dosages and dosing regimens de novo, the present methods may not be performed during the course of the clinical trial. Preferably, the regimen used results in a statistically significant improvement (p.ltoreq.0.05) in at least one sign or symptom (e.g., annual growth rate or Δheight SDS) of the patient in the population relative to a contemporary or historical control population receiving placebo. Improvement in signs or symptoms of disease treatable with CNP in individual patients can also be monitored by comparison to baseline measurements prior to treatment or averages from historical control populations. For example, if the improvement in a patient's sign or symptom exceeds the mean value of the control population and one or both standard deviations of the mean value, the improvement in the patient's sign or symptom may be considered significant. Although clinical trials may be used to determine dosages and dosing regimens de novo, the present methods may not be performed during the course of the clinical trial.

Treatment refers to curing, reducing or inhibiting further worsening of at least one sign or symptom of the disease, or stabilizing at least one sign or symptom of the disease. Treatment may be determined by comparing signs and symptoms of individual patients before (baseline) and after receiving treatment, or by comparing a treated patient population to a control population in a clinical trial or animal model trial.

Prevention refers to preventing the sign or symptom of at least one disease, reducing the phase risk, or delaying the onset of at least one disease in a patient population (or animal model) administered the drug or CNP conjugate of the invention, as compared to a control patient population (or animal model) at risk of disease not treated with the drug or CNP conjugate of the invention. Such an amount is also considered to be prophylactically effective if the results obtained for a single patient treated are more favorable than the average results in a control population of like patients not treated by the method of the invention.

In certain embodiments, the invention is a kit of parts comprising: the unit dosage form of the present invention in a vial (visual), a syringe preloaded with a reconstitution solution, a needle for transferring the reconstitution solution from the syringe to the vial, an administration syringe and an injection needle.

The reconstitution solution is a sterile liquid, such as sterile water.

In certain embodiments, a vial adapter may be used in place of the needle to transfer the reconstituted solution from the syringe into the vial and to transfer the reconstituted unit dosage form from the vial to the administration syringe. In certain embodiments, the vial adapter is a one-piece molded plastic component with a plastic spike or needle for piercing.

The needle used to transfer the reconstituted solution into the vial is a large gauge transfer needle that ensures transfer of the reconstituted solution from the syringe into the vial containing the unit dosage form of the invention. The larger inside diameter of the needle tube ensures an increase in flow rate, thereby reducing the injection force required. In certain embodiments, the needle is a 21Gx25mm needle.

The needle should ensure comfort of subcutaneous injection, preferably with an injection time of less than 10 seconds for the reconstituted unit dosage form. In other words, the injection needle should have a certain gauge and needle length to ensure that the drug is injected into the subcutaneous layer with limited pain. Various injection needles were tested and found to range in length from about 3 to 13mm. Furthermore, it has been found that an injection needle such as a 29G X8 mm needle, a 30G X12 mm needle or a 30G X4 mm needle meets the above requirements.

In certain embodiments, injection of highly concentrated reconstituted unit dosage forms (e.g., unit dosage forms containing about 60.4mg/ml CNP conjugate) can be challenging due to the high viscosity of the CNP conjugate. Applicants have unexpectedly found that even highly concentrated unit dosage forms can be administered using 30G x 12mM or 30G x 4mM needles.

In certain embodiments, the invention is a kit of parts comprising a pen injector or an auto-injector and a vial containing a unit dose of the invention in the form of a prefilled cartridge for use with the pen injector or the auto-injector.

In certain embodiments, the invention is a pen injector or an auto injector comprising the unit dosage form of the invention.

The invention also provides a CNP, CNP conjugate, or a pharmaceutically acceptable salt thereof (CNP or CNP conjugate or pharmaceutically acceptable salt thereof as described herein) or unit dosage form (as described herein) for administration to a patient to provide sustained exposure of an effective level of free plasma CNP (free CNP) between administrations for use in the methods of treatment disclosed herein. Suitably, the CNP, CNP conjugate, or pharmaceutically acceptable salt or unit dosage form thereof is administered via a dosing frequency that maintains the plasma free CNP concentration at a therapeutic level, such as a plasma free CNP concentration of at least about 1pmol/L, between consecutive doses. It will be appreciated that sustained release may be achieved by treatment with repeated administrations of a therapeutically effective amount of a CNP conjugate or a pharmaceutically acceptable salt thereof.

Suitably, the continuous exposure of free CNP is for at least 6 months, such as at least 9 months, such as at least 1 year, via repeated administration of CNP or CNP conjugate or a pharmaceutically acceptable salt or unit dosage form thereof, e.g. once daily, at least once daily, once weekly, at least once weekly, once monthly or at least once monthly.

It will be appreciated that during initial treatment, for example, the first 1,2 or 3 months from the start of treatment (in untreated patients), the minimum free CNP concentration in the plasma may rise gradually (which may be referred to as the break-in period) after each successive administration until a sustained therapeutically effective exposure level is maintained, i.e., the trough and peak values of CNP are in steady state due to the approximately equal amounts of administration and elimination in repeated administration cycles.

Continuous exposure may be obtained when the minimum free CNP concentration (also referred to as trough concentration) in the plasma between successive administrations is maintained at a level of at least about 1pmol/L, or at least about 1.5pmol/L, or at least about 5pmol/L, or about 8pmol/L, or about 15 pmol/L. Or sustained exposure may be obtained when the minimum free CNP concentration (also referred to as trough concentration) in the plasma between successive administrations is maintained at a level of at least about 1.4pmol/L, or at least about 1.6pmol/L, or at least about 6.5pmol/L, or about 9.4pmol/L, or about 19.7 pmol/L.

As demonstrated by the clinical trial data reported herein, such levels of sustained free CNP-38 exposure were found to have therapeutic effects.

In certain embodiments, sustained exposure may be obtained when the minimum free CNP concentration (also referred to as trough concentration) in the plasma between consecutive administrations is, for example, at least about 1.4pmol/L, or at least about 1.6pmol/L, or at least about 6.5pmol/L, or about 9.4pmol/L, or about 19.7 pmol/L.

In certain embodiments, the minimum free CNP concentration (also referred to as trough concentration) in the plasma between consecutive administrations is maintained at a level of at least about 1.8pmol/L, or at least about 2.3pmol/L, or at least about 8.4pmol/L, or at least about 12pmol/L, or at least about 25 pmol/L. In certain embodiments, the minimum free CNP concentration (also referred to as trough concentration) in the plasma between consecutive administrations is maintained at a level of at least about 2.7pmol/L, or at least about 2.4pmol/L, or at least about 9.7pmol/L, or at least about 14.1pmol/L, or at least about 29 pmol/L. In certain embodiments, the minimum free CNP concentration (also referred to as trough concentration) in the plasma between consecutive administrations is maintained at a level of at least about 2.7pmol/L, or at least about 2.4pmol/L, or at least about 9.7pmol/L, or at least about 14.1pmol/L, or at least about 29 pmol/L.

In certain embodiments, the CNP, CNP conjugate or pharmaceutically acceptable salt or unit dosage form thereof is administered according to a dosing regimen (route of administration, frequency and amount) to achieve plasma levels of free CNP, wherein the trough is in the range of 1.8pmol/L to about 29pmol/L and the peak is in the range of about 30pmol/L to about 100pmol/L in steady state. In some embodiments, the trough value ranges from about 8 to about 29pmol/L and the peak value ranges from about 30 to about 50pmol/L. In some embodiments, the trough value ranges from about 18 to about 29pmol/L and the peak value ranges from about 30 to about 50pmol/L. In certain embodiments, the ratio of peak to valley is no more than 1.5:1, 2:1, or 3:1. Optionally, once a week and by subcutaneous route. In certain embodiments, the concentration of free CNP in the patient is measured. The baseline level may be measured prior to treatment and one or more times after initiation of treatment.

The invention also provides a method of reducing the frequency of a cartilage-related adverse event in a patient diagnosed with a cartilage hypoplasia, the method comprising administering a therapeutically effective amount of a CNP, CNP conjugate, or pharmaceutically acceptable salt or unit dosage form thereof, wherein optionally the patient may be a pediatric patient, and/or a patient suffering from open epiphysis.

In certain embodiments, the achondroplasia-related adverse event is selected from the group consisting of: sleep apnea syndrome, ear infections, occipital macropore stenosis and kyphosis. In certain embodiments, the achondroplasia-related adverse event is selected from the group consisting of: sleep apnea syndrome, ear infections, occipital macropore stenosis and kyphosis.

The invention also provides a method of treating sleep apnea syndrome in a patient in need of such treatment, the method comprising administering to the patient a therapeutically effective amount of a CNP, CNP conjugate or pharmaceutically acceptable salt or unit dosage form thereof. For example, the treatment may reduce the incidence of sleep apnea, or reduce the severity of sleep apnea.

The invention also provides a method for treating, or reducing the incidence of, an otic infection in a patient in need of such treatment, the method comprising administering to the patient a therapeutically effective amount of a CNP, CNP conjugate, or pharmaceutically acceptable salt or unit dosage form thereof, thereby treating or reducing the incidence of an otic infection.

The present invention also provides a method for treating occipital macroporous stenosis in a patient in need of such treatment, the method comprising administering to the patient an effective amount of a CNP, CNP conjugate, or pharmaceutically acceptable salt or unit dosage form thereof, thereby treating the occipital macroporous stenosis.

The invention also provides a method of treating kyphosis in a patient in need of such treatment, the method comprising administering to the patient an effective amount of a CNP, CNP conjugate, or pharmaceutically acceptable salt or unit dosage form thereof, thereby treating kyphosis. In certain embodiments, the patient is diagnosed with a bone dysplasia or bone disorder, such as a disorder selected from the group consisting of: achondroplasia, hypoplasia, short stature, noonan syndrome and SHOX deficiency.

In certain embodiments, the patient has been diagnosed with achondroplasia. In certain embodiments, the patient is a pediatric patient with an open epiphysis.

In certain embodiments, the patient is no more than 18 years old or less than 18 years old. In certain embodiments, the patient is no more than 16 years old or less than 16 years old. In certain embodiments, the patient is no more than 14 years old or less than 14 years old. In certain embodiments, the patient is no more than 10 years old or less than 10 years old. In certain embodiments, the patient is no more than 5 years old or less than 5 years old. In certain embodiments, the patient is a hypoplastic cartilage patient aged no more than 2-5 years, such as 2 years, 3 years, or 4 years. In certain embodiments, the patient is a achondroplasia patient at least 6 months of age, such as at least 1 year old or at least 2 years old. In certain embodiments, the patient is a achondroplasia patient with an open epiphysis. In certain embodiments, the patient is a hypoplastic cartilage patient aged no more than 18 years old or less than 18 years old. In certain embodiments, the patient is a hypoplastic cartilage patient aged no more than 16 years or less than 16 years. In certain embodiments, the patient is a achondroplasia patient that is no more than 14 years old or less than 14 years old. In certain embodiments, the patient is a achondroplasia patient that is no more than 10 years old or less than 10 years old. In certain embodiments, the patient is a achondroplasia patient that is no more than 5 years old or less than 5 years old. In certain embodiments, the patient is a hypoplastic cartilage patient aged no more than 2-5 years, such as 2, 3, or 4 years. In certain embodiments, the patient is a achondroplasia patient at least 6 months of age, such as at least 1 year old or at least 2 years old.

The invention is further described by the following non-limiting items.

1. A unit dosage form comprising a therapeutically effective amount of a CNP conjugate, or a pharmaceutically acceptable salt thereof, wherein the CNP moiety is reversibly conjugated to a polymer moiety.

2. The unit dosage form according to claim 1, wherein the unit dosage form comprises a unit dose.

3. The unit dosage form according to claim 2, wherein the unit dosage range is 50-7000 μg CNP.

4. The unit dose according to claim 2 or 3, wherein the unit dose range is 100-5000 μ gCNP.

5. The unit dose according to any one of claims 2-4, wherein the unit dose ranges from about 6 μ gCNP/kg to at least about 100 μg CNP/kg.

6. The unit dose of any one of claims 1-5, wherein the patient has a weight ranging from about 2kg to about 80kg.

7. The unit dose of any one of claims 1-6, wherein the patient has a weight ranging from about 4kg to about 60kg.

8. The unit dosage form of any one of claims 2-7, wherein the unit dosage is about 6 μ gCNP/kg.

9. The unit dosage form of any one of claims 2-7, wherein the unit dosage is about 20 μ gCNP/kg.

10. The unit dosage form of any one of claims 2-7, wherein the unit dosage is about 50 μ gCNP/kg.

11. The unit dosage form of any one of claims 2-7, wherein the unit dosage is about 100 μ gCNP/kg.

12. The unit dosage form of any one of claims 2-7, wherein the unit dosage is about 125 μ gCNP/kg.

13. The unit dosage form of any one of claims 2-12, wherein the unit dosage form is dry or liquid.

14. The unit dosage form of any one of claims 1-13, wherein the unit dosage form is a liquid.

15. The unit dosage form of any one of claims 1-14, wherein the pH of the unit dosage form ranges from about 4 to 6.

16. The unit dosage form according to any one of claims 1-15, wherein the pH of the unit dosage form is about 5.

17. The unit dosage form of any one of claims 2-16, wherein the unit dosage has a volume ranging from about 0.01ml to about 1.1ml.

18. The unit dosage form of any one of claims 2-17, wherein the unit dosage has a volume ranging from about 0.01ml to about 0.75ml.

19. The unit dosage form of any one of claims 2-18, wherein the unit dosage has a volume ranging from about 0.01ml to about 0.50ml.

20. The unit dosage form of any one of claims 1-19, wherein the unit dosage form is contained within a single vial.

21. The unit dosage form according to any of claims 2-20, wherein the incidence of hypotension is less than 10%, preferably less than 8%, most preferably less than 5%, even more preferably less than 3% after administration of the unit dose.

22. The unit dosage form of any one of claims 1-21, wherein the CNP conjugate or pharmaceutically acceptable salt thereof is a compound of formula (Ia) or (Ib):

Wherein the method comprises the steps of

-D is a CNP moiety;

-L ¹ -is a reversible linker moiety;

-L ² -is a single chemical bond or a spacer moiety;

-Z is a polymeric moiety;

Y is an integer selected from the group consisting of: 1.2, 3, 4 and 5.

23. The unit dosage form according to any one of claims 1-22, wherein the CNP conjugate or pharmaceutically acceptable salt is a compound of formula (IIf):

Wherein the method comprises the steps of

Each-Z ^a is

Wherein the method comprises the steps of

Each c1 is an integer independently from 200 to 250.

24. The unit dosage form of any one of claims 1-23, wherein the CNP conjugate is a compound of formula (IIf'):

Wherein the method comprises the steps of

Each Z ^a is

Wherein the method comprises the steps of

Each c1 is independently an integer from 200 to 250.

25. The unit dosage form of any one of claims 1-24, wherein the CNP conjugate is a compound of formula (IIf'):

Wherein the method comprises the steps of

Each Z ^a is

Wherein the method comprises the steps of

Each c1 is an integer independently from 200 to 250.

26. The unit dosage form of any one of claims 1-25, wherein each c1 is independently an integer from 220-245.

27. The unit dosage form of any one of claims 1-26, wherein each c1 is about 225.

28. The unit dosage form according to any one of claims 1-27 for use in the treatment of one or more diseases treatable with CNP.

29. The unit dosage form according to any one of claims 1-28 for use in the treatment of achondroplasia.

30. A method of treating, reducing the risk of, or delaying the progression of a disease treatable with CNP in a human patient, the method comprising the step of administering the unit dosage form of any one of claims 1-27.

31. The method of claim 30, wherein the step of administering a unit dose does not occur during a clinical trial.

32. The method of claim 30, wherein the unit dosage form is administered subcutaneously.

33. The method of claim 30 or 32, wherein the unit dosage form is administered once per week.

34. The method of claim 30 or 32, wherein the unit dosage form is administered once a week for at least one year.

35. The method of claim 30, 33 or 34, wherein the unit dosage form is administered once a week until the patient is over 18 years old.

36. The method of any one of claims 30 or 32-35, wherein the disease is selected from the group consisting of: achondroplasia, hypoplasia, short stature, noonan syndrome and SHOX deficiency.

37. The method of any one of claims 30 or 32-35, wherein the disease is achondroplasia.

38. The method for treating a population of human patients according to any one of claims 30 or 32-37, wherein each patient in the population is administered a unit dosage form of any one of claims 1-27.

The invention is further described by the following non-limiting items.

1. A method of treating achondroplasia in a human patient, the method comprising the step of administering a CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 100 μ gCNP/kg, wherein the CNP conjugate is a compound of formula (IIf'):

Wherein the method comprises the steps of

Each Z ^c is

And wherein

Each c1 is an integer independently from 200 to 250,

Wherein the unit dose is administered once a week.

2. A method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate or pharmaceutically acceptable salt thereof of the following compounds:

(a) Formula (IIf):

Wherein the method comprises the steps of

Each Z ^a is

And wherein

Each c1 is independently an integer from 200 to 250,

Or alternatively

(B) Formula (IIf'):

Wherein the method comprises the steps of

Each Z ^c is

And wherein

Each c1 is independently an integer from 200 to 250,

The unit dose was 100 μg CNP/kg, with the unit dose administered once a week.

3. A method of increasing long bone growth in a human patient, the method comprising the step of administering a CNP conjugate of formula (IIf) or (IIf '), or a pharmaceutically acceptable salt thereof, wherein each of (IIf) and (IIf') is as defined in item 2, in a unit dose of 100 μg CNP/kg, wherein the unit dose is administered once per week.

4. A method of treating skeletal dysplasia, such as achondroplasia, comprising administering a unit dosage form comprising a CNP conjugate of formula (IIf) or (IIf ') or a pharmaceutically acceptable salt thereof, wherein each of (IIf) and (IIf') is as defined in claim 2, the unit dosage is 100 μ gCNP/kg, wherein the unit dosage form is administered once a week to a human patient suffering from open epiphysis, and the treatment reduces the incidence of an adverse event associated with achondroplasia in the human patient.

5. A method of increasing the growth rate of a achondroplasia human patient, the method comprising the step of administering a CNP conjugate of formula (IIf) or (IIf '), or a pharmaceutically acceptable salt thereof, wherein each of (IIf) and (IIf') is as defined in claim 2, administered at a unit dose of 100 μg CNP/kg for at least 52 weeks, wherein the unit dose is administered to a human patient having an open epiphysis, and the treatment reduces the incidence of an adverse event associated with achondroplasia in the human patient.

6. A CNP conjugate, or a pharmaceutically acceptable salt thereof, for use in a method of treating achondroplasia in a human patient, the method comprising the step of administering the CNP conjugate, or a pharmaceutically acceptable salt thereof, at a unit dose of 100 μg CNP/kg, wherein the CNP-conjugate is a compound of formula (IIf'):

Wherein the method comprises the steps of

Each Z ^c is

And wherein

Each c1 is independently an integer from 200 to 250,

Wherein the unit dose is administered once a week.

7. A CNP conjugate or a pharmaceutically acceptable salt thereof for use in a method of increasing the growth rate of a human patient, the method comprising the step of administering a CNP conjugate of the following compound, or a pharmaceutically acceptable salt thereof:

(a) Formula (IIf):

Wherein the method comprises the steps of

Each Z ^a is

And wherein

Each c1 is independently an integer from 200 to 250;

Or alternatively

(B) Formula (IIf'):

Wherein the method comprises the steps of

The unlabeled dashed line represents the nitrogen provided by the formation of an amide bond attached to the lysine side chain at position 26 of the CNP moiety of SEQ ID NO. 24;

Wherein the method comprises the steps of

Each Z ^c is

And wherein

Each c1 is independently an integer from 200 to 250,

The unit dose was 100 μg CNP/kg, with the unit dose administered once a week.

8. A CNP conjugate, or a pharmaceutically acceptable salt thereof, for use in a method of increasing long bone growth in a human patient, the method comprising the step of administering a CNP conjugate of formula (IIf) or (IIf '), or a pharmaceutically acceptable salt thereof, wherein each of (IIf) and (IIf') is as defined in claim 7, in a unit dose of 100 μg CNP/kg, wherein the unit dose is administered once per week.

9. A CNP conjugate, or a pharmaceutically acceptable salt thereof, for use in a method of treating skeletal dysplasia, such as achondroplasia, the method comprising administering a unit dosage form comprising a CNP conjugate of formula (IIf) or (IIf '), or a pharmaceutically acceptable salt thereof, wherein each of (IIf) and (IIf') is as defined in claim 7, the unit dosage amount being 100 μg CNP/kg, wherein the unit dosage form is administered once per week to a human patient having an open epiphysis, and the treatment reduces the incidence of an adverse event associated with achondroplasia in the human patient.

10. A CNP conjugate, or a pharmaceutically acceptable salt thereof, for use in a method of increasing the growth rate of a human patient suffering from achondroplasia, the method comprising the step of administering a CNP conjugate of formula (IIf) or (IIf '), or a pharmaceutically acceptable salt thereof, wherein each of (IIf) and (IIf') is as defined in claim 7, at least 52 weeks at a unit dose of 100 μg CNP/kg, wherein the unit dose is administered to a human patient suffering from open epiphysis, and the treatment reduces the incidence of an adverse event associated with achondroplasia in the human patient.

11. The method of any one of claims 2-5, or the CNP conjugate for use of any one of claims 7-10, or a pharmaceutically acceptable salt thereof, wherein the CNP conjugate or pharmaceutically acceptable salt thereof has the formula (IIf'):

Wherein the method comprises the steps of

Each Z ^c is

And wherein

Each c1 is independently an integer from 200 to 250.

12. The method according to any one of claims 2 to 4, or the CNP conjugate or pharmaceutically acceptable salt thereof for use according to any one of claims 7 to 9, wherein the patient has achondroplasia.

13. The method of any one of claims 1-5, 11 or 12, or the CNP conjugate for use according to any one of claims 6-10, 11 or 12, or a pharmaceutically acceptable salt thereof, wherein the patient is a pediatric patient or infant.

14. The method according to claim 13, or the CNP conjugate or pharmaceutically acceptable salt thereof for use according to claim 13, wherein the pediatric patient is between 2 and 10 years old, such as between 2 and 5 years old, or such as between 5 and 10 years old.

15. The method of any one of claims 1-5, or 11-14, or CNP conjugate for use of any one of embodiments 6-14, or a pharmaceutically acceptable salt thereof, wherein the CNP conjugate or pharmaceutically acceptable salt thereof is administered subcutaneously.

16. The method of any one of claims 1-5, or 11-15, or the CNP conjugate for use of any one of claims 6-15, or a pharmaceutically acceptable salt thereof, wherein the unit dosage form comprises succinic acid and trehalose.

Examples

Materials and methods

All materials are commercially available unless otherwise indicated.

Example 1: synthetic compound (1)

Compound (1) was synthesized as described for conjugate 11i in WO 2017/118693.

Compound (1) may also be represented as follows:

Wherein n ranges from about 200 to about 250, such as 200-250, corresponding to about 10 kDa.

Compound (1) is a long-acting, essentially inactive prodrug consisting of CNP-38 temporarily bound to the carrier molecule mPEG via a reversible linker. mPEG acts as an inert carrier, prolonging the circulation time of CNP-38 in vivo by shielding, thereby minimizing the clearance of CNP-38 and largely inactivating CNP-38 prior to its release. Compound (1) releases active CNP in a controlled manner by autolysis of the reversible linker, depending on physiological pH and temperature. Thus, compound (1) was designed to provide a sustained exposure of active CNP for 7 days, thus achieving the best pharmacokinetic profile used in ACH. The pharmaceutical product formulation comprising compound (1) is a lyophilized powder in a disposable vial, i.e. 3.9mg CNP-38 per vial of compound (1). Before use, the lyophilized powder must be reconstituted with sterile injection water in a prefilled syringe. The concentration after reconstitution was 3.6mg CNP-38/ml. The solution will be administered via a syringe and needle by subcutaneous injection. For compound (1), the dose level CNP/kg or CNP-38/kg refers to the amount of CNP fraction (. Mu.g) present in the dose of compound (1) per kg of patient body weight.

EXAMPLE 2-1 phase Studies

To date, phase 1 randomized, double-blind, placebo-controlled, single escalation dose trials have been completed for healthy adult male subjects. The test was designed to evaluate the safety, tolerability and pharmacokinetics of single subcutaneous injection of compound (1) in unit doses of no more than 150 μg CNP/kg. Compound (1) was well tolerated at all study doses. A single dose of compound (1) delivered free CNP-38 in a dose-dependent manner and had a long and dose-independent average apparent t _1/2 of 120 hours. Among the subjects receiving compound (1), the most common (> 10% of subjects) reported adverse events (TEAE) caused by treatment are headache, contact dermatitis, postural dizziness, (orthostatic) tachycardia, dizziness and orthostatic hypotension. Most TEAEs are mild or moderate. No serious Adverse Events (AEs) or deaths were reported, and no AEs were considered dose limiting or resulted in termination of the trial. The local tolerability findings (finding) were mainly mild to moderate redness. Two adverse events associated with the injection site were reported: mild injection site pain (10 μg CNP/kg) and mild injection site discomfort (75 μg gCNP/kg). No clinically relevant dose-dependent trend of blood pressure, heart rate or safety laboratory test results was observed at all study doses. Furthermore, no evidence of an immunogenic response was observed in any of the subjects.

EXAMPLE 3-2 phase Studies

A multicentric, double blind, randomized, placebo-controlled, dose escalation trial of phase 2 for evaluating the safety, efficacy and pharmacokinetics of a subcutaneous dose of compound (1) once a week for 52 weeks in pre-pubertal children with achondroplasia, followed by an open label extension period. The study recruited at least 60 pre-pubertal boys and girls with ACH aged 2-10 years. The clinical trial is listed on www.clinicaltrials.gov, clinical trials.gov identifier: NCT04085523. The purpose of this study is as follows:

The main components are as follows:

At week 52, in pre-pubertal children with hypochondrogenesis (ACH)

Safety of determining once weekly Subcutaneous (SC) doses of Compound (1)

Evaluating the effect of once weekly subcutaneous doses of compound (1) on annual avatar high speed (AHV) secondary:

Evaluation of the effect of weekly subcutaneous dose of Compound (1) on the body proportion (upper body to lower body ratio) of pre-pubertal ACH children at week 52

Evaluation of the Pharmacokinetic (PK) Properties of Compound (1) at once weekly subcutaneous doses

Evaluation of the potential immunogenic response of Compound (1) at once weekly subcutaneous doses

Inclusion criteria

Clinical diagnosis and Gene validation of ACH

2. The age of the screening visit is 2-10 years (inclusive)

3. Screening visit was pre-pubertal (girl breast developed to stage 1, or girl testicle volume <4 ml)

4. Can stand without help

5. Caregivers would like and be able to study subcutaneous injections of drugs

6. Written informed consent signed by parents or legal guardians of the participants, and written consent provided by the participants in accordance with the requirements of the institutional review board/human research ethics committee/independent ethics board (IRB/HREC/IEC)

Exclusion criteria

1. The clinical significance of screening is found:

the expected need for surgical intervention during the participation trial, or

Is musculoskeletal in nature, such as a Sortt-Harris (Salter-Harris) fracture and severe hip pain, or

Researchers or medical supervisors consider other conditions that may lead to participants unsuitable for receiving study drugs or for receiving trial related procedures

2. Human growth hormone (hGH) or other medications known to affect height or body proportions have been received at any time (> 3 months)

3. Within 6 months prior to screening visit, any doses of drug intended to affect height or body proportions were received

4. Any study drug or device intended to affect height or body proportion was received at any time

5. A history or presence of growth plate damage or disease other than ACH that affects the growth potential of long bones

6. Any bone related surgical history that affects the growth potential of long bones, such as orthopedic reconstructive surgery and osteotomies (limb extension allowing complete recovery with bone healing time of at least 12 months, occipital majorities Kong Jianya and laminectomy allowing complete recovery with bone healing time of at least 6 months, 8-plate diaphyseal fixation history, but the plates must be removed prior to screening and healing time of at least 4 weeks).

7. Has skeletal dysplasia other than ACH, or a medical condition known to lead to short stature or abnormal growth [ such as severe achondroplasia with slow-developing and acanthosis nigricans (SADDAN), hypochondral development, growth hormone deficiency, tener's syndrome, pseudo-achroplasia, inflammatory bowel disease, chronic renal insufficiency, active celiac disease ¹, vitamin D deficiency ², untreated hypothyroidism ³, poor control of diabetes (HbAlc > 8.0%) or diabetic complications ⁴ ]

¹ Patients with celiac disease who are allowed to respond to gluten-free diets

² Vitamin D deficiency or deficiency that allows treatment with the supplement. Vitamin D deficiency is defined as 25 (OH) D levels <20ng/ml (< 49.9 nmol/L) and vitamin D deficiency is defined as 25 (OH) D levels 20-30ng/ml (49.92-74.86 nmol/L). Participants with vitamin D deficiency or deficiency must receive a vitamin D regimen prior to randomization.

³ Participants with hypothyroidism must be in clinical thyroidism 3 months prior to screening visit and, according to the opinion of the investigator, have achieved the catch-up growth expected of thyroxine replacement therapy

⁴ Participants with diabetes must have received a stable medication regimen for 3 months prior to randomization (allowing adjustment of the dose, but not allowing addition or cessation of medication during this period)

8. In addition to basal cell epithelial tumors/cancers or squamous skin cancers that have been completely resected, there is a history of or presence of malignancy, and medical records show no recurrence within 12 months

9. There is the following history or presence of:

Chronic anaemia (iron deficiency anaemia allowed to heal)

At the discretion of the investigator, suffering from severe cardiovascular disease such as congenital heart disease (allowing the repair of uncomplicated arterial catheter patency and atrial or ventricular septal defects), aortic insufficiency, clinically significant arrhythmias, congestive heart failure of NYHA class II or above, or other conditions affecting blood pressure or heart rate regulation

Conditions affecting hemodynamic stability (e.g., autonomic dysfunction, intolerance to erectile dysfunction)

History of chronic renal insufficiency

Chronic or recurrent diseases that can affect hydration or volume status. This may include conditions associated with reduced nutrient intake or increased volume loss

Fracture occurred within 6 months before screening visit (finger fracture is within 2 months)

Researchers believe that any disease or condition may lead to incomplete completion of the test by the participants, may confuse the interpretation of the test results, or present undue risk of receiving the study medication

10. Children have severe electrocardiographic abnormalities, including evidence of past myocardial infarction, left ventricular hypertrophy, T-wave flattening (particularly in the lower wall leads), or multiple slight nonspecific ST-T-wave changes, or:

QRS >90 milliseconds (msec)

QT interval >440 ms corrected using friedresia (friedricia) formula (QTcF)

PR interval >170 ms

Complete right bundle branch block or left bundle branch block

11. Long-term (> 4 weeks) or repeated (more than twice a year) oral corticosteroid therapy (low and medium doses are allowed to inhale corticosteroid; high doses are not allowed to inhale corticosteroid) is required or expected to be required during the participation trial.

12. Drugs known to prolong QT/QTc intervals were used within 7 days or 5 half-lives (whichever was longer) prior to screening visit.

13. Is being treated by any drug which affects blood pressure or heart rate

14. Known allergy to the components of the study drug [ trehalose, tris (hydroxymethyl) aminomethane, succinate and PEG ]

15. Researchers believe that children are hampered from following the test requirements or from any other reason to complete the test

The test is a multi-center test comprising two treatment sessions. One was a 52 week double-blind, randomized, placebo-controlled, dose escalation trial that evaluated the subcutaneous administration of no more than 5 different dose levels of compound (1) per week in pre-pubertal children with ACH between 2 and 10 years of age.

Table 1 Unit dose of CNP

* If necessary, the dosage will be determined based on the newly emerging data

TABLE 2 baseline demographic data-end of year 2021 status

Table 2 describes baseline demographics for groups 1, 2, and 3, each of which also contained placebo subjects. The baseline demographics of groups 1, 2, 3 and 4 and placebo subjects after the trial was completed and blind data was uncovered are shown in table 3.

TABLE 3 baseline demographic data after trial blinding

Example 4 measurement of vital signs

The participants should rest for at least 5 minutes before measuring vital signs, and vital signs should be measured before any blood is drawn. The following vital signs should be measured:

Heart rate (measured in beats per minute)

Blood pressure (sitting blood pressure, measured in mmHg)

Temperature [ measured in degrees Celsius (C.) or Fahrenheit (F.) ]

All vital signs described above were measured at each visit. On the first visit, the upright Heart Rate (HR) and systolic/diastolic blood pressure will also be measured when the participants rest for 5min (preferably supine) and measured again 3min after standing before administration of the study medication to assess orthostatic hypotension. Orthostatic hypotension is defined as a decrease in Systolic Blood Pressure (SBP) of ≡20-mMHg (STEWART ET al., pediatrics,141,1-13,2018). The accompanying tachycardia is defined as an incremental change in heart rate of > 40bpm, and an absolute upright heart rate of > 130bpm (applicable 13 years old and below) (Singer et al, journal of Pediatrics,160,222-226,2012). Furthermore, at visit 1 and visit 7 (during the extended period of open label), all vital signs must be monitored 1 hour and 2 hours after the study drug injection. For participants who underwent blood collection for pharmacokinetic analysis 8, 24 and 48 hours after the first dose (visit 1 at random and visit 7 at open label extension), the systolic/diastolic blood pressure and heart rate were measured prior to each PK blood collection.

Example 5 Security

In a follow-up visit of up to 65 weeks for 57 patients enrolled in the trial, no withdrawal or termination of the trial was reported for any reason. No serious adverse events associated with the test drug occurred. Also, injection site tolerance is generally very good, totaling 13 Adverse Events (AEs) associated with the test drug or procedure, including 8 injection site reactions occurring in 1900 injections. Adverse Events (AEs) were not reported on erectility. Importantly, no anti-CNP antibodies present in the treatment were detected.

EXAMPLE 6 pharmacokinetic data

During the study, all subjects (if body weight allows) were given sparse PK samples before and 8, 24, 48 hours after initial dosing and every 3 months during the study, 1 month (trough), 2 months (any time in the week), 3 months (any time in the week), 6 months (trough), 9 months (any time) and 12 months (trough) +. Sparse PK data was characterized using a population PK model. Dose-related increases in exposure were observed in the four dosing groups (6. Mu.g/kg, 20. Mu.g/kg, 50. Mu.g/kg and 100. Mu.g/kg). Sustained exposure to free CNP was observed, and the half-life of free CNP was estimated to be about 110 hours.

TABLE 4 predicted median free CNP C for subjects _max

TABLE 5 estimated free CNP half-life in subject

Dose μg/kg/week	Estimated half-life (h)
		6	101
20	106
		50	108
100	110

Blood samples for determination of CNP-38 are acidified with a citrate buffer immediately after collection to stabilize the prodrug in the sample, thereby avoiding further release of CNP-38 from the prodrug. CNP-38 in plasma (heparin) was determined by protein precipitation and solid phase extraction and analyzed using liquid chromatography and tandem mass spectrometry detection. CNP-38 (38 amino acid peptide) was used as reference material, and deuterium labeled CNP-38 (D30-CNP-38) was used as Internal Standard (IS). Positive ions were monitored using a multiple reaction monitoring mode. Quantitative analysis was performed using the peak area ratio of analyte to Internal Standard (IS). Calibration curve fitting was performed using 1/concentration weighted linear regression. The calibration range for non-acidified plasma is 1.38-138pmol/1.

TABLE 6 preliminary average free CNP levels for NCT04085523

Observations below the lower limit of quantitation (LLOQ), have been set to LLOQ (1.38 pM)

* All observations were below LLOQ

NOBS: number of observations for a given week

Time (days): median time after dose 1

Subjects not including placebo

Example 7: neutralizing antibodies

All subjects in the trial were monitored for CNP binding and neutralizing antibodies (as relevant). No anti-CNP binding antibodies were detected after repeated weekly exposures to compound (1) for 1-9 months. Samples were taken in all subjects at screening visit, prior to initial dosing, and at 1, 3, 6, 9 months post dosing + every 3 months during the study. Samples were analyzed using a well-validated anti-CNP antibody assay. All post-dosing samples were confirmed negative for anti-CNP antibodies.

Description of the assay

A sensitive assay was developed for detecting, validating and quasi-quantifying binding antibodies to CNP-38 (and CNP-22) and designed to bridge ECL (electrochemiluminescence) immunoassays. Prior to analysis of clinical samples, the assay has been fully validated against EMAA and FDA regulatory requirements. In this method, biotinylated and ruthenized CNP-38 was prepared as master mix. Samples were incubated in the master mix at the lowest required dilution (MRD) to form complexes, then inoculated onto MSD streptavidin plates to which biotinylated CNP-38 could also bind. Samples were then tested by binding to ruthenium labelled CNP-38 in the complex using MSD S600 plate reader. The presence or absence of anti-drug antibodies is determined by comparing the signal to a statistically derived threshold (determination threshold).

The height of the subjects participating in the study of example 3 was measured using a wall-mounted altimeter. All visits to the same child use the same device. All instruments are calibrated prior to use according to local requirements or instruction of the instrument manufacturer. These data demonstrate that subcutaneous treatment of 2-10 year old achondroplasia patients with 100 μg CNP-38/kg for 52 weeks or more per week with compound (1) has a beneficial effect on bone growth, corresponding to a annual growth rate of 5.42 cm/year, which is significantly higher than that of the placebo-treated group, 4.35 cm/year (p=0.0218).

TABLE 7 superiority of Compound (1) 100 μg CNP-38/kg/week in AGV compared to placebo

Also, for the 100 μg CNP-38/kg/week group, an acceleration trend was observed for AGVs during the 6-12 month period compared to AGVs during the 0-6 month period, which were 5.696 and 5.167 cm/year, respectively.

It is also apparent that AGVs between different age groups are comparable and dose dependent (table 8).

TABLE 8 AGV comparison between age groups

Furthermore, compound (1) administered at 100 μg CNP-38/kg/week showed superiority in terms of achondroplasia-specific height SDS changes compared to placebo. A description of the SDS changes in the achondroplasia-specific height has been disclosed previously (Orphanet J. Rare Dis.2021;16 (1): 522).

TABLE 9

* ACH-specific height SDS

TABLE 10 overview of adverse events occurring in treatment

* Unreported TEAE of grade 3 (severe) or grade 4 (life threatening).

* Adverse events reported by researchers and related to underlying diseases

Surprisingly, treatment with compound (1) was associated with a reduction in the reported adverse events occurring in connection with the treatment of hypoplasia, from 60% in the placebo group to 9.1% in the 100 ug/kg/week dose group.

TABLE 11 adverse events associated with treatment

* Injection site reactions include the following preferred terms: injection site reactions, injection site pain, injection site erythema, injection site discoloration, injection site bleeding, and injection site swelling.

The compound (1) has good injection tolerance and low incidence of Injection Site Reaction (ISR). In the case of administration of injections over 2,000 times, 11 ISRs occurred in only 8 patients. This is comparable to the incidence of placebo-group events, with 1 ISR reported per 2 subjects. The severity of all these events was reported as mild.

Example 8: determination of pharmacokinetic data and minimum sustained free CNP exposure

During the test, sparse blood samples were collected at various time points of the dosing interval and the concentration of free CNP-38 was determined using the method described in example 6. Blood samples taken 6.5-7.5 days after the last dose were sub-set and the range of free CNP-38 plasma concentrations is shown in Table 11 (PK data cut-off time: 2022, 11, 15 days). Thus, for weekly administration of compound (1), this represents a sustained minimum plasma free CNP-38 concentration.

TABLE 11

The values are provided in pmol/LCNP-38. The free CNP value can be calculated by a product factor of about 30 to about 50% to consider the degradation products of free CNP-38 and natural CNP that are still functional. The values of the 150 μg/kg/week dose were simulated using the population PK model for compound (1). The lower value in the range is the lowest free CNP-38 concentration, also known as the trough level or trough concentration. Number of subjects enrolled in observations over a specified time interval: 6. 20, 50 and 100 μg/kg dose groups of 4, 8, 10 and 6, respectively.

Claims

1. A unit dosage form comprising a therapeutically effective amount of a CNP conjugate or a pharmaceutically acceptable salt thereof, wherein the CNP portion is reversibly conjugated to a polymer portion, wherein the unit dosage unit comprises a unit dose ranging from about 12.3 nmol CNP/kg to at least about 37 nmol CNP/kg.

2. The unit dosage form according to claim 1, wherein the unit dose range is 12.3 nmol CNP/kg-36.9 nmol CNP/kg.

3. The unit dosage form according to claim 1 or 2, wherein the unit dose is at least 24.6 nmol CNP/kg.

4. The unit dosage form according to any one of claims 1-3, wherein the unit dose is about 24.6 nmol CNP/kg.

5. The unit dosage form according to any one of claims 1 to 4, wherein the unit dose is 24.6 nmol CNP/kg.

6. The unit dosage form according to any one of claims 1-5, wherein the unit dose range is from about 50 μg CNP/kg to about 150 μg CNP/kg.

7. The unit dosage form according to any one of claims 1 to 6, wherein the unit dose range is 50 μg CNP/kg - 150 μg CNP/kg.

8. The unit dosage form according to any one of claims 1-7, wherein the unit dose is about 100 μg CNP/kg.

9. The unit dosage form according to any one of claims 1 to 8, wherein the unit dose is 100 μg CNP/kg.

10. The unit dosage form according to any one of claims 1 to 9, for administration to a patient having a body weight ranging from about 2 kg to about 80 kg.

11. The unit dosage form according to any one of claims 1 to 10, for administration to a patient having a body weight ranging from about 4 kg to about 60 kg.

12. The unit dosage form according to any one of claims 1-11, wherein the unit dosage form is dry or liquid.

13. The unit dosage form according to any one of claims 1-12, wherein the unit dosage form is liquid.

14. The unit dosage form according to any one of claims 1-13, wherein the pH of said unit dosage form is in the range of about 4-6.

15. The unit dosage form according to any one of claims 1-14, wherein the pH of said unit dosage form is about 5.

16. The unit dosage form according to any one of claims 1-15, wherein the volume of the unit dose ranges from about 0.01 ml to about 1.1 ml.

17. The unit dosage form according to any one of claims 1-16, wherein the volume of the unit dose ranges from about 0.01 ml to about 0.75 ml.

18. The unit dosage form according to any one of claims 1-17, wherein the volume of the unit dose ranges from about 0.01 ml to about 0.50 ml.

19. The unit dosage form according to any one of claims 1-18, wherein the unit dosage form is contained in a single vial.

20. The unit dosage form according to any one of claims 1-19, wherein the CNP conjugate or a pharmaceutically acceptable salt thereof is a compound of formula (Ia) or (Ib):

in

-D is the CNP part;

-L ¹ - is a reversible linker moiety;

-L ² - is a single chemical bond or a spacer moiety;

-Z is a polymer moiety;

x is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16; and

y is an integer selected from the group consisting of 1, 2, 3, 4 and 5.

21. The unit dosage form according to any one of claims 1-20, wherein the CNP conjugate or pharmaceutically acceptable salt is a compound of formula (IIf):

in

The unlabeled dashed line represents the connection to the nitrogen provided by the lysine side chain at position 26 of the CNP portion of SEQ ID NO: 24 through formation of an amide bond; and

The dashed line marked with an asterisk indicates connection to -Z with the following structure,

in

Each -Z ^a is

in

Each c1 is independently an integer between 200 and 250.

22. The unit dosage form according to any one of claims 1 to 21, wherein the CNP conjugate is a compound of formula (IIf'):

in

Each ^Za is

in

Each c1 is independently an integer between 200 and 250.

23. The unit dosage form according to any one of claims 1 to 22, wherein the CNP conjugate is a compound of formula (IIf'):

in

Each ^Za is

in

Each c1 is independently an integer between 200 and 250.

24. The unit dosage form according to any one of claims 21-23, wherein each c1 is independently an integer from 220 to 245.

25. The unit dosage form according to any one of claims 21-24, wherein each c1 is about 225.

26. The unit dosage form according to any one of claims 1 to 25, wherein upon administration of said unit dosage form, the incidence of hypotension is less than 10%, preferably less than 8%, most preferably less than 5%, even more preferably less than 3%, such as less than 1% or absent.

27. The unit dosage form according to any one of claims 1 to 26, for use in the treatment of one or more diseases treatable with CNP.

28. The unit dosage form according to any one of claims 1 to 26, for use in the treatment of skeletal dysplasia.

29. The unit dosage form according to any one of claims 1 to 26, for use in the treatment of a disease selected from the group consisting of achondroplasia, hypochondroplasia, short stature, Noonan syndrome and SHOX deficiency.

30. The unit dosage form according to any one of claims 1 to 26, for use in the treatment of achondroplasia.

31. The unit dosage form according to any one of claims 1-26, for use in treating achondroplasia, said unit dosage form comprising at least 24 nmol CNP/kg unit dose of a CNP conjugate or a pharmaceutically acceptable salt thereof, wherein said unit dosage form is administered to a human patient and said administration results in an annualized growth velocity (AGV) of about 5.4 cm/year.

32. The unit dosage form for use according to claim 31, wherein the annualized growth velocity is about 5.42 cm/year.

33. The unit dosage form for use according to claim 31 or 32, wherein the annualized growth velocity is 5.42 cm/year.

34. The unit dosage form for use according to any one of claims 31 to 33, wherein the unit dose is about 24.6 nmol CNP/kg.

35. The unit dosage form for use according to any one of claims 31 to 34, wherein the unit dose is 24.6 nmol CNP/kg.

36. The unit dosage form for use according to any one of claims 31 to 35, wherein the unit dose is about 100 μg CNP/kg, and the CNP conjugate or a pharmaceutically acceptable salt thereof has formula (IIf) or (IIf').

37. The unit dosage form for use according to any one of claims 31 to 36, wherein the unit dose is 100 μg CNP/kg, and the CNP conjugate or a pharmaceutically acceptable salt thereof has formula (IIf').

38. The unit dosage form for use according to any one of claims 31 to 37, wherein the unit dosage form is administered once a week to pediatric patients aged 2 to 10 years.

39. The unit dosage form according to any one of claims 1-26, for use in treating achondroplasia, said unit dosage form comprising at least 24 nmol CNP/kg unit dose of a CNP conjugate or a pharmaceutically acceptable salt thereof, wherein said unit dosage form is administered to a human patient aged 2-5 years, and said administration results in an annualized growth velocity (AGV) of about 5.95 cm/year.

40. The unit dosage form for use according to claim 39, wherein the annualized growth velocity is 5.95 cm/year.

41. The unit dosage form for use according to claim 39 or 40, wherein said administration is performed once a week.

42. The unit dosage form for use according to any one of claims 39 to 41, wherein the unit dose is about 24.6 nmol CNP/kg.

43. The unit dosage form for use according to any one of claims 39 to 42, wherein the unit dose is 24.6 nmol CNP/kg.

44. The unit dosage form for use according to any one of claims 39 to 43, wherein the unit dose is about 100 μg CNP/kg, and the CNP conjugate or a pharmaceutically acceptable salt thereof has formula (IIf) or (IIf').

45. The unit dosage form for use according to any one of claims 39 to 44, wherein the unit dose is 100 μg CNP/kg and the CNP conjugate or a pharmaceutically acceptable salt thereof has formula (IIf').

46. A unit dosage form according to any one of claims 1-26 for use in treating achondroplasia, said unit dosage form comprising a CNP conjugate or a pharmaceutically acceptable salt thereof in a unit dose range of 12.3 nmol CNP/kg-36.9 nmol CNP/kg, wherein said unit dosage form is administered to a human patient with open epiphysis, and said treatment reduces the incidence of adverse events associated with achondroplasia in the human patient.

47. The unit dosage form for use according to claim 46, wherein the unit dose is 24.6 nmol CNP/kg.

48. The unit dosage form for use according to claim 46 or 47, wherein the unit dose is about 100 μg CNP/kg and the CNP conjugate or a pharmaceutically acceptable salt thereof has formula (IIf) or (IIf').

49. The unit dosage form for use according to any one of claims 46 to 48, wherein the unit dose is 100 μg CNP/kg and the CNP conjugate or a pharmaceutically acceptable salt thereof has formula (IIf').

50. A unit dosage form according to any one of claims 1-26 for use in treating achondroplasia, said unit dosage form comprising a CNP conjugate or a pharmaceutically acceptable salt thereof in a unit dose range of 12.3 nmol CNP/kg-36.9 nmol CNP/kg, wherein said unit dosage form is administered once a week by subcutaneous injection to a human patient with open epiphysis, and wherein each administration is associated with a frequency of injection site reactions of less than 3%, such as less than 2%, such as less than 1% or the absence of injection site reactions.

51. The unit dosage form for use according to claim 50, wherein the unit dose is 24.6 nmol CNP/kg.

52. The unit dosage form for use according to claim 50 or 51, wherein the unit dose is about 100 μg CNP/kg, and the CNP conjugate or a pharmaceutically acceptable salt thereof has formula (IIf) or (IIf').

53. The unit dosage form for use according to any one of claims 50-52, wherein the unit dose is 100 μg CNP/kg, and the CNP conjugate or a pharmaceutically acceptable salt thereof has formula (IIf').

54. The unit dosage form for use according to any one of claims 50-53, wherein the injection site reaction comprises injection site pain, injection site erythema, injection site discoloration, injection site bleeding or injection site swelling.

55. A kit comprising the unit dosage form of any one of claims 1-26, a syringe prefilled with a reconstitution solution, a needle for transferring the reconstitution solution from the syringe to the vial, an administration syringe, and an injection needle.

56. A method of treating achondroplasia in a human patient, the method comprising the step of administering a CNP conjugate or a pharmaceutically acceptable salt thereof at a unit dose of about 6 μg CNP/kg to about 150 μg CNP/kg.

57. The method of claim 56, wherein the unit dose is 100 μg CNP/kg.

58. The method of claim 56 or 57, wherein the CNP conjugate or a pharmaceutically acceptable salt thereof is administered once a week.

59. The method of any one of claims 56-58, wherein the CNP conjugate or a pharmaceutically acceptable salt thereof is administered subcutaneously.

60. The method of any one of claims 56-59, wherein the CNP conjugate or a pharmaceutically acceptable salt thereof is administered to a human patient with open epiphysis once a week by subcutaneous injection, and wherein each administration is associated with a frequency of injection site reactions of less than 3%, such as less than 2%, such as less than 1% or the absence of injection site reactions.

61. The method of any one of claims 56-60, wherein the treatment reduces the incidence of adverse events associated with achondroplasia in a human patient.

62. The method of claim 61, wherein the adverse event associated with achondroplasia is selected from the group consisting of sleep apnea syndrome, ear infection, foramen magnum stenosis, and kyphosis.

63. The method of any one of claims 56-62, wherein the CNP conjugate or a pharmaceutically acceptable salt thereof has formula (IIf'):

in

The unlabeled dashed line represents the connection to the nitrogen provided by the lysine side chain at position 26 of the CNP portion of SEQ ID NO: 24 by formation of an amide bond;

in

Each Z ^c is

And among them

Each c1 is independently an integer between 200 and 250.