Extended release hydrogel conjugates of c-natriuretic peptides

Abstract

To provide longer-acting forms of C-type natriuretic peptide (CNP) and analogs thereof in more convenient forms for the treatment of various conditions and diseases, such as dwarfism and achondroplasia, which allow maintenance of therapeutic levels of peptide between administrations so as to provide therapeutic peptide levels for a sufficient time in a daily administration schedule without the need for overdosing.SOLUTION: Provided herein are extended release hydrogel conjugates of c-natriuretic peptides, methods of preparation thereof, and methods of use thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 118,568, filed November 25, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Statement Regarding Sequence Listing Submission This application is accompanied by an electronic Sequence Listing. The Sequence Listing is provided in a file named 670572002640SeqList.txt, created on November 19, 2021, and is 3,047 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Provided herein are sustained-release hydrogel conjugates of c-natriuretic peptide, methods for making same, and methods for using same. [Background technology]

[0004] C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family, which includes atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). It is released in response to hypertension and hypervolemic states, promoting natriuresis, resulting in sodium and water loss and lowering blood volume and blood pressure. CNP was first isolated in 1990 and was the last of the three major natriuretic peptides to be discovered. However, it is the most widely expressed of this family, particularly in the brain, chondrocytes, and endothelial cells. Exogenous CNP, which normally acts as a local paracrine / autocrine regulator, has been shown to be a potent dilator of arteries and veins in vitro and to lower blood pressure in vivo in humans. Among its many non-cardiovascular effects, CNP plays a major role in regulating bone growth and may also play a role in neuronal development and protection. Genetic disruption of CNP production in mice causes dwarfism through reduced bone length, with femurs, tibias, and vertebrae being 50–80% shorter than those in wild-type mice. As a result, CNP and its analogs have attracted attention for the treatment of dwarfism and achondroplasia.

[0005] Natriuretic peptides are characterized by a core 17-amino acid disulfide-bonded ring that is critical for receptor binding. This ring structure is conserved among family members and species. The initial product of the CNP gene (Nppc) is a 126-amino acid preproCNP. Cleavage of the signal peptide generates the proCNP peptide, which is further processed by furin proprotein convertase to generate the 53-amino acid CNP-53. CNP-53 is further processed by an as yet unidentified protease to generate the major active species, the 22-amino acid CNP (CNP-22). CNP activity is tightly regulated by two major degradation pathways, resulting in a very short plasma half-life (approximately 3 minutes). CNP has a high affinity for NPR-C, a clearance receptor that transports the peptide into lysosomes for degradation. It is also proteolyzed by neutral endopeptidase (NEP) in plasma and on endothelial cell surfaces.

[0006] Three specific natriuretic peptide receptors, NPR-A, NPR-B, and NPR-C, have been characterized. The low affinity of CNP for NPR-A suggests that it may not be important in mediating physiological responses to CNP. CNP is thought to be the sole endogenous ligand for NPR-B and can also bind to and activate NPR-C. CNP binds to NPR-B at physiological concentrations (picomolar) with 50-500-fold higher affinity than ANP and BNP. Genetic knockout of NPR-B results in impaired endochondral ossification, resulting in longitudinal shortening of vertebral and limb bones. This model further suggests a role for NPR-B in the development of female reproductive organs. NPR-B is primarily present in veins but also in arteries. NPR-C binds all three natriuretic peptides with high affinity. Genetic knockout of NPR-C also results in skeletal abnormalities and increased basal bone turnover, presumably due to a shift in CNP clearance from NPR-C to NPR-B.

[0007] Achondroplasia is a genetic disorder characterized by dwarfism caused by mutations that result in overactivity of fibroblast growth factor receptor 3 (FGFR3). It is the most common form of dwarfism, affecting approximately 1 in 27,500 individuals. The primary phenotype is short stature, averaging approximately 4 feet, with a large head and prominent forehead. Associated complications include sleep apnea, recurrent ear infections, obesity, hydrocephalus, and spinal stenosis. Growth hormone therapy is ineffective in patients with achondroplasia. FGFR3 downregulates cartilage and bone growth by inhibiting the development of chondrocytes, the cells that normally generate and maintain the cartilage matrix required for bone growth. Therefore, overactivity of FGFR3 leads to reduced bone growth and achondroplasia. Binding of fibroblast growth factor receptor 3 (FGFR3) to FGFR3 initiates a signaling cascade via the MAPK / ERK pathway. This cascade can be disrupted by activation of NPR-B, which interferes with the RAF-1 protein in the MAPK / ERK pathway. Therefore, CNP or CNP analogs may be useful for the treatment of achondroplasia.

[0008] Several CNP analogs have been disclosed (U.S. Patent Nos. 8,198,242, 8,377,884, and 9,266,939). Vosoritide (BMN-111), a CNP analog with increased NEP resistance due to increased peptide chain length, has a slightly extended half-life (20 minutes) and has shown promise as a once-daily treatment for achondroplasia in phase 3 clinical trials and is currently under FDA approval. Various analogs and PEGylated conjugates of CNP are also being investigated (Wendt, J Pharmacol Exp Ther 353:132-149, April 2015). Controlled-release conjugates of CNP have been disclosed (PCT Publication Nos. WO2016 / 110577, WO2017 / 118703, WO2017 / 118693, WO2017 / 118698, WO2017 / 118700, WO2017 / 118704, and WO2017 / 118707). Known side effects of BMN-111 include increased heart rate and decreased arterial blood pressure, which become more pronounced as the dose increases. Because BMN-111 has a short half-life, relatively high doses are required to provide therapeutic peptide levels for a sufficient period of time with a daily dosing schedule. Significantly increasing the half-life would allow for the maintenance of therapeutic levels of the peptide between doses without the need for such overdosing.

[0009] CNP prodrugs that extend the half-life of CNP through releasable conjugation have been disclosed (Breinholt et al., 2019 J. Pharmacol Exp Ther 370: 459-71; PCT Publication Nos. WO2016 / 110577, WO2017 / 118698, and WO2019 / 0022237). Combination therapies using controlled-release CNP analogs have also been disclosed (PCT Publication No. WO2018 / 060314). The disclosed conjugates release CNP via a hydrolytic mechanism, which is disadvantageous because it is difficult to avoid premature release from the conjugate during storage, leading to deterioration in the presence of moisture and a shortened shelf life, necessitating the development of dry formulations (PCT Publication No. WO2020 / 165081). However, such formulations require reconstitution before use and may not be suitable for insoluble conjugates such as microparticle hydrogels. Thus, there remains a need for more convenient forms of longer acting CNPs for the treatment of various ailments and diseases. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows two linker-CNPs of formula (II). In both embodiments, Z = azide, n = 1, R = H, each R = methyl, and E is [(Gln6,14)CNP38] attached to the linker through the α-amine of the N-terminal glycine. In the first linker-peptide, R = isopropyl-SO2-, and the linker releases the peptide with a half-life of 260 hours at pH 7.4 and 37°C after conjugation to a hydrogel microsphere. In the second linker-peptide, R = (N,N-dimethylamino)-SO2-, and the linker releases the peptide with a half-life of 1200 hours at pH 7.4 and 37°C after conjugation to a hydrogel microsphere.

[0011] [Figure 2]FIG. 2 is a diagram illustrating a method for preparing a conjugate of formula (IV), where M is a hydrogel containing degradable crosslinks, which comprises contacting a hydrogel of formula (III) containing a reactive connecting group Z′ with a linker-peptide of formula (II) containing an associated reactive group Z under conditions where the connecting functional group Z reacts with the connecting functional group Z′ to conjugate the linker-peptide to the hydrogel via the remaining functional group Z*.

[0012] [Figure 3] Figure 3 shows the structure of the crosslinks in the conjugates of formula (I) shown in Example 3, where M is an insoluble hydrogel. In each case, the interpolymer crosslinks include a linker with B* = triazole, C* = carboxamide, q = 1, R1a = (N,N-dimethylamino)sulfonyl, R2a = H, and R4a = methyl. In the first case, the linker-CNP of formula (II) [where Z = azide, n = 1, R1 = isopropyl-SO2-, R2 = H, each R4 = methyl, and E is (Gln6,14)CNP38 attached to the linker via the alpha amine of the N-terminal glycine] is conjugated via a carbamoylbicyclononynyl group to a hydrogel of formula (III), where x = 0, y = 4, and z = 0. In the second case, a linker-CNP of formula (II) where Z = azide, n = 1, R = (N,N-dimethylamino)SO -, R = H, each R = methyl, and E is (Gln6,14)CNP attached to the linker via the alpha amine of the N-terminal glycine is conjugated via a carbamoylbicyclononynyl group to a hydrogel of formula (III) where x = 4, y = 0, and z = 0.

[0013] [Figure 4] FIG. 4 shows one method for preparing a linker-CNP of formula (II): a protected CNP peptide is prepared on a solid support using standard methods, the linker is attached by reaction with succinimidyl carbonate, and the peptide is then deblocked, cleaved from the resin, and a disulfide is formed.

[0014] [Figure 5] Figure 5 shows an idealized structure depicting the arrangement of P1 (filled circles) and P2 (open circles) and a linker drug LE (filled circles) in a crosslinked matrix M. The two polymers are arranged alternately in the matrix by their linkage through the associated groups Z and Z' to prevent self-association, and each crosslink contains a linker drug. In practice, some crosslinks may be missing, for example, due to missing arms in commercial production of the polymer or the formation of multiple crosslinks between individual P1 and P2 units.

[0015] [Figure 6] Figure 6 shows the results of the stability study of Example 1. Vosoritide and (Gln6,Gln14)CNP38 were maintained at pH 7.4 and 37°C and monitored by anion-exchange HPLC. (Gln6,Gln14)CNP38 showed superior stability to vosoritide.

[0016] [Figures 7A-7B] Figures 7A and 7B show the results of a pharmacokinetic experiment in mice treated with the conjugate of Example 3 (see also Figure 3). Panel A shows the concentration of (Gln6,14)CNP38 in the plasma of mice after subcutaneous injection of the conjugate of Example 3, where R1 = isopropylsulfonyl. Panel B shows the concentration of (Gln6,14)CNP38 in the plasma of mice after subcutaneous injection of the conjugate of Example 3, where R1 = (N,N-dimethylamino)sulfonyl.

[0017] [Figure 8]Figure 8 shows the results of a pharmacokinetic study in juvenile cynomolgus monkeys treated with the conjugate of Example 3, where R1 = isopropylsulfonyl. Figure 8 shows the plasma concentration of [Gln6,14]CNP-38 after a single subcutaneous administration of 1.3 μmol / kg of the conjugate of Example 3, where R1 = isopropylsulfonyl, to juvenile cynomolgus monkeys. Data are the average of three animals. This conjugate provided continuous exposure to >100 pM [Gln6,14]CNP-38 for approximately one month.

[0018] [Figure 9] Figure 9 shows the results of a pharmacokinetic study in juvenile cynomolgus monkeys treated with the conjugate of Example 3, where R1 = (N,N-dimethylamino)sulfonyl. Plasma concentrations of [Gln6,14]CNP-38 after a single subcutaneous administration of 1.2 μmol / kg of the conjugate of Example 3, where R1 = (N,N-dimethylamino)sulfonyl, to juvenile cynomolgus monkeys. Data are the average of three animals. This conjugate provided sustained exposure to >100 pM of [Gln6,14]CNP-38 for over three months.

[0019] [Figure 10] Figure 10 shows measurements of total length (tail length + nose-to-anus length, TL) in mice treated with a fixed dose per mouse at each dosing interval: [Gln6,14]CNP-38 and the conjugate of Example 3, where R1 = isopropylsulfonyl (4A) or R1 = (N,N-dimethylamino)sulfonyl (4B). From left to right: black (solid line), vehicle control; brown (empty), QD[Gln6,14]CNP-38; blue (horizontal stripes), QWk 4A 20 nmol / mouse; red (upward diagonal line), QWk 4A 50 nmol / mouse; green (downward diagonal line), single dose 4B 85 nmol / mouse. Plotted as percent of initial total length (TL) versus time; values ​​are expressed as mean ± SD.

[0020] [Figure 11]FIG. 11 shows measurements of total length (tail length + nose-to-anus length, TL) in mice treated with [Gln6,14]CNP-38 and the conjugates of Example 3 where R1 = isopropylsulfonyl (4A) or (R1 = (N,N-dimethylamino)sulfonyl (4B) using weight-adjusted doses (μmol / kg). From left to right: black (solid line), vehicle control; brown (open), QD[Gln6,14]CNP-38; blue (horizontal line), QWk 4A 1.5 μmol / kg; red (rising diagonal line), QWk 4A 2.2 μmol / kg; green (descending diagonal line), Q2Wk 2.2 μmol / kg; purple (checkered), single dose 4B 6.1 μmol / kg; orange (vertical line), single dose 4B. 40 μmol / kg. Plotted as percent of initial total length (TL) against time; values ​​are presented as mean ± SD.

[0021] [Figure 12] Figure 12 shows photographs of representative mice treated with [Gln6,14]CNP-38 or conjugate 4A (Example 3, where R1 = isopropylsulfonyl) for 5 weeks. A) Vehicle control; B) 70 nmol QD of [Gln6,14]CNP-38 peptide; C) 2.2 μmol / kg biweekly (Q2Wk) conjugate 4A; D) 2.2 μmol / kg weekly (QWk) conjugate 4A; and E) 1.5 μmol / kg weekly (QWk) conjugate 4A. Anesthetized mice were first positioned for measurement with their heads extended, noses aligned with a horizontal guideline, and tails straightened. The distance between the top guideline and the end of the tail best represents total length (TL).

[0022] [Figures 13A-13B]Figures 13A and 13B show peptide release and hydrogel degradation at pH 9.4 and 37°C for the conjugates of Example 3 where R1 = isopropylsulfonyl (Figure 13A) and R1 = (N,N-dimethylamino)sulfonyl (Figure 13B). In Figure 13A, when R1 = isopropylsulfonyl, total solubilized peptide (solid line) and solubilized PEG (dashed line) resulted in a (Gln6,14)CNP38 release t = 6.1 hours, which corresponds to 610 hours under physiological conditions (pH 7.4, 37°C). In Figure 13B, when R1 = (N,N-dimethylamino)sulfonyl, total solubilized peptide (solid line) and solubilized PEG (dashed line) resulted in a (Gln6,14)CNP38 release t = 15.8 hours, which corresponds to 1580 hours under physiological conditions (pH 7.4, 37°C). Data are means of n=6, and error bars are shown as standard deviations. Summary of the Invention

[0023] The present disclosure relates to conjugates that provide sustained, low-level release of C-natriuretic peptides (CNPs), supporting weekly, monthly, or even less frequent administration of these peptides, and are expected to be useful in treating diseases and conditions such as achondroplasia.

[0024] In one aspect, the present disclosure provides a sustained-release conjugate comprising an insoluble hydrogel matrix having multiple covalently attached linker-peptides, wherein the linkers are cleaved via a beta-elimination mechanism under physiological pH and temperature conditions to release free CNP peptides. The conjugates of the present invention can be represented schematically as formula (I): M-(LE) a (I) where M is an insoluble hydrogel matrix linked to multiple CNP peptides E via a cleavable linker L, L is a linker that cleaves via a pH-dependent beta-elimination mechanism, such as the linkers disclosed in U.S. Pat. No. 8,680,315, and a is an integer representing the number of LE moieties that will produce an appropriate concentration of E in a given volume of matrix. Suitable concentrations range from 0.01 to 50 mg of peptide per mL of matrix, preferably from 1 to 25 mg of peptide per mL of matrix. The linker L releases free CNP peptides over the desired administration period with a suitable half-life, typically 150 to 2500 hours measured in vitro at pH 7.4 and 37°C, and preferably 250 to 1500 hours in vitro.

[0025] In a second aspect, the present invention provides a compound of formula (II) [ka] (II) [In the formula, n = 0–6; R 1 and R 2 are independently H, alkyl, CN, NO, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR 3 , -SOR 3 , or -SO2R 3 where: R 3 is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 5 , or -NR 5 2, where: Each R 5 are independently H or optionally substituted alkyl, or both R 5groups taken together with the nitrogen to which they are attached form a heterocyclic ring; where R 1 and R 2 may be taken together with the carbons to which they are attached to form a 3- to 8-membered ring, where R 1 and R 2 Only one of may be H or alkyl; Each R 4 are independently H or C1-C3 alkyl, or both R 4 form a 3- to 6-membered ring together with the carbon to which they are attached; Z is a functional group that mediates coupling to the support M via the associated functional group Z'; and NH is the amino residue of CNP peptide E. A linker-CNP(LE) is provided, which is represented by: DETAILED DESCRIPTION OF THE INVENTION

[0026] The term "alkyl" is understood to include linear, branched, or cyclic saturated hydrocarbon groups having 1 to 20, 1 to 12, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. In some embodiments, alkyl is linear or branched. Examples of linear or branched alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. In some embodiments, alkyl is cyclic. Examples of cyclic alkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, and the like.

[0027] The term "alkoxy" is understood to include alkyl groups attached to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and the like.

[0028] The term "alkenyl" is understood to include a non-aromatic unsaturated hydrocarbon having a carbon-carbon double bond and having 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0029] The term "alkynyl" is understood to include a non-aromatic unsaturated hydrocarbon having a carbon-carbon triple bond and having 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0030] The term "aryl" is understood to include aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. The term "heteroaryl" includes aromatic rings of 3 to 15 carbons, preferably 3 to 7 carbons, containing at least one N, O, or S atom, including pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and the like.

[0031] In some cases, the alkenyl, alkynyl, aryl, or heteroaryl moiety can be coupled to the remainder of the molecule via an alkyl bond. In such situations, the substituent is referred to as alkenylalkyl, alkynylalkyl, arylalkyl, or heteroarylalkyl, indicating that an alkylene moiety is between the alkenyl, alkynyl, aryl, or heteroaryl moiety and the molecule to which it is attached.

[0032] The term "halogen" or "halo" is understood to include bromo, fluoro, chloro, and iodo.

[0033] The term "heterocyclic ring" or "heterocyclyl" is understood to mean a 3-15 membered aromatic or non-aromatic ring containing at least one N, O, or S atom. Examples include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, as well as the exemplary groups provided above for the term "heteroaryl." In some embodiments, the heterocyclic ring or heterocyclyl is non-aromatic. In some embodiments, the heterocyclic ring or heterocyclyl is aromatic.

[0034] "Optionally substituted" is understood to mean that, unless otherwise specified, a group may be unsubstituted or substituted with one or more (e.g., 1, 2, 3, 4, or 5) identical or different substituents. Examples of substituents include, but are not limited to, alkyl, alkenyl, alkynyl, halogen, -CN, -OR. aa , -SR aa , -NR aa R bb , -NO2, -C=NH(OR aa ), -C(O)R aa , -OC(O)R aa , -C(O)OR aa , -C(O)NR aa R bb , -OC(O)NR aa R bb , -NR aa C(O)R bb , -NR aa C(O)OR bb , -S(O)R aa , -S(O)2R aa , -NR aa S(O)R bb , -C(O)NR aa S(O)R bb , -NR aa S(O)2R bb , -C(O)NR aa S(O)2R bb , -S(O)NR aa R bb , -S(O)NR aaR bb , -P(O)(OR aa )(OR bb ), heterocyclyl, heteroaryl, or aryl, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl are each independently optionally selected from R cc and optionally substituted with, where R aa and R bb are each independently H, alkyl, alkenyl, alkynyl, heterocyclyl, heteroaryl, or aryl; R aa and R bb taken together with the nitrogen atom to which they are attached form a heterocyclyl optionally substituted with alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, or -CN, wherein Each R cc is independently alkyl, alkenyl, alkynyl, halogen, heterocyclyl, heteroaryl, aryl, -CN, or -NO2.

[0035] As used herein, unless otherwise stated, the use of the terms "a," "an," etc. means one or more.

[0036] The drug release rate is determined by the group R 1 and R 2 It can be adjusted by selecting an appropriate R 1 and R 2 For a description of the group R 1 and R 2 Each of the intervening R 1 R 2The CH protons may be independently substituted with electron-donating and / or electron-withdrawing substituents that alter the acidity of the protons, thereby allowing for great flexibility and control over the rate of drug elimination. Electron-withdrawing groups are defined as groups with a Hammett's sigma value greater than 0 (see, e.g., Hansch et al. 1991 Chemical Reviews 91: 165-195). The term "electron-donating group" refers to a group in which R 1 R 2 refers to a substituent that reduces the acidity of the CH; electron-donating groups typically have a negative Hammett σ or Tuft σ * The Hammett constants are associated with constants that are well known in the field of physical organic chemistry. (Hammett constants represent aryl / heteroaryl substituents, while Taft constants represent substituents on non-aromatic moieties.) Examples of suitable electron-donating substituents include, but are not limited to, lower alkyl, lower alkoxy, lower alkylthio, amino, alkylamino, dialkylamino, and silyl. Similarly, an "electron-withdrawing group" is defined as any group such as R 1 R 2 It refers to a substituent that increases the acidity of the CH group, and electron-withdrawing groups typically have a positive Hammett σ or Taft σ * It is related to the constant R, which is well known in physical organic chemistry. 1 and R 2 For a description of suitable electron-donating and electron-withdrawing substituents that can be used to adjust the .alpha.-methyl group, see US Pat. No. 9,649,385.

[0037] In some embodiments, R 1 and R 2 In some embodiments, at least one of R 1 and R 2 At least one of R is -NO. 1 and R 2 At least one of R is an optionally substituted aryl containing 6 to 10 carbons. For example, in some embodiments, R 1and R 2 At least one of R is phenyl, naphthyl, or anthracenyl, each of which may be optionally substituted. 1 and R 2 At least one of R is an optionally substituted heteroaryl containing 3 to 7 carbons and at least one N, O, or S atom. For example, in some embodiments, R 1 and R 2 At least one of R is pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl, each of which may be optionally substituted. 1 and R 2 At least one of R is an optionally substituted alkenyl containing 2 to 20 carbon atoms. 1 and R 2 At least one of R is an optionally substituted alkynyl containing 2 to 20 carbon atoms. 1 and R 2 At least one of the following is -COR 3 , -SOR 3 , or -SO2R 3 where R 3 is H, optionally substituted alkyl containing 1 to 20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 5 , or -NR 5 2, where each R 5 are independently H or optionally substituted alkyl containing 1 to 20 carbon atoms, or both R 5 The groups, taken together with the nitrogen to which they are attached, form a heterocyclic ring.

[0038] In some embodiments, R1 and R 2 At least one of -CN, -SOR 3 , or -SO2R 3 In some embodiments, R 1 and R 2 At least one of is -CN or -SO2R 3 In some embodiments, R 1 and R 2 At least one of is -CN or -SO2R 3 where R 3 is optionally substituted alkyl, optionally substituted aryl, or —NR 5 2. In some embodiments, R 1 and R 2 At least one of is -CN, -SON(CH), -SOCH, -SOphenyl, -SO(chlorophenyl), -SO(4-methylphenyl), -SON(CHCH)O, -SON(CHCH)S, -SOCH(CH), -SON(CH)(CHCH), or -SON(CHCHOCH). In some embodiments, R 1 and R 2 One of the two is -CN or -SO2R 3 where R 3 is optionally substituted alkyl, optionally substituted aryl, or —NR 5 2; and the other is H. In some embodiments, R 1 and R 2 one of is -CN, -SON(CH)2, -SOCH3, -SOphenyl, -SO(chlorophenyl), -SO(4-methylphenyl), -SON(CHCH)2O, -SON(CHCH)2S, -SOCH(CH)2, -SON(CH)(CHCH), or -SON(CHCHOCH)2; and the other is H.

[0039] In some embodiments, each R 4is independently C1-C3 alkyl. In some embodiments, R 4 are both methyl.

[0040] In some embodiments, n is an integer from 1 to 6. In some embodiments, n is an integer from 1 to 3. In some embodiments, n is an integer from 0 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0041] In some embodiments, R 1 is the CN or SO2R defined above 3 and R 2 is H, and each R 4 is C1-C3 alkyl and n=1 to 3.

[0042] The connecting group Z can be any group capable of selective reaction in the presence of the CNP peptide. Exemplary groups include, but are not limited to, azides, where the associated group Z' on M is an alkyne or cycloalkyne and the remaining connecting group is a triazole; aminoethers, where the associated group Z' on M is a carbonyl and the remaining connecting group is an oxime; trans-cyclooctene or cyclopropane, where the associated group Z' on M is a 1,2,5,6-tetrazine and the remaining functional group is a pyridazine; and thiols, where the associated group Z' on M is a maleimide or halocarbonyl and the remaining connecting group is a thioether. In a preferred embodiment, Z is azide and Z' is cyclooctyne. Exemplary cyclooctynes ​​known in the art include, but are not limited to, 5-hydroxycyclooctyne (5HCO), 1-fluorocyclooct-2-yne-1-carboxylate (MFCO), and bicyclo[6.1.0]non-4-yne (BCN) (see Dommerholt et al., Top Curr Chem (Z) (2016) 374:16).

[0043] The term "CNP" includes all peptides characterized by their ability to bind to NPR-B and thereby regulate chondrocyte growth, proliferation, and differentiation. This includes, but is not limited to, the sequences listed in PCT Publications 2009 / 067639, 2010 / 135541, and 2016 / 110577, and Morozumi et al. (2019) PLoS ONE 14(2): e0212680. Preferably, the term "CNP" refers to peptides set forth in SEQ ID NOS: 1-6 and their stabilized analogs. Particularly preferred are CNP analogs in which specific amino acid residues have been substituted to improve peptide stability. Such stabilized analogs include peptides in which asparagine residues have been replaced with residues less susceptible to deamidation, such as glutamine or alanine, and peptides in which oxidation-sensitive residues have been replaced, such as methionine replaced with norleucine. Exemplary embodiments of CNP are provided below in SEQ ID NOS: 1-6.

[0044] Sequence number 1 ([CNP22]) GLSKGCFGLKLDRIGSMSGLGC C 6 -C 22 Disulfides

[0045] SEQ ID NO: 2 ([vosoritide]) PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC C 23 -C 39 Disulfides

[0046] SEQ ID NO: 3 ([CNP38]) LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC C 22 -C 38 Disulfides

[0047] SEQ ID NO: 4 ([(Gln 6,14 )CNP38]) LQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC C 22-C 38 Disulfides

[0048] SEQ ID NO: 5 ([(Gln 6,14 ,Nle 33 )CNP38]) LQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSXSGLGC C 22 -C 38 Disulfides

[0049] Sequence number 6 ([ASB20123]) GLSKGCFGLKLDRIGSMSGLGCVQQRKDSKKPPAKLQPR C 6 -C 22 Disulfides

[0050] In some embodiments, the stabilized CNP comprises (Gln 6,14 )-CNP38 (SEQ ID NO: 4), or (Gln 6,14 ,Nle 33 ) CNP38 (SEQ ID NO: 5).

[0051] In the description herein, it is understood that any description, variation, embodiment, or aspect of a moiety can be combined with any description, variation, embodiment, or aspect of any other moiety, as if each and every combination of descriptions were specifically and individually set forth. For example, all descriptions, variations, embodiments, or aspects provided herein with respect to n in formula (II) can be combined with R 1 , R 2 , R 4 , Z, and E. It is also understood that all descriptions, variations, embodiments, or aspects of a formula such as formula (I), (II), (III), (IV), or (V), where applicable, apply to other formulas described herein as well, and that each and every description, variation, embodiment, or aspect is set forth in the same manner as if set forth separately and individually for all formulas.

[0052] In some embodiments, M is a water-insoluble hydrogel carrier. In preferred embodiments, M is a compound represented by formula (III): [ka] (III) [In the formula, q = 0 to 6; R 1a and R 2a are independently H, alkyl, CN, NO, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR 3a , -SOR 3a , or -SO2R 3a where: R 3a is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 5a , or -NR 5a 2, where: Each R 5a are independently H or optionally substituted alkyl, or both R 5a groups taken together with the nitrogen to which they are attached form a heterocyclic ring; where R 1a and R 2a may be taken together with the carbons to which they are attached to form a 3- to 8-membered ring, where R 1a and R 2a Only one of may be H or alkyl; Each R 4a are independently H or C1-C3 alkyl, or both R 4a form a 3- to 6-membered ring together with the carbon to which they are attached, Z' is a functional group that mediates coupling of the linker-CNP of formula (II) disclosed herein to the associated functional group Z; x and y are each independently 0 to 6; B * and C * are each independently a connecting group; and P 1 and P 2 are independently r-arm polymers having an average molecular weight of 1 to 40 kDa, where r is an integer from 2 to 8. In a preferred embodiment, P 1 and P 2 is a poly(ethylene glycol) with an r-arm.

[0053] The degradation rate of the hydrogel of formula (III) is R 1 and R 2 As discussed herein, the group R 1a and R 2a can be adjusted by appropriate selection of

[0054] In some embodiments, R 1a and R 2a In some embodiments, at least one of R 1a and R 2a At least one of R is -NO. 1a and R 2a At least one of R is an optionally substituted aryl containing 6 to 10 carbons. For example, in some embodiments, R 1a and R 2a At least one of R is phenyl, naphthyl, or anthracenyl, each of which may be optionally substituted. 1a and R 2a At least one of R is an optionally substituted heteroaryl containing 3 to 7 carbons and at least one N, O, or S atom. For example, in some embodiments, R1a and R 2a At least one of R is pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl, each of which may be optionally substituted. 1a and R 2a At least one of R is an optionally substituted alkenyl containing 2 to 20 carbon atoms. 1a and R 2a At least one of R is an optionally substituted alkynyl containing 2 to 20 carbon atoms. 1a and R 2a At least one of the following is -COR 3a , -SOR 3a , or -SO2R 3a where R 3a is H, optionally substituted alkyl containing 1 to 20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 5a , or -NR 5a 2, where each R 5a are independently H or optionally substituted alkyl containing 1 to 20 carbon atoms, or both R 5a The groups, taken together with the nitrogen to which they are attached, form a heterocyclic ring.

[0055] In some embodiments, R 1a and R 2a At least one of -CN, -SOR 3a , or -SO2R 3a In some embodiments, R 1a and R 2a At least one of is -CN or -SO2R 3a In some embodiments, R 1a and R2a At least one of is -CN or -SO2R 3a where R 3a is optionally substituted alkyl, optionally substituted aryl, or —NR 5a 2. In some embodiments, R 1a and R 2a At least one of is -CN, -SON(CH), -SOCH, -SOphenyl, -SO(chlorophenyl), -SO(4-methylphenyl), -SON(CHCH)O, -SON(CHCH)S, -SOCH(CH), -SON(CH)(CHCH), or -SON(CHCHOCH). In some embodiments, R 1a and R 2a One of the two is -CN or -SO2R 3a where R 3a is optionally substituted alkyl, optionally substituted aryl, or —NR 5a 2; and the other is H. In some embodiments, R 1a and R 2a one of is -CN, -SON(CH)2, -SOCH3, -SOphenyl, -SO(chlorophenyl), -SO(4-methylphenyl), -SON(CHCH)2O, -SON(CHCH)2S, -SOCH(CH)2, -SON(CH)(CHCH), or -SON(CHCHOCH)2; and the other is H.

[0056] In some embodiments, each R 4a is independently C1-C3 alkyl. In some embodiments, R 4a are both methyl.

[0057] In some embodiments, q is an integer from 1 to 6. In some embodiments, q is an integer from 2 to 3. In some embodiments, q is an integer from 1 to 3. In some embodiments, q is an integer from 0 to 3. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, q is 4. In some embodiments, q is 5. In some embodiments, q is 6.

[0058] In some embodiments, x is an integer from 1 to 6. In some embodiments, x is an integer from 2 to 3. In some embodiments, x is an integer from 1 to 3. In some embodiments, x is an integer from 0 to 3. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 3. In some embodiments, x is 4. In some embodiments, x is 5. In some embodiments, x is 6.

[0059] In some embodiments, y is an integer from 1 to 6. In some embodiments, y is an integer from 2 to 3. In some embodiments, y is an integer from 1 to 3. In some embodiments, y is an integer from 0 to 4. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, y is 6.

[0060] P 1 and P 2Hydrogels of formula (III), in which B is a poly(ethylene glycol) having an r-arm, can be prepared by the methods described, for example, in Henise et al. (2015) Bioconj. Chem. 26: 270-8; Henise et al., Int. J. Polymer Sci. Vol. 2019, Article ID 9483127; and Henise et al. (2020) Engineering Reports 2020;2:e12213. These hydrogels are provided to adjust the drug release rate and the subsequent dissolution rate of the hydrogel. * and C * Typical connecting groups for B include, but are not limited to, triazole, carboxamide, carbamate, oxime, and thioether. When the hydrogel polymerization is via azide / cyclooctyne cycloaddition, B * and / or C * The connecting group of B is triazole as described above. * is a triazole, and C * is a carboxamide. 1 and P 2 are synthetic or natural polymers, such as poly(ethylene glycol), dextran, or hyaluronic acid. In these hydrogels, polymer chains are crosslinked to form an insoluble three-dimensional matrix (Figure 6), with each crosslink having a point of attachment to a linker-CNP of formula (II). The crosslinks are primarily formed by groups R 1a and R 2a These hydrogels are slowly cleaved by non-hydrolytic beta-elimination at a rate governed by the cleavage time, ultimately yielding soluble polymer fragments. These hydrogels are characterized by the presence of a connecting group Z formed by the reaction of the corresponding groups Z and Z' on Formula (II) and Formula (III), as shown in Figure 2. * to form a conjugate of formula (I), which is an insoluble hydrogel comprising a linker-peptide conjugated at each bridge, more specifically a conjugate of formula (IV), 1 , R2 , R 4 , E, n, Z * ,y,P 1 , P 2 , B * , q, r, C * , x, R 1a , R 2a , and R 4a are as disclosed herein.]. [ka] (IV)

[0061] An exemplary structure of a crosslink in such a conjugate of formula (IV) is shown in Figure 3. Figure 2 further illustrates the hydrogel of formula (III) containing a connecting group Z', where Z and Z' react to form a residue Z that connects the linker-peptide to the hydrogel. * A method for preparing a conjugate of Formula (IV) is shown, comprising contacting a linker-peptide of Formula (II) containing the relevant linking group Z with M under conditions that form a conjugate of Formula (IV). When Z and Z' are azide / cyclooctyne, such conditions are temperatures of 0-100°C, preferably 0-50°C, and more preferably 25-50°C. The solvent in which the linker-peptide and hydrogel are suspended or dissolved can be water, an organic solvent, or a mixed solvent, depending on the solubility of the linker-peptide. Typical solvents are aqueous buffers with a pH of 2-7, preferably 2-5, optionally mixed with a water-miscible cosolvent such as methanol, ethanol, 2-propanol, tert-butanol, acetonitrile, dimethylformamide, acetonitrile, or tetrahydrofuran. When M is an insoluble matrix, the conjugate of Formula (I) can be optionally isolated by washing to remove unreacted linker-peptide and reaction by-products. The reaction procedure for formulas (II) and (III) is similar to that reported by Henise et al. (2020) Engineering Reports 2020;2:e12213.

[0062] Linker-CNP of formula (II) can be prepared by reacting the CNP peptide or a protected version thereof with a linker reagent of formula (V), where X is a leaving group such as chloride, O-succinimidyl, O-nitrophenyl, etc., and the remaining groups are as disclosed herein for formula (II). [ka] (V)

[0063] The linker can be attached to either the N-terminal α-amine or lysine ε-amine group of the CNP peptide using chemistry such as that described in U.S. Patent No. 8,680,315. When the linker is attached to the N-terminal α-amine, this is preferably done during synthesis of the peptide on a solid support, as shown in Figure 3. After synthesis of the protected peptide sequence on a solid support, the linker is attached by reaction with an activated carbonate, such as succinimidyl carbonate, as described in the Examples below, and the resulting intermediate is deblocked, cleaved from the resin, and disulfide formed to yield the linker-CNP of formula (II).

[0064] In a third aspect, the present invention relates to protocols for formulating and administering the conjugates of Formula (I). In one embodiment, the conjugates are prepared as hydrogel microspheres suitable for subcutaneous injection using a narrow-gauge needle. These microspheres can be formulated into any solution suitable for injection and can include any excipients necessary to maintain the stability of the conjugate, such as buffers, antimicrobial agents, antioxidants, density and osmolality adjusters, and agents to promote suspension and prevent microsphere aggregation. Given the pH-sensitive nature of the beta-elimination mechanism of linker cleavage in these conjugates, stability is enhanced by the use of low-pH buffers, preferably pH 2-7, more preferably pH 3-7, and even more preferably pH 4-5. Suitable buffers are those known in the art for pharmaceutical use, including acetate, citrate, malate, maleate, phosphate, and other buffers effective in these pH ranges.

[0065] Administration can be by any route, such as subcutaneous, intramuscular, or intraarticular. It is expected that the conjugates of the invention will be useful in the treatment of diseases and conditions in both humans and animals that respond to CNP, such as achondroplasia, when administered weekly, monthly, or more frequently.

[0066] All references cited herein are incorporated by reference in their entirety. The following examples are provided to illustrate the present invention, but are not intended to limit the scope of the invention. [Example]

[0067] The following examples are intended to illustrate, but not limit, the scope of the present disclosure, and all references cited therein are incorporated herein by reference with respect to particular aspects of their disclosures, including those aspects specifically and those cited generally.

[0068] Manufacturing A Preparation of linker of formula (V) [ka] 4-Azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate A 1.43 M solution of n-butyllithium in hexanes (70 mL, 100 mmol) was added to a stirred solution of N,N-dimethylmethanesulfonamide (12.33 g, 100 mmol) in 200 mL of anhydrous THF maintained at −50° C. under an inert atmosphere. The mixture was warmed to −20° C. over 1 h and then recooled to −50° C. before adding methyl 3-azido-2,2-dimethylpropionate (prepared according to Kim, Synthetic Communications; 7.70 g, 50 mmol). The mixture was warmed to +10° C. over 2 h and then quenched with 20 mL of 6N HCl. The mixture was diluted with methyl t-butyl ether (MTBE, 200 mL), washed with water (2×100 mL) and brine (1×100 mL), dried over MgSO4, filtered, and concentrated to give 14.05 g of crude ketone product. Chromatography on SiO2 (220 g) using a step gradient of 0, 20, 30, 40, and 50% EtOAc / hexanes afforded purified 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butanone (10.65 g, 86%) as a crystalline solid.

[0069] The ketone was dissolved in 200 mL of methanol, cooled on ice, and treated with sodium borohydride (0.96 g, 25 mmol) for 15 min, then quenched with 4 mL of 6 N HCl and concentrated. The resulting slurry was diluted with methyl t-butyl ether (MTBE, 200 mL), washed with water (1 × 100 mL) and brine (1 × 100 mL), dried over MgSO, filtered, and concentrated to give 10.0 g of crystalline 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butanol.

[0070] Pyridine (10.6 mL, 132 mmol) was added over 10 minutes to a stirred mixture of N-hydroxysuccinimide (6.90 g, 60 mmol) and triphosgene (5.93 g, 20 mmol) in 250 mL of dichloromethane cooled on ice. The mixture was stirred on ice for 15 minutes and then allowed to warm to ambient temperature over 30 minutes. A solution of 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butanol (10.0 g, 40 mmol) in 20 mL of dichloromethane was added, and the mixture was further stirred at ambient temperature for 1 hour. After cooling on ice, the mixture was treated with 100 mL of water, and the phases were separated. The organic phase was washed twice with water, once with 5% KHSO4, and once with brine, dried over MgSO4, filtered, and concentrated. The crude product was crystallized from 100 mL of 30% EtOAc / hexanes to give 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (11.1 g, 71%) as a white crystalline solid.

[0071] 4-Azido-1-(isopropylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate A 1.38 M solution of n-butyllithium in hexanes (14.5 mL, 20 mmol) was added to a solution of diisopropylamine (2.96 mL, 21 mmol) in 80 mL of THF at −78° C. The mixture was warmed briefly to ambient temperature and then recooled to −78° C. Isopropyl methyl sulfone (2.69 g, 22 mmol) was added dropwise over 5 minutes, followed by methyl 3-azido-2,2-dimethylpropionate (1.57 g, 10 mmol). The mixture was allowed to warm slowly to ambient temperature over 1 hour and then quenched by the addition of 3.47 mL of 6 N HCl (20.5 mmol). The mixture was diluted with ethyl acetate, washed with water (2 × 100 mL) and brine (1 × 100 mL), dried over MgSO4, filtered, and concentrated to give 2.9 g of crude ketone product as a yellow liquid. Chromatography on SiO2 (25 g) using a gradient of 0-70% EtOAc / hexanes afforded purified 4-azido-1-(isopropylsulfonyl)-3,3-dimethyl-2-butanone (1.73 g, 70%) as a crystalline solid.

[0072] The ketone was dissolved in 14 mL of methanol, cooled on ice, and treated with sodium borohydride (0.13 g, 3.5 mmol) for 15 min, then quenched with 1.2 mL of 6 N HCl and concentrated. The resulting slurry was diluted with methyl t-butyl ether (MTBE, 200 mL), washed with water (1 × 100 mL) and brine (1 × 100 mL), dried over MgSO, filtered, and concentrated to give 1.51 g of 4-azido-1-(isopropylsulfonyl)-3,3-dimethyl-2-butanol as a colorless oil.

[0073] Pyridine (0.93 mL, 11.6 mmol) was added over 1 minute to a stirred mixture of the alcohol (1.44 g, 5.78 mmol) and triphosgene (2.92 g, 9.83 mmol) in 45 mL of THF. After 30 minutes, the suspension was filtered and concentrated. The residue was redissolved in 25 mL of THF, treated with N-hydroxysuccinimide (1.97 g, 17.1 mmol) and pyridine (1.38 mL, 17.1 mmol), stirred for 30 minutes, then diluted with ethyl acetate, washed with 5% KHSO, water, and brine, dried over MgSO, filtered, and evaporated. Chromatography on SiO2 (25 g) using a gradient of 0–80% EtOAc / hexanes afforded purified 4-azido-1-(isopropylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (1.46 g) as a white crystalline solid. Linkers prepared according to these procedures include, but are not limited to: 4-azido-1-(methylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein R 1 =MeSO2, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 1. 4-Azido-1-(isopropylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein R 1 = i PrSO2, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 1. 4-Azido-1-(phenylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein R 1 = PhSO2, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 1. 4-azido-1-(4-methylphenylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein R 1 = (4-methylphenyl)SO2, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 1. 4-azido-1-(chlorophenylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein R 1 = (4-chlorophenyl)SO2, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 1. 4-azido-1-cyano-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein R 1 =CN, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 1. 4-Azido-1-(N,N-dimethylaminosulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein R 1 =(Me2N)SO2, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 1. 4-Azido-1-(morpholinosulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein R 1 =(O(CH2CH2)2NSO2, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 1. 4-Azido-1-(thiomorpholinosulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein R 1 =(S(CH2CH2)2NSO2, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 1. Starting from ethyl 4-chloro-2,2-dimethylbutyrate, the following linkers were prepared: 5-Azido-1-(chlorophenylsulfonyl)-4,4-dimethyl-2-pentyl succinimidyl carbonate (wherein R 1 = (4-chlorophenyl)SO2, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 2. 5-Azido-1-cyano-4,4-dimethyl-2-pentyl succinimidyl carbonate (wherein R 1 =CN, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 2. 5-azido-1-(phenylsulfonyl)-4,4-dimethyl-2-pentyl succinimidyl carbonate (wherein R 1 = PhenylSO2, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 2. 5-azido-1-(methylsulfonyl)-4,4-dimethyl-2-pentyl succinimidyl carbonate (wherein R 1 =MeSO2, R 2 =H, each R 4 = Me, Z = azide, X = O-succinimidyl, and n = 2.

[0074] Manufacturing B Hydrogel of formula (III) Hydrogels of formula (III) were prepared as sterile injectable microspheres as described in Henise et al (2020) Engineering Reports 2(8): e12213. Hydrogels prepared included: (a)P 1 and P 2 are 10 kDa four-arm poly(ethylene glycol); B * = triazole; C *= carboxamide; q=1; R 1a = (N,N-dimethylamino)sulfonyl; R 2a =H; each R 4a = methyl; x = 4; y = 0; and Z' = NH-CO-O-(bicyclo[6.1.0]non-4-yn-9-ylmethyl). (b)P 1 and P 2 are 10 kDa four-arm poly(ethylene glycol); B * = triazole; C * = carboxamide; q=1; R 1a = (N,N-dimethylamino)sulfonyl; R 2a =H; each R 4a = methyl; x = 0; y = 4; and Z' = NH-CO-O-(bicyclo[6.1.0]non-4-yn-9-ylmethyl). (c)P 1 and P 2 are 20 kDa four-arm poly(ethylene glycol); B * = triazole; C * = carboxamide; q=1; R 1a = (N,N-dimethylamino)sulfonyl; R 2a =H; each R 4a = methyl; x = 4; y = 0; and Z' = NH-CO-O-(bicyclo[6.1.0]non-4-yn-9-ylmethyl). (d)P 1 and P 2 are 20 kDa four-arm poly(ethylene glycol); B * = triazole; C * = carboxamide; q=1; R 1a = (N,N-dimethylamino)sulfonyl; R 2a =H; each R 4a = methyl; x = 0; y = 4; and Z' = NH-CO-O-(bicyclo[6.1.0]non-4-yn-9-ylmethyl). (e)P 1 and P 2 are 20 kDa four-arm poly(ethylene glycol); B *= triazole; q=4; R 1a =CN;R 2a =H; each R 4a =H; x=0; y=4; and Z'=NH-CO-O-(4-cyclooctynyl). 1 and P 2 are 20 kDa 4-arm poly(ethylene glycol); B * = triazole; C * = carboxamide; q=1; R 1a = (N,N-dimethylamino)sulfonyl; R 2a =H; each R 4a = methyl; x = 4; y = 0; and Z' = NH-CO-O-(4-cyclooctynyl). (f)P 1 and P 2 are 20 kDa four-arm poly(ethylene glycol); B * = triazole; C * = carboxamide; q=4; R 1a =CN;R 2a =H; each R 4a =H; x=0; y=4; and Z'=NH-CO-O-(4-cyclooctynyl).

[0075] Example 1 (Gln 6,14 Stability studies of CNP38 and vosoritide In a 1.5 mL glass vial, 100 μM [Gln 6,14 ]-CNP38 or 100 μM vosoritide was treated with 10 mM sodium phosphate (pH 7.4) containing 0.13 M NaCl. The reaction was maintained at 37°C, and aliquots were periodically removed and stored at -20°C until analysis. Samples were analyzed on a Dionex BioLC ProPac SCX-10, 250 x 4 mm column using a gradient of 0 to 100% Buffer B in 10 min at 1 mL / min at a column temperature of 40°C. Peaks were monitored at 220 nm. Buffer A: 10 mM MES, pH 6.2, at 22°C; Buffer B: 10 mM MES, 1 M NaCl, pH 6.2, at 22°C. Significant degradation of vosoritide was observed within 1 week, whereas [Gln6,14 ]-CNP38 was extremely stable throughout the 4-week stability experiment (Figure 6).

[0076] Example 2 Preparation of Linker-CNP of Formula (II) [ka] The linker-CNP of formula (II) was prepared by solid-phase peptide synthesis. A method for preparing CNP by suppressing the racemization of the C-terminal cysteine ​​is described in Fujiwara et al., Chem. Pharm. Bull. (1996) 44(7): 1326-31. (Gln 6,14 After the peptide sequence of CNP was prepared on the resin, the linker was attached as the final residue using either 4-azido-3,3-dimethyl-1-(isopropylsulfonyl)-2-butyl succinimidyl carbonate or 4-azido-3,3-dimethyl-1-((N,N-dimethylamino)sulfonyl)-2-butyl succinimidyl carbonate. After disulfide formation, deblocking, and cleavage from the resin, the linker-CNP was isolated by reverse-phase HPLC.

[0077] Example 3 Preparation of CNP-loaded hydrogel microspheres of formula (IV) Activated degradable hydrogel microspheres of formula (III), wherein P 1 and P 2 = 10 kDa 4-arm poly(ethylene glycol), B * = triazole, C * = carboxamide, q = 1, R 1a = (N,N-dimethylamino)SO2, R 2a =H, each R 4a= methyl, x = 4, y = 0, r = 4, and Z' = (bicyclo[6.1.0]non-4-yn-9-yl)CH2-O-CO-NH ("BCN-O-CO-NH"), were prepared as described in Henise et al. (2020) Engineering Reports 2020;2:e12213. A slurry of activated microspheres (2.7 mL of slurry, 15.0 μmol of BCN) in reaction solvent (100 mM citrate in 1:1 iPrOH:HO, pH 3.0) was added to 1.2 equivalents of R to BCN (4.8 μmol in 2.4 mL of reaction solvent). 1 The loaded microspheres were mixed with linker-CNP of formula (II) (Example 2) = (N,N-dimethylamino)SO and incubated at 37°C for 16 hours with stirring. The loaded microspheres were washed four times with 12 mL of reaction solvent (OD of the final wash). 280 The loaded CNP was washed four times with 12 mL of isotonic acetate buffer (10 mM Na acetate, 143 mM NaCl, 0.05% polysorbate 20 (w / v), pH 5.0). The loaded CNP concentration and fraction were determined by dissolving approximately 30 μL of the loaded slurry (approximately 30 mg) in nine volumes (approximately 270 μL, 1 μL:1 mg slurry) of 125 mM borate pH 9.4 at 37°C for 24 h twice. The peptide content was measured by measuring the absorbance at 276 nm (ε = 5800 M). -1 cm -1 ) The PEG content in each slurry was measured using the PEG assay described previously. The percent loading of the microsphere slurry was determined as the ratio of peptide concentration to theoretical PEG reactive end groups based on the PEG assay. Similarly, activated degradable hydrogel microspheres of formula (III), where P 1 and P 2 = 10 kDa 4-arm poly(ethylene glycol), B * = triazole, C * = carboxamide, q = 1, R 1a = (N,N-dimethylamino)SO2, R 2a =H, each R 4a= methyl, x = 0, y = 4, r = 4, and Z' = (bicyclo[6.1.0]non-4-yn-9-yl)CH2-O-CO-NH ("BCN-O-CO-NH"), 1 = isopropyl-SO2 (Example 2) conjugate was prepared.

[0078] Example 4 Kinetics of peptide release A sample of loaded microspheres (approximately 30 mg) was placed in a 1.5 mL screw-cap microcentrifuge tube, and peptide release was initiated by adding 9 volumes (approximately 0.27 mL) of 100 mM Na-borate buffer (pH 9.4). The reaction was incubated in a 37°C water bath. At t=0 and various time points, the microsphere slurry was pelleted at 10,000 × g, 5 μL of the reaction supernatant was removed, and the samples were stored at -20°C. The (Gln 6,14 The concentration of CNP38 was measured by absorbance at 280 nm using Nanodrop UV-Vis. 280 was plotted and fitted to a single exponential function using Prism 8.0 software to determine the release rate of each peptide. 1 For R = isopropyl-SO2, a release half-life of 6.1 hours was observed, which corresponds to a release half-life of 610 hours at pH 7.4 and 37°C. 1 For =(N,N-dimethylamino)-SO2, a release half-life of 21 hours was observed, which corresponds to a release half-life of 2100 hours at pH 7.4 and 37°C.

[0079] Example 5 Pharmacokinetic studies in mice CD-1 mice (male, 7 weeks old, weighing ∼30 g) were administered a slurry of the hydrogel microspheres of Example 3 by subcutaneous injection suspended in a pH 5.0 buffer containing 10 mM sodium acetate, 143 mM NaCl, 10 mM methionine, 0.05% polysorbate 20 (w / v), and 1.2% sodium hyaluronate 60 kDa (w / v). 1Hydrogel microsphere conjugates of α-(N,N-dimethylamino)SO2 were administered at 0.7 μmol per mouse. HALT protease inhibitor was added and serum samples were collected at the following time points: 0, 8, 24, 48, 96, 168, 240, 336, 408, 04, 576, 672, 840, 1008, 1176, 344, 1512, 1680, 1848, and 2016 hours. The complete time course was collected using two groups of four mice each, with serum samples collected at alternating time points from each group. 1 The hydrogel microsphere conjugate, α = isopropyl-SO2, was administered at 0.22 μmol per mouse. HALT protease inhibitor was added and serum samples were collected at the following time points: 0, 8, 24, 48, 72, 120, 168, 240, 336, 432, 504, 600, and 672 hours. A complete time course was collected using three groups of four mice each, with serum samples collected every three time points across the groups. Serum samples were analyzed by absorbance (ε 280 =1681.8M -1 cm -1 ) as a standard quantified by (Gln 6,14 ) CNP38 was used to analyze the results by ELISA. The results are shown in Figure 7. The data were fit to a two-phase model. 1 For isopropyl-SO2, half-lives of 39 and 212 hours were observed. 1 For =(N,N-dimethylamino)-SO2, half-lives of 58 and 607 hours were observed.

[0080] Example 6 Pharmacokinetics in cynomolgus monkeys [Gln 6,14 ] CNP-38 loaded microspheres were prepared as described in Example 3, which 1 = isopropylsulfonyl, and R 1The loading slurries contained 3.1 μmol and 3.6 μmol of peptide / mL for (N,N-dimethylamino)sulfonyl, respectively. Microspheres were formulated in isotonic acetate (10 mM Na acetate, 143 mM NaCl) (pH 5.0), 0.05% Tween 20 buffer containing 1.2% sodium hyaluronate. A syringe (0.3 mL U-100 insulin syringe with a fixed 29 g × 1 / 2 inch needle, BD#34702) was filled with 1 mL of formulated microspheres under aseptic conditions.

[0081] Normal juvenile cynomolgus monkeys (3 monkeys per group, 1 female and 2 males) weighing 1.8 to 2.6 kg and over 18 months of age at the start of the experiment were administered the conjugate of Example 3 at a dose of 1.3 μmol / kg (R 1 = isopropylsulfonyl) or 1.2 μmol / kg (R 1 = (N,N-dimethylamino)-sulfonyl) was administered subcutaneously. Unwanted material was expelled from the prefilled syringe, and the remaining volume was administered. Blood samples (approximately 1 mL) were collected from a peripheral vein of monkeys administered 4A and 4B before administration and at 8, 24, 48, 96, 168, 240, 336, 408, 504, 576, 672, 744, and 840 hours post-dose. For monkeys administered 4B, blood was also collected at 1008, 1176, 1344, 1680, and 2160 hours post-dose. Blood samples were processed to plasma by collecting them in tubes containing K2EDTA and protease inhibitors (Halt protease inhibitor, ThermoFisher Scientific) and divided into two equal volumes for storage at -80°C. Beginning one day after the first dose, animals were examined at least weekly for general signs of toxicity, including fecal and urine quality, at the injection site, head, neck, limbs, trunk, tail, body orifices, and genitals. During handling, each animal was observed for changes in skin, hair, eyes, mucous membranes, secretions and excretions, lacrimation, piloerection, pupil size, and abnormal breathing patterns. Animal cages were also inspected for abnormal feces, urine, vomit, or other excretions or excretions.

[0082] Plasma [Gln 6,14 CNP-38 concentrations were measured using a CNP-22 fluorescent EIA kit (cat# FEK-012-03) from Phoenix Pharmaceuticals, Inc. and read on a Molecular Devices Spectramax i3 plate reader. Plasma samples were diluted with blank cynomolgus monkey plasma and substituted [Gln 6,14 ] Concentrations were obtained over the range of a standard curve (2 pM to 2 nM) generated using CNP-38. The analysis was repeated by two operators on separate days using two different sample aliquots.

[0083] Figures 8 and 9 show the R 1 Example 3 (FIG. 8) where R = isopropylsulfonyl, or 1 9 shows plasma concentrations in juvenile cynomolgus monkeys treated with Example 3 (FIG. 9), where R is (N,N-dimethylamino)-sulfonyl. 1 The conjugate of Example 3, in which [Gln = isopropylsulfonyl] was found to inhibit 100 pM or more of [Gln 6,14 ] provided continuous exposure to CNP-38. 1 The conjugate of Example 3, where [Gln] = (N,N-dimethylamino)-sulfonyl, demonstrated 100 pM or more of [Gln] over 3 months. 6,14 ] provided continuous exposure to CNP-38.

[0084] Example 7 Pharmacokinetics in young mice Three-week-old wild-type male mice (FVB / nJ; Charles River Laboratory, Inc.) were administered the conjugate of Example 3 by subcutaneous injection for 35 days at either a weight-adjusted dose (nmol / kg) or a fixed dose (nmol / animal, regardless of initial body weight). In the fixed dose / animal experiment, R 1 The conjugate of Example 3 (Conjugate 4A), where R = isopropylsulfonyl, was administered at either 20 or 50 nmol / mouse, and R 1The conjugate of Example 3 (Conjugate 4B), where [Gln] = (N,N-dimethylamino)sulfonyl, was administered at either 85 or 600 nmol / mouse. In weight-adjusted dose / animal experiments, Conjugate 4A was administered at 1.5 or 2.2 μmol / kg, and Conjugate 4B was administered at 6.1 μmol / kg. Doses were calculated based on body weight immediately prior to administration and administered at approximately the same time of day. Control animals received [Gln] in 30 mM acetic acid (pH 4.0) containing 10% (w / v) sucrose and 1% (v / v) benzyl alcohol (Wendt; Breinholt). 6,14 ] CNP-38 or vosoritide was administered at 70 nmol / kg, and control animals received the formulation buffer (isotonic acetate (10 mM Na acetate, 143 mM NaCl) (pH 5.0), 0.05% Tween 20 buffer containing 1.2% sodium hyaluronate). Daily weight-adjusted doses were calculated based on body weight immediately before dosing and administered at approximately the same time each day.

[0085] Free [Gln 6,14 For CNP-38 analysis, protease inhibitors (Halt Protease Inhibitor Cocktail, ThermoScientific) were added, and blood was collected via the tail vein and treated with K2EDTA to obtain approximately 15–20 μL of plasma. For mice treated with conjugate 4A, blood was collected at 0, 24, 168, 192, 336, 360, 504, 528, 672, and 840 h. For mice treated with conjugate 4B, blood was collected at 0, 24, 168, 336, 504, 672, and 840 h.

[0086] Body weight, tail length, and nose-anal length were collected at the start of the study and measured weekly throughout the treatment period. Mice were anesthetized using isoflurane and placed on a supine table. The tail was pulled straight and a metal ruler was used to gently depress the mouse, straightening the spinal curvature and allowing the mouse to fully extend. The nose-anal length (distance from the tip of the nose to the anus) and tail length (distance from the anus to the tip of the tail) were measured and recorded. The same ruler was used for all measurements, and all measurements were taken and recorded in centimeters. Total length (TL) was calculated by adding the measured tail length and nose-anal length.

[0087] On day 35, mice were anesthetized and bone lengths were measured by digital X-ray (spine, right femur, right tibia, humerus and ulna) in addition to measurements of nasoanal length and tail length. After deep anesthesia with isoflurane, mice were placed prone, and the lengths of the spine (distance from the cranial-most edge of the C1 vertebra to the caudal-most edge of the S4 vertebra in a lateral view, with manual consideration of curvature), right femur (distance from the center of the most proximal femoral head to the center of the most distal femoral head in a dorsoventral view), right tibia (distance from the center of the most proximal femoral head to the center of the most distal femoral head in a dorsoventral view), right humerus (distance from the center of the most proximal femoral head to the center of the most distal femoral head in a dorsoventral view), and right ulna (distance from the center of the most proximal femoral head to the center of the most distal femoral head in a dorsoventral view) were measured by radiography using a Spectral Imaging Instruments AMI HTX and associated Aura software.

[0088] Figures 10 and 11 show the weight-adjusted tail length measurements, respectively. Figure 12 shows the effect of 70 nmol of QDs on the total tail length after 5 weeks of administration: A) vehicle control; B) [Gln 6,14 Photographs of anesthetized mice treated with CNP-38 peptide; C) 2.2 μmol / kg of conjugate 4A every other week (Q2Wk); D) 2.2 μmol / kg of conjugate 4A every week (QWk); and E) 1.5 μmol / kg of conjugate 4A every week (QWk).

[0089] Table 1 shows the R 1 = isopropylsulfonyl (4A) or R 1 = (N,N-dimethylamino)sulfonyl (4B). Measurements were taken by X-ray. [Standard deviation] (% change from vehicle).

[0090] As a result, 1.5 μmol / kg of [Gln 6,14 ] weekly (QWk) administration of 4A containing CNP-38 or 2.2 μmol / kg of [Gln 6,14 Its biweekly (Q2Wk) administration with [Gln ]CNP-38 increased growth compared to vehicle control, and daily (QD) administration of [Gln 6,14 A single dose of 6.1 μmol / kg of conjugate 4B also demonstrated a significant improvement in serotonin activity compared with daily [Gln 6,14 ] supported growth enhancement comparable to that of CNP-38. These results suggest that appropriate regimens of long-acting 4A and 4B administered weekly, biweekly, or monthly can achieve growth promotion in young mice that is comparable to or superior to daily administration of vosoritide or other CNP variants. [Table 1]

[0091] Example 8 Kinetics of peptide release and hydrogel degradation Example 3 (R 1 = isopropylsulfonyl, or R 1 = (N,N-dimethylamino)sulfonyl) prepared according to (Gln 6,14 Biodegradable hydrogel microspheres loaded with CNP38 were prepared under accelerated conditions at pH 9.4 and 37°C according to a published method (Henise et al. (2020) Engineering Reports 2020;2:e12213). 6,14) The release of CNP38 and dissolved PEG was analyzed.

[0092] The solubilized peptides were analyzed by OD 280 The solubilized PEG was quantified by OD, and the solubilized PEG was quantified using a BaCl / I / NaI colorimetric assay using PEG8000 as the standard. The initial degradation of the hydrogel microspheres may dissolve the PEG fragments still carrying the peptides. 280 The total solubilized peptide measured by OD may be the sum of the free peptide released from the microspheres and the solubilized PEGylated peptide. Therefore, the data were analyzed by correcting for the amount of solubilized peptide, assuming a constant first-order cleavage rate of the drug linker. Assuming that the solubilized PEG is representative of the bulk hydrogel, the total OD 280 The amount of solubilized PEG peptide contributed to the (PEG-peptide) sol = PEG(t)·exp(-kt) where PEG(t) = amount of solubilized PEG present at time t, and k = first order rate constant for linker-peptide cleavage. This amount is OD 280 The amount of free peptide released from the hydrogel at time t is obtained by subtracting OD from the total peptide measured by 280 The experimental data for and PEG were normalized according to the total amount in the assay, as determined by the plateau value, and the normalized data were then fitted using an iterative process to determine the value of k that gave the best fit according to the residual sum of squares.

[0093] Peptide release and hydrogel degradation were measured using OD 280 The normalized values ​​for α and β solubilized PEG were averaged and measured in six replicates. The results are shown in Figures 13A and 13B.

[0094] As shown in Figure 13A, R 1 For the conjugate of Example 3, where k is 0.05, the optimum value for k is t at pH 9.4.1 / 2 = 6.1 hours, which is estimated to be 610 hours at pH 7.4 and 37°C. The hydrogel microspheres were completely dissolved by approximately 30 hours at this pH. As shown in Figure 13B, R 1 For the conjugate where t = (N,N-dimethylamino)sulfonyl, the optimum value for k is t at pH 9.4. 1 / 2 = 15.8 hours, which translates to 1580 hours at pH 7.4 and 37° C. The hydrogel microspheres were completely dissolved by approximately 30 hours at this pH.

Claims

1. Formula (I) M-(L-E) ａ (I) [In the formula, M is a hydrogel matrix; L is a linker; E is C-natriuretic peptide; and a is an integer representing the number of LE moieties that produce the appropriate concentration of E in a given volume of matrix. Hydrogel conjugate of C-natriuretic peptide, shown as

2. 2. The hydrogel conjugate of claim 1, wherein E is selected from the group consisting of SEQ ID NOs: 1-6.

3. E is (Gln ６，１４ 2. The hydrogel conjugate of claim 1, wherein the amino acid sequence is CNP38 (SEQ ID NO: 4).

4. M is a group represented by formula (III) 【Chemistry 1】 (III) [In the formula, q=0 to 6; R １ａ and R ２ａ are independently H, alkyl, CN, NO ２ , optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR ３ａ , -SOR ３ａ , or -SO ２ R ３ａ where: R ３ａ is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ５ａ , or -NR ５ａ ２ where: Each R ５ａ are independently H or optionally substituted alkyl, or both R ５ａ the groups, taken together with the nitrogen to which they are attached, form a heterocyclic ring; Here, R １ａ and R ２ａ may be taken together with the carbons to which they are attached to form a 3- to 8-membered ring, where R １ａ and R ２ａ Only one of may be H or alkyl; Each R ４ａ are independently H or C １ -C ３ alkyl, or both R ４ａ form a 3- to 6-membered ring together with the carbons to which they are attached; Z' is a functional group that mediates coupling to the linker-LE; x and y are each independently 0 to 6; B ＊ and C ＊ are each independently a connecting group; and P １ and P ２ are independently r-arm polymers having an average molecular weight of 1 to 40 kDa, where r is an integer from 2 to 8. The hydrogel conjugate according to any one of claims 1 to 3, which is a biodegradable hydrogel represented by the formula:

5. L is the following formula: 【Chemistry 2】 [In the formula, n=0 to 6; R １ and R ２ are independently H, alkyl, CN, NO ２ , optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR ３ , -SOR ３ , or -SO ２ R ３ where: R ３ is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ５ , or -NR ５ ２ where: Each R ５ are independently H or optionally substituted alkyl, or both R ５ the groups, taken together with the nitrogen to which they are attached, form a heterocyclic ring; Here, R １ and R ２ may be taken together with the carbons to which they are attached to form a 3- to 8-membered ring, where R １ and R ２ Only one of may be H or alkyl; Each R ４ are independently H or C １ -C ３ alkyl, or both R ４ form a 3- to 6-membered ring together with the carbons to which they are attached; Z ＊ is a connecting group; and NH is the residue of the amino group of the CNP peptide. The hydrogel conjugate of any one of claims 1 to 4, comprising a residue represented by:

6. R １ CN or SO ２ R ３ 6. The hydrogel conjugate of claim 5, wherein:

7. Each R ４ But independently, C １ -C ３ alkyl, or both R ４ The hydrogel conjugate of claim 5 or 6, wherein, together with the carbon to which they are attached, form a 3- to 6-membered ring.

8. The hydrogel conjugate is represented by formula (IV): 【Transformation 3】 (IV) [In the formula, n=0 to 6; R １ and R ２ are independently H, alkyl, CN, NO ２ , optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR ３ , -SOR ３ , or -SO ２ R ３ where: R ３ is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ５ , or -NR ５ ２ where: Each R ５ are independently H or optionally substituted alkyl, or both R ５ the groups, taken together with the nitrogen to which they are attached, form a heterocyclic ring; Here, R １ and R ２ may be taken together with the carbons to which they are attached to form a 3- to 8-membered ring, where R １ and R ２ Only one of may be H or alkyl; Each R ４ are independently H or C １ -C ３ alkyl, or both R ４ form a 3- to 6-membered ring together with the carbons to which they are attached; Z ＊ , B ＊ , and C ＊ are each independently a connecting group; E is a CNP peptide; q=0 to 6; R １ａ and R ２ａ are independently H, alkyl, CN, NO ２ , optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR ３ａ , -SOR ３ａ , or -SO ２ R ３ａ where: R ３ａ is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ５ａ , or -NR ５ａ ２ where: Each R ５ａ are independently H or optionally substituted alkyl, or both R ５ａ the groups, taken together with the nitrogen to which they are attached, form a heterocyclic ring; Here, R １ａ and R ２ａ may be taken together with the carbons to which they are attached to form a 3- to 8-membered ring, where R １ａ and R ２ａ Only one of may be H or alkyl; Each R ４ａ are independently H or C １ -C ３ alkyl, or both R ４ａ form a 3- to 6-membered ring together with the carbon to which they are attached, x and y are each independently 0 to 6; and P １ and P ２ are independently r-arm polymers having an average molecular weight of 1 to 40 kDa, where r is an integer from 2 to 8. The hydrogel conjugate of any one of claims 1 to 7, wherein

9. R １ CN or SO ２ R ３ and R １ａ CN or SO ２ R ３ａ 9. The hydrogel conjugate of claim 8, wherein:

10. R １ CN or SO ２ R ３ and R ２ is H; each R ４ is methyl; n=1-2; E is SEQ ID NO: 4; Z ＊ and B ＊ is a triazole; C ＊ is a carboxamide; x=0-4; y=0-4; R １ａ CN or SO ２ R ３ａ and R ２ａ is H; each R ４ａ is methyl; q=1 to 2; P １ and P ２ and r=4-8. The hydrogel conjugate of claim 8, wherein each of

11. R １ But SO ２ R ３ and R ３ The hydrogel conjugate of claim 10, wherein is optionally substituted alkyl.

12. R １ But SO ２ R ３ and R ３ However, (R ５ ) ２ The hydrogel conjugate of claim 10, wherein N is N.

13. 10. A method for producing the hydrogel conjugate of claim 1, comprising: 【Chemistry 4】 (II) [In the formula, n=0 to 6; R １ and R ２ are independently H, alkyl, CN, NO ２ , optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR ３ , -SOR ３ , or -SO ２ R ３ where: R ３ is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ５ , or -NR ５ ２ where: Each R ５ are independently H or optionally substituted alkyl, or both R ５ the groups, taken together with the nitrogen to which they are attached, form a heterocyclic ring; Here, R １ and R ２ may be taken together with the carbons to which they are attached to form a 3- to 8-membered ring, where R １ and R ２ Only one of may be H or alkyl; Each R ４ are independently H or C １ -C ３ alkyl, or both R ４ form a 3- to 6-membered ring together with the carbons to which they are attached; Z is a functional group that mediates coupling to the hydrogel of formula (III) via the associated functional group Z′; and NH is the amino group residue of CNP peptide E. The linker-CNP peptide represented by the formula: ＊ under conditions to form a compound of formula (III) 【Transformation 5】 (III) [In the formula, q=0 to 6; R １ａ and R ２ａ are independently H, alkyl, CN, NO ２ , optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR ３ａ , -SOR ３ａ , or -SO ２ R ３ａ where: R ３ａ is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ５ａ , or -NR ５ａ ２ where: Each R ５ａ are independently H or optionally substituted alkyl, or both R ５ａ the groups, taken together with the nitrogen to which they are attached, form a heterocyclic ring; Here, R １ａ and R ２ａ may be taken together with the carbons to which they are attached to form a 3- to 8-membered ring, where R １ａ and R ２ａ Only one of may be H or alkyl; Each R ４ａ is H or C １ -C ３ alkyl, or both R ４ａ form a 3- to 6-membered ring together with the carbons to which they are attached; Z′ is a functional group that mediates coupling to an associated functional group Z; x and y are each independently 0 to 6; B ＊ and C ＊ are each independently a connecting group; and P １ and P ２ are independently r-arm polymers having an average molecular weight of 1 to 40 kDa, where r is an integer from 2 to 8. and optionally isolating the resulting conjugate.

14. A pharmaceutical composition comprising the hydrogel conjugate of any one of claims 1 to 12 and a pharmaceutically acceptable buffer, wherein the pharmaceutical composition has a pH of 3 to 7.

15. 15. Use of the pharmaceutical composition of claim 14 in the treatment of a disease or condition requiring treatment with a CNP peptide in a patient in need of treatment.

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