C-Natriuretic Peptide Sustained-Release Hydrogel Conjugate

Abstract

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

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and interest to U.S. Provisional Application No. 63 / 118,568, filed on 25 November 2020, the disclosures of which are incorporated herein by reference in their entirety.

[0002] Statement regarding the submission of sequence listings This application is filed with an electronic sequence listing. The sequence listing is provided as a file named 670572002640SeqList.txt, created on November 19, 2021, and has a size of 3,047 bytes. The electronic information of the sequence listing is incorporated in its entirety by reference.

[0003] This specification provides a sustained-release hydrogel conjugate of c-natriuretic peptide, a method for producing the same, and a method for using the same. [Background technology]

[0004] C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which are released in response to hypertension and hypervolemic states, promoting natriuresis and, as a result of diuresis, causing the loss of sodium and water, thereby lowering blood volume and blood pressure. CNP was first isolated in 1990, making it the last of the three major natriuretic peptides to be discovered, but it is the most widely expressed of this family, found particularly in the brain, chondrocytes, and endothelial cells. Exogenous CNP, which usually acts as a local paracrine / autocrine regulator, is a potent arterial and venous dilator in vitro and has been shown to lower blood pressure in the human body. 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 the femur, tibia, and vertebrae being 50–80% shorter than in wild-type mice. As a result, CNP and its analogues are attracting attention for the treatment of dwarfism and achondroplasia.

[0005] Natriuretic peptides (NAPs) are characterized by a core 17-amino acid disulfide linkage ring crucial for receptor binding. This cyclic structure is conserved among family members and species. The first product of the CNP gene (Nppc) is a 126-amino acid prepro-CNP, which is cleaved by the signal peptide to produce a pro-CNP peptide, further processed by frin proprotein convertase to produce 53-amino acid CNP-53. CNP-53 is further processed by an unidentified protease to produce the major active species, 22-amino acid CNP (CNP-22). CNP activity is tightly controlled 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 takes the peptide into lysosomes for degradation. It is also proteolytic by neutral endopeptidases (NEPs) in plasma and on the surface of endothelial cells.

[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 the physiological response to CNP. CNP is thought to be a sole endogenous ligand for NPR-B and can also bind to and activate NPR-C. CNP binds to NPR-B at physiological concentrations (picomoles) with an affinity 50 to 500 times higher than ANP and BNP. Gene knockout of NPR-B results in impaired endochondral ossification, resulting in longitudinal shortening of the vertebrae and limb bones. This model further suggests a role for NPR-B in the development of female reproductive organs. NPR-B is primarily found in veins but is also present in arteries. NPR-C binds to all three natriuretic peptides with high affinity. NPR-C gene knockout also results in skeletal abnormalities and increased basal bone metabolism, which is likely due to a change 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 people. The main phenotype is short stature, averaging about 4 feet, with a large head and a protruding forehead. Associated complications include sleep apnea syndrome, recurrent ear infections, obesity, hydrocephalus, and spinal stenosis. Growth hormone therapy is ineffective in patients with achondroplasia. FGFR3 normally downregulates cartilage and bone growth by inhibiting the development of chondrocytes, which are cells that produce and maintain the cartilage matrix necessary for bone growth. Therefore, overactivity of FGFR3 causes reduced bone growth and achondroplasia. When fibroblast growth factor binds to FGFR3, a signaling cascade occurs via the MAPK / ERK pathway. This cascade can be interrupted 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 in the treatment of achondroplasia.

[0008] Several CNP analogs have been disclosed (U.S. Patents 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 a Phase 3 clinical trial; it is currently under review by the FDA. Various CNP analogs and PEGylated conjugates are also being studied (Wendt, J Pharmacol Exp Ther 353:132-149, April 2015). Controlled release conjugates of CNP have been disclosed (PCT publication numbers WO2016 / 110577, WO2017 / 118703, WO2017 / 118693, WO2017 / 118698, WO2017 / 118700, WO2017 / 118704, and WO2017 / 118707). Known side effects of BMN-111 are increased heart rate and decreased arterial blood pressure, which become more pronounced with increasing doses. Due to the short half-life of BMN-111, relatively high doses are required to provide sufficient time-therapeutic peptide levels in a daily dosing schedule. A significantly increased half-life would allow for the maintenance of therapeutic peptide levels between dosings without requiring such overdoses.

[0009] CNP prodrugs that extend the half-life of CNP through releaseable conjugations have been disclosed (Breinholt et al., 2019 J. Pharmacol Exp Ther 370: 459-71; PCT publication numbers, WO2016 / 110577, WO2017 / 118698, and WO2019 / 0022237). Combination therapies using controlled-release CNP analogs have also been disclosed (PCT publication number, WO2018 / 060314). The disclosed conjugates release CNP via a hydrolysis mechanism, which is disadvantageous because it is difficult to avoid premature release from the conjugate during storage, leading to degradation in the presence of moisture and a shortened shelf life; therefore, the development of dry formulations is necessary (PCT publication number, WO2020 / 165081). However, such formulations need to be reconstituted before use and may not be suitable for insoluble conjugates such as microparticle hydrogels. Therefore, there is still a need for more convenient, longer-acting forms of CNP for the treatment of various diseases and illnesses. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows two linker-CNPs of formula (II). In both embodiments, Z = azide, n = 1, R2 = H, each R4 = methyl, and E is [(Gln6,14)CNP38], bound to the linker via an α-amine of the N-terminal glycine. In the first linker-peptide, R1 = isopropyl-SO2-, and the linker releases the peptide after conjugation to hydrogel microspheres with a half-life of 260 hours at pH 7.4 and 37°C. In the second linker-peptide, R1 = (N,N-dimethylamino)-SO2-, and the linker releases the peptide after conjugation to hydrogel microspheres with a half-life of 1200 hours at pH 7.4 and 37°C.

[0011] [Figure 2]Figure 2 shows a method for producing a conjugate of formula (IV), where M is a hydrogel containing a degradable crosslink, which includes the step of contacting a hydrogel of formula (III) containing a reactive conjugate Z' with a linker-peptide of formula (II) containing the associated reactive group Z, under conditions in which the conjugate functional group Z reacts with the conjugate functional group Z' to conjugate the linker-peptide to the hydrogel via the residual functional group Z*.

[0012] [Figure 3] Figure 3 shows the crosslinking structure in the conjugate of formula (I) shown in Example 3, where M is an insoluble hydrogel. In each case, the interpolymer crosslinking includes a linker represented by 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) [wherein Z=azide, n=1, R1=isopropyl-SO2-, R2=H, each R4=methyl, and E is (Gln6,14)CNP38 bonded to the linker via an α-amine of the N-terminal glycine] is conjugated to the hydrogel of formula (III) with x=0, y=4, and z=0 via a carbamoylbicyclononinyl group. In the second case, the linker-CNP of formula (II) [wherein Z=azide, n=1, R1=(N,N-dimethylamino)SO2-, R2=H, each R4=methyl, and E is (Gln6,14)CNP38 bonded to the linker via an α-amine of the N-terminal glycine] is conjugated to the hydrogel of formula (III), where x=4, y=0, and z=0, via a carbamoylbicyclononinyl group.

[0013] [Figure 4] Figure 4 shows one method for producing the linker-CNP of formula (II), where the protected CNP peptide is produced on a solid support using a standard method, the linker is bonded by reaction with succinimidyl carbonate, and then the peptide is deblocked and cleaved from the resin to form a disulfide.

[0014] [Figure 5] Figure 5 shows an ideal structure representing the arrangement of P1 (filled circles), P2 (white circles), and linker drug LE (black circles) in the crosslinking matrix M. The two polymers are alternately arranged in the matrix by their linkage via related groups Z and Z', preventing self-bonding, and each crosslink contains the linker drug. In practice, some crosslinks may be missing, for example, due to missing arms in the commercial production of the polymer, or due to the formation of multiple crosslinks between individual P1 and P2 units.

[0015] [Figure 6] Figure 6 shows the results of the stability test for 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 better stability than vosoritide.

[0016] [Figures 7A-7B] Figures 7A and 7B show the results of pharmacokinetic experiments in mice treated with the conjugate of Example 3 (see also Figure 3). Figure 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. Figure 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 pharmacokinetic experiments in young cynomolgus monkeys treated with the Example 3 conjugate, where R1 = isopropylsulfonyl. Figure 8 shows the plasma concentrations of [Gln6,14]CNP-38 after a single subcutaneous administration of 1.3 μmol / kg of the Example 3 conjugate, where R1 = isopropylsulfonyl, to young cynomolgus monkeys. The data are the average of three animals. This conjugate provided continuous exposure to [Gln6,14]CNP-38 at ≥100 pM for approximately one month.

[0018] [Figure 9] Figure 9 shows the results of pharmacokinetic experiments in young cynomolgus monkeys treated with the Example 3 conjugate, where R1 = (N,N-dimethylamino)sulfonyl. The plasma concentrations of [Gln6,14]CNP-38 after a single subcutaneous administration of 1.2 μmol / kg of the Example 3 conjugate (R1 = (N,N-dimethylamino)sulfonyl) to young cynomolgus monkeys are shown. The data are averages from three animals. This conjugate provided continuous exposure to [Gln6,14]CNP-38 at ≥100 pM for more than 3 months.

[0019] [Figure 10] Figure 10 shows the results of measuring the total length (tail length + nose-to-anus length, TL) of mice treated with a constant amount of [Gln6,14]CNP-38 and the conjugate of Example 3, where R1 = isopropylsulfonyl (4A) or R1 = (N,N-dimethylamino)sulfonyl (4B), per mouse at each dosing interval. From left to right: Black (solid line), vehicle control; Brown (light blue), QD[Gln6,14]CNP-38; Blue (horizontal stripe), QWk 4A 20 nmol / mouse; Red (rising line), QWk 4A 50 nmol / mouse; Green (descending line), single dose 4B 85 nmol / mouse. Plotted as a percentage of initial total length (TL) against time; values ​​are shown as mean ± SD.

[0020] [Figure 11]Figure 11 shows the total length (tail length + nose-to-anus length, TL) measurements of mice treated with [Gln6,14]CNP-38 and the conjugate 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 (light blue), QD[Gln6,14]CNP-38; Blue (horizontal line), QWk 4A 1.5 μmol / kg; Red (rising line), QWk 4A 2.2 μmol / kg; Green (descending line), Q2Wk 2.2 μmol / kg; Purple (checkerboard pattern), single dose 4B 6.1 μmol / kg; Orange (vertical line), single dose 4B 40 μmol / kg. Plotted as a percentage of the initial total volume (TL) against time; values ​​are shown as mean ± SD.

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

[0022] [Figures 13A-13B]Figures 13A and 13B show peptide release and hydrogel degradation in the conjugates of Example 3, where R1 = isopropylsulfonyl (Figure 13A) and R1 = (N,N-dimethylamino)sulfonyl (Figure 13B), at pH 9.4 and 37°C. In Figure 13A, with R1 = isopropylsulfonyl, a release time of (Gln6,14)CNP38 t1 / 2 = 6.1 hours was obtained by total solubilized peptide (solid line) and solubilized PEG (dashed line), which corresponds to 610 hours under physiological conditions (pH 7.4, 37°C). In Figure 13B, with R1 = (N,N-dimethylamino)sulfonyl, a release time of (Gln6,14)CNP38 t1 / 2 = 15.8 hours was obtained by total solubilized peptide (solid line) and solubilized PEG (dashed line), which corresponds to 1580 hours under physiological conditions (pH 7.4, 37°C). The data represents the mean of n=6, and the error bars are shown as the standard deviation. [Overview of the Initiative]

[0023] This disclosure relates to a conjugate of C-natriuretic peptide (CNP) that provides sustained, low-level release, supporting weekly, monthly, or even less frequent administration of these peptides, and is expected to be useful in the treatment of diseases and conditions such as achondroplasia.

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

[0025] In a second aspect, the present invention provides a compound of formula (II)

Chemical formula

[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, alkyls are 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, and n-decyl. In some embodiments, alkyls are cyclic. Examples of cyclic alkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, and cyclohexyl.

[0027] The term "alkoxy" is understood to include alkyl groups bonded to oxygen, such as methoxy, ethoxy, isopropoxy, cyclopropoxy, and cyclobutoxy.

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

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

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

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

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

[0033] The terms “heterocyclic ring” or “heterocyclyl” are understood to mean a 3- to 15-membered aromatic or non-aromatic ring containing at least one N, O, or S atom. Examples, but not limited to, include piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, as well as the illustrative 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, unless otherwise specified, that the 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, but not limited to, include alkyl, alkenyl, alkynyl, halogen, -CN, and -OR substituents. 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)2NR aaR bb , -P(O)(OR aa )(OR bb Examples include heterocyclyl, heteroaryl, or aryl, where alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl are each independently and optionally R cc It may be replaced with, here, R aa and R bb Each of these is independently H, alkyl, alkenyl, alkynyl, heterocyclyl, heteroaryl, or aryl, R aa and R bb These, together with the nitrogen atom to which they are bonded, form a heterocycline which may optionally be substituted with alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, or -CN, where, Each R cc These are independently alkyl, alkenyl, alkynyl, halogen, heterocyclyl, heteroaryl, aryl, -CN, or -NO2.

[0035] In this specification, unless otherwise specified, the use of terms such as "a" and "an" means one or more.

[0036] The drug release rate is based on R 1 and R 2 It is adjustable by appropriately selecting the appropriate R. 1 and R 2 For a description, see U.S. Patent No. 8,680,315. In some embodiments, base R 1 and R 2 Each of these is an intervening R 1 R 2The CH proton may be independently substituted with electron-donating and / or electron-withdrawing substituents that alter its acidity, resulting in very high flexibility and control over the rate of drug elimination. Electron-withdrawing groups are defined as groups with a Hammett rule sigma value greater than 0 (see, e.g., Hansch et al. 1991 Chemical Reviews 91: 165-195). The term "electron-donating group" is used in R 1 R 2 This refers to substituents that reduce the acidity of CH, and electron-donating groups are typically negative Hammett σ or Tuft σ * These are associated with constants and are well known in the field of physical organic chemistry. (Hammett constants represent aryl / heteroaryl substituents, and Taft constants represent substituents in the non-aromatic moiety). Suitable electron-donating substituents include, but are not limited to, lower alkyl, lower alkoxy, lower alkylthio, amino, alkylamino, dialkylamino, and silyl groups. Similarly, "electron-withdrawing groups" are R 1 R 2 This refers to substituents that increase the acidity of the CH group, and electron-withdrawing groups are typically positive Hammett σ or Tuft σ * It is associated with a constant and is well known in the field of 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 coefficient, see U.S. Patent No. 9,649,385.

[0037] In some embodiments, R 1 and R 2 At least one of them is -CN. In some embodiments, R 1 and R 2 At least one of them is -NO2. In some embodiments, R 1 and R 2 At least one of them is an aryl compound containing 6 to 10 carbon atoms, which may be optionally substituted. For example, in some embodiments, R 1and R 2 At least one of them is phenyl, naphthyl, or anthracenyl, each of which may be optionally substituted. In some embodiments, R 1 and R 2 At least one of them is optionally substituted heteroaryl containing 3 to 7 carbons and containing at least one N, O or S atom. For example, in some embodiments, R 1 and R 2 At least one of them is pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl, each of which may be optionally substituted. In some embodiments, R 1 and R 2 At least one of them is optionally substituted alkenyl containing 2 to 20 carbon atoms. In some embodiments, R 1 and R 2 At least one of them is optionally substituted alkynyl containing 2 to 20 carbon atoms. In some embodiments, R 1 and R 2 At least one of them 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 is independently H, or optionally substituted alkyl containing 1 to 20 carbon atoms, or both R 5 groups 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 them is -CN, -SOR 3 , or -SO2R 3 In some embodiments, R 1 and R 2 At least one of them is -CN, or -SO2R 3 In some embodiments, R 1 and R 2 At least one of them is -CN, or -SO2R 3 And here, 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 them is -CN, -SO2N(CH3)2, -SO2CH3, -SO2phenyl, -SO2(chlorophenyl), -SO2(4-methylphenyl), -SO2N(CH2CH2)2O, -SO2N(CH2CH2)2S, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2. In some embodiments, R 1 and R 2 One of them is -CN, or -SO2R 3 And here, R 3 is optionally substituted alkyl, optionally substituted aryl, or -NR 5 It is 2; and the other is H. In some embodiments, R 1 and R 2 One of them is -CN, -SO2N(CH3)2, -SO2CH3, -SO2phenyl, -SO2(chlorophenyl), -SO2(4-methylphenyl), -SO2N(CH2CH2)2O, -SO2N(CH2CH2)2S, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2; and the other is H.

[0039] In some embodiments, each R 4R is independently a C1-C3 alkyl group. In some embodiments, R 4 Both are 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 This refers to CN or SO2R as defined above. 3 And R 2 H is H, and each R 4 The group consists of C1-C3 alkyl groups, 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. Typical groups, but not limited to them, include azides, in which case the related group Z' on M is an alkyne or cycloalkyne, and the remaining connecting group is a triazole; also, amino ethers, in which case the related group Z' on M is a carbonyl, and the remaining connecting group is an oxime; also, trans-cyclooctene or cyclopropane, in which case the related group Z' on M is 1,2,5,6-tetrazine, and the remaining functional group is pyridazine; and also, thiols, in which case the related group Z' on M is a maleimide or halocarbonyl, and the remaining connecting group is a thioether. In preferred embodiments, Z is an azide and Z' is cyclooctine. Typical cyclooctins known in the art include, but are not limited to, 5-hydroxycyclooctin (5HCO), 1-fluorocycloocto-2in-1-carboxylate (MFCO), and bicyclo[6.1.0]nona-4-in (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" means the peptides of SEQ ID NOs: 1-6 and their stabilized analogs. Particularly preferred are CNP analogs in which specific amino acid residues are substituted to improve the stability of the peptide. Such stabilized analogs include peptides in which the asparagine residue is replaced with a residue less susceptible to deamidation, such as glutamine or alanine, and peptides in which an oxidation-sensitive residue is substituted, such as peptides in which methionine is substituted with norleucine. Exemplary embodiments of CNP are provided in SEQ ID NOs: 1-6 below.

[0044] Sequence ID 1 ([CNP22]) GLSKGCFGLKLDRIGSMSGLGC C 6 -C 22 disulfide

[0045] Sequence ID 2 ([Bosolitide]) PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC C 23 -C 39 disulfide

[0046] Sequence ID 3 ([CNP38]) LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC C 22 -C 38 disulfide

[0047] Sequence ID 4([(Gln 6,14 )CNP38]) LQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC C 22-C 38 disulfide

[0048] Sequence ID 5([(Gln 6,14 ,Nle 33 )CNP38]) LQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSXSGLGC C 22 -C 38 disulfide

[0049] Sequence ID 6 ([ASB20123]) GLSKGCFGLKLDRIGSMSGLGCVQQRKDSKKPPAKLQPR C 6 -C 22 disulfide

[0050] In some embodiments, stabilized CNP is (Gln 6,14 )-CNP38 (Sequence ID 4), or (Gln 6,14 ,Nle 33 This is CNP38 (sequence number 5).

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

[0052] In some embodiments, M is a water-insoluble hydrogel carrier. In preferred embodiments, M is a material of formula (III) [ka] (III) [In the formula, q = 0 to 6; R 1a and R 2a These are independently H, alkyl, CN, NO2, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, and -COR 3a -SOR 3a , or -SO2R 3a And here, 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, and here, Each R 5a R is independently an alkyl group that is H or optionally substituted, or both R 5a The groups, together with the nitrogen atoms to which they are bonded, form a heterocyclic ring; Here, R 1a and R 2a These may form a 3- to 8-membered ring together with the carbon atoms to which they are bonded, where R 1a and R 2a Only one of them may be H or alkyl; Each R 4a These are independently either H or C1-C3 alkyl, or both R 4a These, together with the carbon atoms to which they are bonded, form a 3-6 membered ring. Z' is a functional group that mediates the coupling of the linker-CNP of formula (II) disclosed herein to the relevant functional group Z; x and y are independently between 0 and 6; B * and C * Each is an independent connective; and, P 1 and P 2 These are independently r-arm polymers with an average molecular weight of 1 to 40 kDa, where r is an integer between 2 and 8. This is a degradable hydrogel represented by [the symbol]. In a preferred embodiment, P 1 and P 2 It is a poly(ethylene glycol) having an r-arm.

[0053] The degradation rate of the hydrogel in equation (III) is R 1 and R 2 As will be discussed in this specification, base R 1a and R 2a It can be adjusted by the appropriate selection of [the appropriate element].

[0054] In some embodiments, R 1a and R 2a At least one of them is -CN. In some embodiments, R 1a and R 2a At least one of them is -NO2. In some embodiments, R 1a and R 2a At least one of them is an aryl compound containing 6 to 10 carbon atoms, which may be optionally substituted. For example, in some embodiments, R 1a and R 2a At least one of them is phenyl, naphthyl, or anthracenyl, each of which may be optionally substituted. In some embodiments, R 1a and R 2a At least one of them is an optionally substituted heteroaryl containing 3 to 7 carbon atoms and at least one N, O, or S atom. For example, in some embodiments, R1a and R 2a At least one of them is pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl, each of which may be optionally substituted. In some embodiments, R 1a and R 2a At least one of them is an optionally substituted alkenyl containing 2 to 20 carbon atoms. In some embodiments, R 1a and R 2a At least one of them is an optionally substituted alkynyl containing 2 to 20 carbon atoms. In some embodiments, R 1a and R 2a At least one of them is -COR 3a -SOR 3a , or -SO2R 3a And here, R 3a 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 R is independently H, or an optionally substituted alkyl group containing 1 to 20 carbon atoms, or both R 5a The groups, together with the nitrogen atoms to which they are bonded, form a heterocyclic ring.

[0055] In some embodiments, R 1a and R 2a At least one of them is -CN, -SOR 3a , or -SO2R 3a In some embodiments, R 1a and R 2a At least one of them is -CN, or -SO2R 3a In some embodiments, R 1a and R2a At least one of them is -CN, or -SO2R 3a And here, 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 them is -CN, -SO2N(CH3)2, -SO2CH3, -SO2phenyl, -SO2(chlorophenyl), -SO2(4-methylphenyl), -SO2N(CH2CH2)2O, -SO2N(CH2CH2)2S, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2. In some embodiments, R 1a and R 2a One of them is -CN, or -SO2R 3a And here, R 3a is optionally substituted alkyl, optionally substituted aryl, or -NR 5a It is 2; and the other is H. In some embodiments, R 1a and R 2a One of them is -CN, -SO2N(CH3)2, -SO2CH3, -SO2phenyl, -SO2(chlorophenyl), -SO2(4-methylphenyl), -SO2N(CH2CH2)2O, -SO2N(CH2CH2)2S, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2; and the other is H.

[0056] In some embodiments, each R 4a R is independently a C1-C3 alkyl group. In some embodiments, R 4a Both are 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 2However, hydrogels of formula (III) that are poly(ethylene glycol) having an r-arm are prepared by methods described, for example, 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 control the drug release rate and the subsequent hydrogel dissolution rate. * and C * Typical connecting groups include, but are not limited to, triazoles, carboxamides, carbamates, oximes, and thioethers. When hydrogel polymerization is carried out by azide / cyclooctin cyclization, B * and / or C * The connecting group is triazole, as described above. In some embodiments, B * It is a triazole, and C * It is carboxamide. 1 and P 2 These are synthetic or natural polymers such as poly(ethylene glycol), dextran, and hyaluronic acid. In these hydrogels, polymer chains are crosslinked to form an insoluble three-dimensional matrix (Figure 6), where each crosslink has a binding point to the linker-CNP of formula (II). The crosslinks are mainly formed by the group R 1a and R 2a At a rate dominated by , the polymer is slowly cleaved by non-hydrolyzable beta-elimination, ultimately yielding soluble polymer fragments. These hydrogels are formed by the reaction of the relevant groups Z and Z' on formulas (II) and (III), as shown in Figure 2, forming connecting groups Z * This enables binding with the linker drug via the linker, thereby generating a conjugate of formula (I), where M is an insoluble hydrogel containing a linker-peptide conjugated at each crosslink, which more specifically corresponds to formula (IV) [wherein R 1 , R2 , R 4 E, n, Z * , y, P 1 , P 2 B * , q, r, C * x, R 1a , R 2a , and R 4a This is as disclosed herein. [ka] (IV)

[0061] Figure 3 shows an exemplary structure of crosslinking in such a conjugate of formula (IV). Figure 2 further shows a hydrogel of formula (III) containing the linking group Z', where Z and Z' react to link the linker peptide residue Z to the hydrogel. * A method for producing a conjugate of formula (IV) is shown, comprising contacting a linker-peptide of formula (II) containing the relevant connecting group Z under conditions that form a conjugate of formula (IV). If Z and Z' are azide / cyclooctine, such conditions are a temperature of 0 to 100°C, preferably 0 to 50°C, more preferably 25 to 50°C. The solvent in which the linker-peptide and hydrogel are suspended or dissolved may be water, an organic solvent, or a mixed solvent, depending on the solubility of the linker-peptide. A typical solvent is an aqueous buffer with a pH of 2 to 7, preferably 2 to 5, which may optionally be mixed with a water-miscible cosolvent such as methanol, ethanol, 2-propanol, tert-butanol, acetonitrile, dimethylformamide, acetonitrile, or tetrahydrofuran. If M is an insoluble matrix, the conjugate of formula (I) may optionally be isolated by washing to remove unreacted linker-peptides and reaction byproducts. The reaction procedures for equations (II) and (III) are similar to those reported by Henise et al. (2020) Engineering Reports 2020;2:e12213.

[0062] The linker-CNP of formula (II) can be prepared by reacting a CNP peptide or a protected derivative thereof with a linker reagent of formula (V) [wherein X is a leaving group such as chloride, O-succinimidyl, or O-nitrophenyl, 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 a chemical method 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 in the synthesis of the peptide on a solid support, as shown in Figure 3. After the synthesis of the protected peptide sequence on the solid support, the linker is attached by reaction with an active carbonate such as succinimidyl carbonate, as described in the examples below. The resulting intermediate is deblocked, cleaved from the resin, and a disulfide is formed to obtain the linker-CNP of formula (II).

[0064] In a third aspect, the present invention relates to a protocol for formulating and administering a conjugate of formula (I). In one embodiment, the conjugate is 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 may contain any excipients necessary to maintain the stability of the conjugate, such as buffers, antimicrobial agents, antioxidants, density and osmotic pressure modifiers, and agents that promote suspension and prevent microsphere aggregation. In particular, given the pH-sensitive nature of the beta-desorption mechanism of linker cleavage in these conjugates, stability is enhanced by using a buffer, preferably a low pH buffer, preferably pH 2-7, more preferably pH 3-7, and even more preferably pH 4-5. Suitable buffers are those known in the art with respect to pharmaceutical applications, including acetates, citrates, malates, maleates, phosphates, and other buffers effective in these pH ranges.

[0065] Administration can be via any route, such as subcutaneous, intramuscular, or intra-articular. The conjugate of the present invention is expected to be useful in treating diseases and conditions in both humans and animals that respond to CNP, such as achondroplasia, at weekly, monthly, or more frequent administration frequencies.

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

[0067] The following examples are illustrative and not limit the scope of this disclosure. All references cited herein, including those cited in particular and those cited in general, are incorporated herein by reference with respect to specific aspects of their disclosure.

[0068] Manufacturing A Manufacturing of a 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 hexane (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 heated to -20°C over 1 hour, then recooled to -50°C, and methyl 3-azido-2,2-dimethylpropionate (prepared according to Kim, Synthetic Communications; 7.70 g, 50 mmol) was added. The mixture was heated to +10°C over 2 hours 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 obtain 14.05 g of crude ketone product. Chromatography of SiO2 (220 g) using step gradients of 0, 20, 30, 40, and 50% siRNA / hexane yielded purified 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butanone (10.65 g, 86%) as a crystalline solid.

[0069] The above ketone was dissolved in 200 mL of methanol, cooled on ice, treated with sodium borohydride (0.96 g, 25 mmol) for 15 minutes, then quenched with 4 mL of 6N 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 MgSO4, filtered, and concentrated to obtain 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 mixture of N-hydroxysuccinimide (6.90 g, 60 mmol) and triphosgene (5.93 g, 20 mmol) stirred in 250 mL of dichloromethane cooled on ice. The mixture was stirred on ice for 15 minutes and then warmed 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 stirred further at ambient temperature for 1 hour. After cooling on ice, the mixture was treated with 100 mL of water to separate the phases. 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% siRNA / hexane to obtain 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 hexane (14.5 mL, 20 mmol) was added at -78°C to a 2.96 mL solution of diisopropylamine (21 mmol) in 80 mL of THF. The mixture was briefly warmed to ambient temperature and then recooled to -78°C. Isopropylmethylsulfone (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 slowly warmed to ambient temperature over 1 hour, and then quenched with 3.47 mL of 6N 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 obtain 2.9 g of crude ketone product as a yellow liquid. Chromatography of SiO2 (25g) using a 0-70% siRNA / hexane gradient yielded purified 4-azido-1-(isopropylsulfonyl)-3,3-dimethyl-2-butanone (1.73g, 70%) as a crystalline solid.

[0072] The above ketone was dissolved in 14 mL of methanol, cooled on ice, treated with sodium borohydride (0.13 g, 3.5 mmol) for 15 minutes, 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 MgSO4, filtered, and concentrated to obtain 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 mixture of the aforementioned alcohol (1.44 g, 5.78 mmol) and triphosgene (2.92 g, 9.83 mmol) stirred in 45 mL of THF. After 30 minutes, the suspension was filtered and concentrated. The residue was redissolved in 25 mL of THF and 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% KHSO4, water, and brine, dried over MgSO4, filtered, and evaporated. Chromatography of SiO2 (25 g) using a 0-80% siRNA / hexane gradient yielded purified 4-azido-1-(isopropylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (1.46 g) as a white crystalline solid. Linkers manufactured according to these procedures include, but are not limited to, the following: 4-Azido-1-(methylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (in formula (V), R 1 =MeSO2, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=1. 4-Azido-1-(isopropylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (in formula (V), R 1 = i PrSO2, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=1. 4-Azido-1-(phenylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (in formula (V), R 1 =PhSO2, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=1. 4-Azido-1-(4-methylphenylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (in formula (V), R 1 =(4-methylphenyl)SO2, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=1. 4-Azido-1-(chlorophenylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (in formula (V), R 1 =(4-chlorophenyl)SO2, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=1. 4-Azido-1-cyano-3,3-dimethyl-2-butyl succinimidyl carbonate (in formula (V), R 1 =CN, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=1. 4-Azido-1-(N,N-dimethylaminosulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (in formula (V), R 1 =(Me2N)SO2, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=1. 4-Azido-1-(morpholinosulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (in formula (V), R 1 =(O(CH2CH2)2NSO2, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=1. 4-Azido-1-(thiomorpholinosulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (in formula (V), R 1 =(S(CH2CH2)2NSO2, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=1. The following linkers were prepared starting from ethyl 4-chloro-2,2-dimethylbutyrate. 5-Azido-1-(chlorophenylsulfonyl)-4,4-dimethyl-2-pentyl succinimidyl carbonate (in formula (V), R 1 =(4-chlorophenyl)SO2, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=2. 5-Azido-1-cyano-4,4-dimethyl-2-pentyl succinimidyl carbonate (in formula (V), R 1 =CN, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=2. 5-Azido-1-(phenylsulfonyl)-4,4-dimethyl-2-pentyl succinimidyl carbonate (in formula (V), R 1 =phenyl SO2, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=2. 5-Azido-1-(methylsulfonyl)-4,4-dimethyl-2-pentyl succinimidyl carbonate (in formula (V), R 1 =MeSO2, R 2 =H, each R 4 =Me, Z=azid, X=O-succinimidil, and n=2.

[0074] Manufacturing B Hydrogel of formula (III) The hydrogels of formula (III) were prepared as sterile, injectable microspheres according to the instructions in Henise et al (2020) Engineering Reports 2(8): e12213. The prepared hydrogels include the following: (a)P 1 and P 2 These are, respectively, 10kDa 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-(bicyclo[6.1.0]nona-4-in-9-ylmethyl). (b)P 1 and P 2 These are, respectively, 10kDa 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=0; y=4; and Z'=NH-CO-O-(bicyclo[6.1.0]nona-4-in-9-ylmethyl). (c)P 1 and P 2 These are, respectively, 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-(bicyclo[6.1.0]nona-4-in-9-ylmethyl). (d)P 1 and P 2 These are, respectively, 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=0; y=4; and Z'=NH-CO-O-(bicyclo[6.1.0]nona-4-in-9-ylmethyl). (e)P 1 and P 2 These are, respectively, 20 kDa 4-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-cyclooctinyl). P 1 and P 2 These are 20 kDa 4-arm poly(ethylene glycol) respectively; 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-cyclooctinyl). (f)P 1 and P 2 These are, respectively, 20 kDa 4-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-cyclooctinyl).

[0075] Example 1 (Gln 6,14 Stability studies of CNP38 and bosolitide 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 mixture was maintained at 37°C, and aliquots were periodically removed and stored at -20°C until analysis. Samples were analyzed using Dionex BioLC, ProPac SCX-10, 250 × 4 mm; 0–100% buffer B for 10 minutes; 1 mL / min; column temperature 40°C. Peaks were monitored at 220 nm. Buffer A: 10 mM MES, pH 6.2, @22°C; Buffer B: 10 mM MES, 1 M NaCl, pH 6.2, @22°C. Significant degradation of vosoritide was observed within one week, compared to [Gln6,14 ]-CNP38 was extremely stable throughout the entire 4-week stability experiment period (Figure 6).

[0076] Example 2 Manufacturing of linker-CNP of formula (II) [ka] The linker-CNP of formula (II) was prepared by solid-phase peptide synthesis. A method for producing CNP while suppressing racemization of the C-terminal cysteine ​​is described in Fujiwara et al., Chem. Pharm. Bull. (1996) 44(7): 1326-31. (Gln 6,14 After preparing the peptide sequence of CNP on a resin, a 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 hydrogel microspheres supported by CNP of formula (IV) Activated degradable hydrogel microspheres of formula (III), wherein P 1 and P 2 = 10kDa 4-arm poly(ethylene glycol), B * = Triazole, C * =carboxamide, q=1, R 1a = (N,N-dimethylamino)SO2, R 2a =H, each R 4aHydrogel microspheres with methyl, x=4, y=0, r=4, and Z'=(bicyclo[6.1.0]nonano-4-in-9-yl)CH2-O-CO-NH ("BCN-O-CO-NH") were prepared according to the description in Henise et al. (2020) Engineering Reports 2020;2:e12213. A slurry (2.7 mL slurry, 15.0 μmol BCN) containing activated microspheres in a reaction solvent (100 mM citrate in 1:1 iPrOH:H2O, pH 3.0) was mixed with R at a ratio of 1.2 equivalents relative to BCN (4.8 μmol in 2.4 mL of reaction solvent). 1 The microspheres were mixed with the linker-CNP of formula (II) = (N,N-dimethylamino)SO2 (Example 2) 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 washing solution). 280 The samples were washed four times with 12 mL of isotonic acetate buffer (10 mM sodium acetate, 143 mM NaCl, 0.05% polysorbate 20 (w / v), pH 5.0) until the concentration was below detectable. The concentration and fraction of the supported CNP were determined by dissolving approximately 30 μL of packed slurry (approximately 30 mg) twice at 37°C for 24 hours in 9 times the volume (approximately 270 μL, 1 μL: 1 mg slurry) of 125 mM borate pH 9.4. Peptide content was determined by absorbance at 276 nm (ε = 5800 M). -1 cm -1 The PEG content in each slurry was determined by the PEG assay described above. The loading percentage of the microsphere slurry was determined as the ratio of the peptide concentration to the theoretical PEG-reactive terminal group based on the PEG assay. Similarly, activated degradable hydrogel microspheres of formula (III), where P 1 and P 2 = 10kDa 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]nonano-4-in-9-yl)CH2-O-CO-NH ("BCN-O-CO-NH"), R 1 A conjugate containing isopropyl-SO2 (Example 2) was prepared.

[0078] Example 4 Peptide release dynamics A sample of the supported microspheres (approximately 30 mg) was placed in a 1.5 mL screw-cap microcentrifuge tube, and peptide release was initiated by adding 9 times (approximately 0.27 mL) of 100 mM sodium borate buffer (pH 9.4). The reaction mixture 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 collected, and the sample was stored at -20°C. (Gln) 6,14 The concentration of CNP38 was measured using Nanodrop UV-Vis by absorbance at 280 nm. 280 The data was plotted and fitted to a single exponential function using Prism 8.0 software to determine the release rate of each peptide. 1 =In the case of 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 In the case of (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 A slurry of hydrogel microspheres from Example 3, 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), was administered by subcutaneous injection to CD-1 mice (male, 7 weeks old, body weight ~30 g). 1Hydrogermic sphere conjugates, specifically (N,N-dimethylamino)SO2, were administered at a dose of 0.7 μmol per mouse. 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, with the addition of a HALT protease inhibitor. The complete time course was collected using two groups of four mice each, with serum samples collected alternately from each group at different time points. 1 Hydrogel sphere conjugate, which is isopropyl-SO2, was administered at a dose of 0.22 μmol per mouse. Serum samples were collected at the following time points: 0, 8, 24, 48, 72, 120, 168, 240, 336, 432, 504, 600, and 672 hours, with the addition of a HALT protease inhibitor. The complete time course was collected using three groups of four mice each, with serum samples collected every three time points, alternating between groups. Serum samples were measured by absorbance (ε). 280 = 1681.8M -1 cm -1 (Gln) is used as a standard quantified by 6,14 The analysis was performed using CNP38 by ELISA. The results are shown in Figure 7. The data were fitted to a two-phase model. 1 In the case of isopropyl-SO2, half-lives of 39 hours and 212 hours were observed. 1 In the case of (N,N-dimethylamino)-SO2, half-lives of 58 hours and 607 hours were observed.

[0080] Example 6 Pharmacokinetics in cynomolgus monkeys [Gln 6,14 Microspheres supported with CNP-38 were prepared according to the description in Example 3, and this is R 1 =Isopropylsulfonyl, and R 1The packed slurry contained 3.1 μmol and 3.6 μmol peptides / mL of (N,N-dimethylamino)sulfonyl, respectively. The microspheres were formulated with 0.05% Tween 20 buffer containing isotonic acetate (10 mM sodium acetate, 143 mM NaCl) (pH 5.0) and 1.2% sodium hyaluronate. One mL of the formulated microspheres was filled into a syringe (0.3 mL U-100 insulin syringe, BD#34702 with a fixed 29 g × 1 / 2 inch needle) under sterile conditions.

[0081] At the start of the experiment, normal young cynomolgus monkeys (3 in each group, 1 female and 2 males) weighing 1.8-2.6 kg and over 18 months old were administered the conjugate from Example 3 at a concentration of 1.3 μmol / kg (R) based on their current body weight. 1 = isopropylsulfonyl) or 1.2 μmol / kg (R 1 (N,N-dimethylamino)-sulfonyl was administered subcutaneously. Unnecessary material was drained from the pre-filled syringe, and the remaining amount was administered. Blood samples (approximately 1 mL) were collected from the peripheral veins 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 after administration. For monkeys administered 4B, blood was also collected at 1008, 1176, 1344, 1680, and 2160 hours after administration. The blood samples were processed into plasma by collecting them in tubes containing K2EDTA and a protease inhibitor (Halt protease inhibitor, ThermoFisher Scientific), and divided into two equal volumes for storage at -80°C. From day 1 after the initial administration, animals were examined at least once a week for general signs of toxicity, including the quality of feces and urine, at the injection site, head, neck, limbs, torso, tail, body openings, and genitals. During handling, changes in each animal were observed, including skin, hair, eyes, mucous membranes, secretion and excretion, lacrimation, piloerection, pupil size, and abnormal respiratory patterns. The animal cages were also examined for abnormal feces, urine, vomit, and other excretions and secretions.

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

[0083] Figures 8 and 9 are R 1 =Isopropylsulfonyl, as in Example 3 (Figure 8), or R 1 The plasma concentrations of young cynomolgus monkeys treated with Example 3 (Figure 9), which is (N,N-dimethylamino)-sulfonyl, are shown. 1 = The conjugate of Example 3, which is isopropylsulfonyl, was steeped for about 1 month at a concentration of 100 pM or higher [Gln 6,14 This provided continuous exposure to CNP-38. 1 The conjugate of Example 3, which is (N,N-dimethylamino)-sulfonyl, was found to have a concentration of 100 pM or higher for more than 3 months. 6,14 This provided continuous exposure to CNP-38.

[0084] Example 7 Pharmacokinetics of young mice Three-week-old wild-type male mice (FVB / nJ; Charles River Laboratory, Inc.) were administered the conjugate from Example 3 by subcutaneous injection for 35 days, either in a weight-adjusted amount (nmol / kg) or a fixed amount (nmol / animal, unrelated to initial body weight). In the fixed-amount / animal experiment, R 1 The conjugate of Example 3 (Conjugate 4A), which is isopropylsulfonyl, was administered to mice at either 20 or 50 nmol / mouse, and R 1The conjugate of Example 3 (conjugate 4B), which is (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 before administration and administered at approximately the same time each day. Comparison animals were given [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 vosolitide was administered at 70 nmol / kg, while control animals were given the formulation's buffer (isotonic acetate (10 mM sodium acetate, 143 mM NaCl) (pH 5.0), 0.05% Tween 20 buffer containing 1.2% sodium hyaluronate). The daily weight-adjusted dose was calculated based on the body weight immediately before administration and administered at approximately the same time each day.

[0085] Free [Gln 6,14 For the analysis of CNP-38, a protease inhibitor (Halt protease inhibitor cocktail, ThermoScientific) was 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 hours. For mice treated with conjugate 4B, blood was collected at 0, 24, 168, 336, 504, 672, and 840 hours.

[0086] Body weight, tail length, and nose-to-anus length were collected at the start of the study and measured weekly throughout the treatment period while the mice were alive. Mice were anesthetized with isoflurane, placed on a supine table, their tails were pulled straight, and the mice were gently pressed down with a metal ruler to straighten the spinal curvature and allow the mice to fully extend. The nose-to-anus 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 performed and recorded in centimeters. Total length (TL) was calculated by adding the measured tail length and nose-to-anus length.

[0087] On day 35, the mice were anesthetized, and in addition to measuring nasal and anal length and tail length, bone lengths were measured by digital X-ray (vertebrae, right femur, right tibia, humerus, and ulna). After deep anesthesia with isoflurane, the mice were placed face down, and the lengths of the following bones were measured by X-ray using a Spectral Imaging Instruments AMI HTX and associated Aura software: the spine (distance from the most cranial end of the C1 vertebra to the most caudal end of the S4 vertebra when viewed from the side, with manual consideration of curvature), the right femur (distance from the nearest center of the femoral head to the most distal center when viewed dorsoventrally), the right tibia (distance from the nearest center of the bone to the most distal center when viewed dorsoventrally), the right humerus (distance from the nearest center of the bone to the most distal center when viewed dorsoventrally), and the right ulna (distance from the nearest center of the bone to the most distal center when viewed dorsoventrally).

[0088] Figures 10 and 11 show the measured total tail length of the weight-adjusted doses, respectively. Figure 12 shows the following at 5 weeks: A) Vehicle control; B) QD of 70 nmol [Gln 6,14 The images show anesthetized mice treated with CNP-38 peptide: C) 2.2 μmol / kg conjugate 4A every other week (Q2Wk); D) 2.2 μmol / kg conjugate 4A every week (QWk); and E) 1.5 μmol / kg conjugate 4A every week (QWk).

[0089] Table 1 is R 1 = Isopropylsulfonyl (4A) or R 1 The bone length after 5 weeks of treatment with the conjugate of Example 3, which is (N,N-dimethylamino)sulfonyl (4B), is shown. Measurements were taken by X-ray. [Standard deviation] (percentage change from vehicle).

[0090] As a result, 1.5 μmol / kg [Gln 6,14 Weekly (QWk) administration of 4A containing CNP-38, or 2.2 μmol / kg of [Gln 6,14 [CNP-38] administered every other week (Q2Wk) increased growth compared to daily (QD) [Gln 6,14 It showed that it was equivalent to or better than that of CNP-38 or vosolitide. A single dose of 6.1 μmol / kg conjugate 4B was also effective, and daily [Gln] was used for at least 3 weeks until stabilization. 6,14 This supported growth increases comparable to those of CNP-38. These results suggest that appropriate regimens of long-acting 4A and 4B administered weekly, bi-weekly, or monthly can achieve growth enhancement in young mice equivalent to or better than daily administration of bosolitide or other CNP variants. [Table 1]

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

[0092] Solubilized peptides are OD 280 The quantification was performed by [method], and the solubilized PEG was quantified using a BaCl2 / I2 / NaI colorimetric assay with PEG8000 as the standard. Due to the initial degradation of hydrogel microspheres, there is a possibility that PEG fragments still bound to peptides may dissolve, so OD 280 The total solubilized peptide measured by this method may be the sum of the free peptide released from the microspheres and the solubilized PEGylated peptide. Therefore, assuming a constant primary cleavage rate of the drug linker, the data were analyzed by correcting for the amount of solubilized peptide. Assuming that the solubilized PEG is representative of the bulk hydrogel, the total OD 280 The amount of solubilized PEG peptide contributing to this can be easily given by the following formula. (PEG-peptide) sol = PEG(t)·exp(-kt) In the formula, PEG(t) = the amount of solubilized PEG present at time t, and k = the first-order rate constant of linker-peptide cleavage. This amount OD 280 By subtracting from the total peptide measured by OD, the amount of free peptide released from the hydrogel at time t is obtained. 280 The experimental data for PEG were normalized according to the total volume of the assay determined by the plateau value, and the normalized data were then fitted using an iterative process to determine the value of k that gives the best fit according to the sum of squared residuals.

[0093] Peptide release and hydrogel degradation are OD 280 The normalized values ​​of the solubilized PEG were averaged and measured six times. The results are shown in Figures 13A and 13B.

[0094] As shown in Figure 13A, R 1 = In the case of the conjugate of Example 3, which is isopropylsulfonyl, the optimal value of k is at pH 9.4, and t1 / 2 = 6.1 hours, which is estimated to be 610 hours at pH 7.4 and 37°C. Hydrogel microspheres were completely dissolved at this pH within approximately 30 hours. As shown in Figure 13B, R 1 For the conjugate =(N,N-dimethylamino)sulfonyl, the optimal value of k is at pH 9.4, and t 1 / 2 This corresponds to 15.8 hours, which is estimated to be 1580 hours at pH 7.4 and 37°C. Hydrogel microspheres completely dissolved within approximately 30 hours at this pH.

Claims

1. Formula (IV) 【Chemistry 1】 (IV) [In the formula, n = 0 to 6; R1 is -SO2R3, R2 is H, alkyl, CN, NO2, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR3, -SOR3, or -SO2R3, 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 And here, Each R ５ R is independently H or optionally substituted alkyl, or both R ５ The groups, together with the nitrogen atoms to which they are bonded, form a heterocyclic ring; Here, R １ and R ２ may together with the carbon to which they are attached form a 3- to 8-membered ring, where only one of R １ and R ２ may be H or alkyl; Each R ４ H or C １ -C ３ Alkyl or both R ４ These, together with the carbon atoms to which they are bonded, form a 3- to 6-membered ring; Z ＊ , B ＊ , and C ＊ Each of them is independently a connecting element; E is a type C natriuretic peptide; q = 0 to 6; R1a is -SO2R3a, R 2a is 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 And here, Each R ５ａ R is independently H or optionally substituted alkyl, or both R ５ａ The groups, together with the nitrogen atoms to which they are bonded, form a heterocyclic ring; Each R ４ａ H or C １ -C ３ Alkyl or both R ４ａ These, together with the carbon atoms to which they are bonded, form a 3- to 6-membered ring. x and y are independently between 0 and 6; and, P １ and P ２ This is a poly(ethylene glycol) with an r-arm having an average molecular weight of 1 to 40 kDa, where r is an integer from 2 to 8. A hydrogel conjugate, as shown by [formula].

2. R2 is H; each R ４ However, it is methyl; n = 1 to 2; E is sequence number 4; Z ＊ and B ＊ However, it is a triazole; C ＊ However, it is carboxamide; x = 0 to 4; y = 0 to 4; R 2a is H; each R ４ａ However, it is methyl; q = 1 to 2; P １ and P ２ The hydrogel conjugate according to claim 1, wherein each is independently a poly(ethylene glycol) having an r-arm of 1 to 40 kDa; and r = 4 to 8.

3. The hydrogel conjugate according to claim 2, wherein R3 is an optionally substituted alkyl group.

4. R ３ The hydrogel conjugate according to claim 3, wherein the methyl

5. R 3 is (R ５ ) ２ N is such that each R ５ The hydrogel conjugate according to claim 2, wherein the atoms are independently H or optionally substituted alkyl.

6. Each R ４ａ The hydrogel conjugate according to any one of claims 1 to 5, wherein the alkyl group is independently optionally substituted.

7. Both R ４ａ The hydrogel conjugate according to claim 6, wherein the methyl

8. A hydrogel conjugate according to any one of claims 1 to 7, wherein q is an integer from 1 to 3.

9. A hydrogel conjugate according to any one of claims 1 to 8, wherein x is an integer from 1 to 3.

10. A hydrogel conjugate according to any one of claims 1 to 9, wherein y is an integer from 1 to 3.

11. The hydrogel conjugate according to any one of claims 1 to 10, wherein E is selected from the group consisting of Sequence ID Nos. 1 to 6.

12. E is (Gln ６，１４ A hydrogel conjugate according to any one of claims 1 to 10, wherein the compound is CNP38 (SEQ ID NO: 4).

13. formula 【Chemistry 2】 [In the formula, n = 0 to 6; R1 is -SO2R3, R ２ 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 And here, 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 And here, Each R ５ R is independently H or optionally substituted alkyl, or both R ５ The groups, together with the nitrogen atoms to which they are bonded, form a heterocyclic ring; Each R ４ H or C １ -C ３ Alkyl or both R ４ These, together with the carbon atoms to which they are bonded, form a 3- to 6-membered ring; 【Transformation 3】 This indicates the binding point of the linker drug unit to the polymer carrier; and, E is a type C natriuretic peptide. A hydrogel conjugate, as shown by [formula].

14. The hydrogel conjugate according to claim 13, wherein E is selected from the group consisting of Sequence ID Nos. 1 to 6.

15. E is (Gln ６，１４ The hydrogel conjugate according to claim 13, wherein the compound is CNP38 (SEQ ID NO: 4).

16. R2 is H; each R ４ The hydrogel conjugate according to any one of claims 13 to 15, wherein n is methyl; n = 1 to 2; and E is sequence number 4.

17. The hydrogel conjugate according to claim 16, wherein R3 is an optionally substituted alkyl group.

18. R ３ The hydrogel conjugate according to claim 17, wherein the methyl group is present.

19. R 3 is (R ５ ) ２ N is such that each R ５ The hydrogel conjugate according to claim 16, wherein the atoms are independently H or optionally substituted alkyl.

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