Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/2242—Atrial natriuretic factor complex: Atriopeptins, atrial natriuretic protein [ANP]; Cardionatrin, Cardiodilatin

Definitions

• C-type natriuretic peptide is a member of a family of natriuretic peptides including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) that are released in response to hypertensive and hypervolaemic states, and promote natriuresis and diuresis resulting in loss of sodium and water thereby lowering blood volume and pressure. While CNP was first isolated in 1990 and was the last of the three main natriuretic peptides to be discovered, it is the most widely expressed of the family, being found especially in brain, chondrocytes, and endothelial cells.
• exogenous CNP is a potent dilator of arteries and veins ex vivo, and has been shown to lower blood pressure in vivo in humans.
• CNP has a primary role in the regulation of bone growth and may also play a role in neuronal development and protection.
• Genetic disruption of CNP production in mice leads to dwarfism through the reduction of bone length; femurs, tibiae, and vertebrae are 50-80% shorter than in wild-type mice.
• CNP and analogs have attracted attention for the treatment of dwarfism and achondroplasia.
• Natriuretic peptides are characterized by a core 17-amino acid disulfide-linked ring that is critical for receptor binding. This cyclic structure is conserved across members of the family and between species.
• the initial product of the CNP gene (Nppc) is a 126-amino acid prepro-CNP; cleavage of a signal peptide yields a pro-CNP peptide that is further processed by the furin proprotein convertase to yield the 53-amino acid CNP-53.
• CNP-53 is further processed by as-yet unidentified proteases to provide the predominant active species, the 22-amino acid CNP (CNP-22).
• CNP activity is tightly regulated by two major degradative pathways resulting in an extremely short plasma half-life ( ⁇ 3 minutes).
• CNP has high affinity for a clearance receptor, NPR-C, that internalizes the peptide into the lysosome for degradation. It is also proteolyzed by neutral endopeptidase (NEP) in plasma and on endothelial cell surfaces.
• NEP neutral endopeptidase
• NPR-A Three specific natriuretic peptide receptors have been characterized, NPR-A, NPR-B, and NPR-C.
• CNP appears to be the sole endogenous ligand for NPR-B and can also bind and activate NPR-C.
• CNP binds NPR-B at physiological concentrations (picomolar) with an affinity 50-500 times greater than that of ANP and BNP.
• Genetic knock-out of NPR-B results in impairment of endochondrial ossification and resulting longitudinal shortening of vertebrae and limb bones. This model has further suggested a role for NPR-B in development of the female reproductive tract.
• NPR-B is found primarily on veins, but also on 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 clearance of CNP from NPR-C to NPR-B.
• Achondroplasia is a genetic disorder characterized by dwarfism caused by a mutation that results in an overactive fibroblast growth factor receptor 3 (FGFR3). It is the most prevalent form of dwarfism, affecting about 1 in 27,500 people. The predominant phenotype is short height, on average about 4 feet, an enlarged head and prominent forehead. Associated complications include sleep apnea, recurrent ear infections, obesity, hydrocephalus, and spinal stenosis. Growth hormone therapy is not effective for patients with achondroplasia.
• FGFR3 overactive fibroblast growth factor receptor 3
• FGFR3 normally down-regulates cartilage and bone growth by inhibiting the development of chrondrocytes, cells that produce and maintain the cartilaginous matrix necessary for bone growth; hyperactive FGFR3 thus results in diminished bone growth and achondroplasia.
• Binding of finbroblast growth factors to FGFR3 results in a signaling cascade 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.
• CNP or CNP analogs may find utility in the treatment of achondroplasia. [0008]
• CNP analogs have been disclosed (US Patent Nos. 8,198,242, 8,377,884, and 9,266,939).
• Various analogs as well as PEGylated conjugates of CNP have also been 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,
• BMN-111 WO2017/118700, WO2017/118704, and WO2017/118707.
• Known side-effects of BMN-111 are increased heart rate and a drop in arterial blood pressure, which become more prominent as the dose is increased.
• the short half-life of BMN-111 requires a relatively high dose in order to provide therapeutic peptide levels for a sufficient time given a daily administration schedule. A significant increase in the half-life would allow for maintenance of therapeutic levels of peptide between dosings without the need for such over-dosing.
• 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 W02019/0022237). Combination therapy using controlled-release CNP analogs has also been disclosed (PCT Publication No. WO20 18/060314).
• the disclosed conjugates release CNP through a hydrolytic mechanism, which is disadvantageous as it is difficult to avoid premature release from the conjugate during storage, leading to degradation and shortened shelf-life in the presence of moisture; this has necessitated development of dry formulations (PCT Publication No.
• Figure 1 illustrates two linker-CNPs of Formula (II).
• Z azide
• n l
• R 2 H
• each R 4 methyl
• E is [(Gln 6,14 )CNP38] attached to the linker via the alpha-amine of the N-terminal glycine.
• R 1 isopropyl-S0 2 - and the linker releases the peptide with a half-life of 260 hours at pH 7.4, 37 °C after conjugation to hydrogel microspheres.
• R 1 (N.N-dimethylamino)-S0 2 - and the linker releases the peptide with a half-life of 1200 hours at pH 7.4, 37 °C after conjugation to hydrogel microspheres.
• FIG. 2 diagrams a method for producing a conjugate of Formula (IV) wherein M is a hydrogel comprising degradable crosslinks, comprising the steps of contacting a hydrogel of Formula (III) comprising a reactive connecting group Z’ with a linker-peptide of Formula (II) comprising a cognate reacting group Z under conditions wherein connecting functionality Z reacts with connecting functionality Z’ so as to conjugate the linker-peptide to the hydrogel through residual functionality Z*.
• Figure 3 illustrates the structures of crosslinks in conjugates of Formula (I) wherein M is an insoluble hydrogel, as illustrated in Example 3.
• Figure 4 illustrates one method to prepare a linker-CNP of Formula (II) wherein the protected CNP peptide is prepared on a solid support using standard methods, the linker is attached by reaction with a succinimidyl carbonate, and the peptide is then deblocked, cleaved from the resin, and the disulfide is formed.
• Figure 5 illustrates an idealized structure showing the disposition of P 1 (filled circles) and P 2 (open circles) and linker-drug L-E (black circles) in the crosslinked matrix M.
• the two polymers alternate in the matrix due to their connection via cognate groups Z and Z’, which prevents self-connection, and each crosslink comprises a linker-drug.
• some crosslinks may be missing, for example due to missing arms in commercial preparations of the polymers or due to formation of multiple crosslinks between individual P 1 and P 2 units.
• Figure 6 shows the results of the stability study of Example 1. Vosoritide and (Gln 6 ,Gln 14 )CNP38 were kept at pH 7.4, 37 °C and monitored by anion-exchange HPLC. (Gln 6 ,Gln 14 )CNP38 showed enhanced stability over vosoritide.
• Figures 7A-7B show the results of pharmacokinetic experiments in mice treated with the conjugates of Example 3 (also Figure 3).
• Anesthetized mice were initially positioned for measurements with their heads extended, noses aligned to the horizontal guide line and tails pulled straight; the distance between the top guide line and the end of the tail best represents total length (TL).
• R 1 isopropylsulfonyl
• CNPs C-natriuretic peptides
• the present disclosure provides extended release conjugates comprising an insoluble hydrogel matrix with a multiplicity of covalently attached linker- peptides, wherein the linkers cleave via a beta-elimination mechanism under physiological conditions of pH and temperature to release free CNP peptides.
• the conjugates of the invention can be illustrated schematically as formula (I)
• M-(L-E) a (I) wherein M is an insoluble hydrogel matrix connected to a multiplicity of CNP peptides E through cleavable linker L, L is a linker that cleaves by a pH-dependent beta-elimination mechanism, such as a linker as disclosed in US Patent 8,680,315, and a is an integer that represents the number of L-E moieties that yield a suitable concentration of E in a given volume of the matrix. Suitable concentrations are 0.01-50 mg peptide per mL of matrix, preferably 1-25 mg of peptide per mL.
• the linker L releases free CNP peptides with a half- life suitable for the desired period of administration, typically between 150 and 2500 hours as measured in vitro , preferably between 250 and 1500 hours in vitro at pH 7.4, 37 °C.
• R 1 and R 2 are independently H, alkyl, CN, NO2, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR 3 , - SOR 3 , or -SO2R 3 , wherein
• 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, wherein each R 5 is independently H or optionally substituted alkyl, or both R 5 groups are taken together with the nitrogen to which they attach to form a heterocyclic ring; wherein R 1 and R 2 may be taken together with the carbon to which they attach to form a 3-8 membered ring, and wherein one and only one of R 1 and R 2 may be H or alkyl; each R 4 is independently H or C1-C3 alkyl, or both R 4 are taken together with the carbon to which they attach to form a 3-6 membered ring; Z is a functional group for mediating coupling to the carrier M through a cognate functional group Z’ ; and
• NH is the residue of an amino group of CNP peptide E.
• alkyl includes linear, branched, or cyclic saturated hydrocarbon groups of 1-20, 1-12, 1-8, 1-6, or 1-4 carbon atoms.
• an alkyl is linear or branched.
• linear or branched alkyl groups include, without limitation, 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.
• an alkyl is cyclic.
• cyclic alkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, and the like.
• alkoxy includes alkyl groups bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and the like.
• alkenyl includes non-aromatic unsaturated hydrocarbons with carbon-carbon double bonds and 2-20, 2-12, 2-8, 2-6, or 2-4 carbon atoms.
• alkynyl includes non-aromatic unsaturated hydrocarbons with carbon-carbon triple bonds and 2-20, 2-12, 2-8, 2-6, or 2-4 carbon atoms.
• aryl includes aromatic hydrocarbon groups of 6-18 carbons, preferably 6-10 carbons, including groups such as phenyl, naphthyl, and anthracenyl.
• heteroaryl includes aromatic rings comprising 3-15 carbons containing at least one N, O or S atom, preferably 3-7 carbons containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and the like.
• alkenyl, alkynyl, aryl or heteroaryl moieties may be coupled to the remainder of the molecule through an alkyl linkage.
• the substituent will be 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 the alkenyl, alkynyl, aryl or heteroaryl is coupled.
• halogen or “halo” includes bromo, fluoro, chloro and iodo.
• heterocyclic ring refers to a 3-15 membered aromatic or non-aromatic ring comprising at least one N, O, or S atom.
• examples include, without limitation, piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, as well as the exemplary groups provided for the term “heteroaryl” above.
• a heterocyclic ring or heterocyclyl is non-aromatic.
• a heterocyclic ring or heterocyclyl is aromatic.
• R aa and R bb are each independently H, alkyl, alkenyl, alkynyl, heterocyclyl, heteroaryl, or aryl, or
• R aa and R bb are taken together with the nitrogen atom to which they attach to form a heterocyclyl, which is optionally substituted by alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, or -CN, and wherein: each R cc is independently alkyl, alkenyl, alkynyl, halogen, heterocyclyl, heteroaryl, aryl, -CN, or -N0 2 .
• each of groups R 1 and R 2 may be independently substituted by electron- donating and/or electron- withdrawing substituents that alter the acidity of the intervening R 1 R 2 CH proton so that enormous flexibility and control over the rate of drug elimination can be achieved.
• Electron-withdrawing groups are defined as groups having a Hammett sigma value greater than 0 (see, for example, Hansch et al. 1991 Chemical Reviews 91: 165-195).
• electron-donating group is meant a substituent resulting in a decrease in the acidity of the R 1 R 2 CH; electron-donating groups are typically associated with negative Hammett s or Taft s* constants and are well-known in the art of physical organic chemistry.
• Hammett constants refer to aryl/heteroaryl substituents
• Taft constants refer to substituents on non-aromatic moieties.
• suitable electron-donating substituents include but are not limited to lower alkyl, lower alkoxy, lower alkylthio, amino, alkylamino, dialkylamino, and silyl.
• electron-withdrawing group is meant a substituent resulting in an increase in the acidity of the R 1 R 2 CH group; electron-withdrawing groups are typically associated with positive Hammett s or Taft s* constants and are well-known in the art of physical organic chemistry. A description of suitable electron-donating and electron- withdrawing substituents that can be used to modulate R 1 and R 2 can be can be found in U.S. Patent No. 9,649,385.
• At least one of R 1 and R 2 is -CN. In some embodiments, at least one of R 1 and R 2 is -NO2. In some embodiments, at least one of R 1 and R 2 is optionally substituted aryl containing 6-10 carbons. For instance, in some embodiments, at least one of R 1 and R 2 is phenyl, naphthyl, or anthracenyl, each of which is optionally substituted. In some embodiments, at least one of R 1 and R 2 is optionally substituted heteroaryl comprising 3-7 carbons and containing at least one N, O, or S atom.
• At least one of R 1 and R 2 is pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl, each of which is optionally substituted.
• At least one of R 1 and R 2 is optionally substituted alkenyl containing 2-20 carbon atoms. In some embodiments, at least one of R 1 and R 2 is optionally substituted alkynyl containing 2-20 carbon atoms.
• At least one of R 1 and R 2 is - COR 3 , -SOR 3 , or -SO2R 3 , wherein R 3 is H, optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 5 or -NR 5 2, wherein each R 5 is independently H or optionally substituted alkyl containing 1-20 carbon atoms, or both R 5 groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring.
• At least one of R 1 and R 2 is -CN, -SOR 3 or -SO2R 3 . In some embodiments, at least one of R 1 and R 2 is -CN or -SO2R 3 . In some embodiments, at least one of R 1 and R 2 is -CN or -SO2R 3 , wherein R 3 is optionally substituted alkyl, optionally substituted aryl, or -NR 5 2.
• R 1 and R 2 is -CN, - S0 2 N(CH 3 )2, -SO2CH3, -S0 2 Phenyl, -S0 2 (chlorophenyl), -S0 2 (4-methylphenyl), - S0 2 N(CH 2 CH 2 ) 2 0, -S0 2 N(CH 2 CH 2 ) 2 S, -S0 2 CH(CH 3 ) 2 , -S0 2 N(CH 3 )(CH 2 CH 3 ), or - S0 2 N(CH 2 CH 2 0CH 3 ) 2 .
• one of R 1 and R 2 is -CN or -S0 2 R 3 , wherein R 3 is optionally substituted alkyl, optionally substituted aryl, or -NR 5 2 ; and the other is H.
• one of R 1 and R 2 is -CN, -S0 2 N(CH 3 ) 2 , -S0 2 CH 3 , -S0 2 Phenyl, - S 0 2 (chlorophenyl) , -S0 2 (4-methylphenyl), -S0 2 N(CH 2 CH 2 ) 2 0, -S0 2 N(CH 2 CH 2 ) 2 S, - S0 2 CH(CH 3 ) 2 , -S0 2 N(CH 3 )(CH 2 CH 3 ), or -S0 2 N(CH 2 CH 2 0CH 3 ) 2 ; and the other is H.
• each R 4 is independently C1-C3 alkyl. In some embodiments, both R 4 are methyl.
• 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.
• Connecting group Z may be any group capable of selective reaction in the presence of a CNP peptide.
• Typical groups include, without limitation, azide, in which case cognate group Z’ on M is an alkyne or cycloalkyne and the residual connecting group is a triazole; aminoether, in which case cognate group Z’ on M is a carbonyl and the residual connecting group is an oxime; trans-cyclooctene or cyclopropane, in which case cognate group Z’ on M is a 1,2,5,6-tetrazine and the residual functionality is a pyridazine; or a thiol, in which case cognate group Z’ on M is a maleimide or halocarbonyl and the residual connecting group is a thioether.
• Z is azide and Z’ is a cyclooctyne.
• Typical cyclooctynes known in the art include, without limitation, 5-hydroxycyclooctyne (5HCO), l-fluorocyclooct-2yne-l-carboxylate (MFCO), bicyclo[6.1.0]non-4-yne (BCN) (see Dommerholt et al., Top Curr Chem (Z) (2016) 374:16).
• CNP includes all peptides characterized by the ability to bind NPR-B and thereby regulate the growth, proliferation, and differentiation of chondrocytes. This includes, without limitation, sequences listed in PCT Publications 2009/067639, 2010/135541, and 2016/110577; and Morozumi et al. (2019) PLoS ONE 14(2): e0212680.
• CNP refers to a peptide of SEQ ID No: 1-6 and stabilized analogs thereof. Particularly preferred are CNP analogs wherein particular amino acid residues have been replaced so as to improve the stability of the peptide.
• Such stabilized analogs include peptides wherein asparagine residues have been replaced by residues less susceptible towards deamidation, for example glutamine or alanine, and peptides wherein oxidation-sensitive residues have been replaced, for example methionine replaced by norleucine.
• Exemplary embodiments of CNP are provided in SEQ ID Nos: 1-6 below.
• the stabilized CNP is (Gln 6,14 )-CNP38 (SEQ ID No: 4) or (Gin 6, 14 ,Nle 33 )CNP38 (SEQ ID No: 5).
• M is a water-insoluble hydrogel carrier.
• R la and R 2a are independently H, alkyl, CN, NO2, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR 3a , -SOR 3a , or -S02R 3a , wherein
• 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 , wherein each R 5a is independently H or optionally substituted alkyl, or both R 5a groups are taken together with the nitrogen to which they attach to form a heterocyclic ring; wherein R la and R 2a may be taken together with the carbon to which they attach to form a 3-8 membered ring, and wherein one and only one of R la and R 2a may be H or alkyl; each R 4a is independently H or C1-C3 alkyl, or both R 4a are taken together with the carbon to which they attach to form a 3-6 membered ring;
• Z’ is a functional group for mediating coupling to cognate functional group Z of a linker- CNP of Formula (II) as disclosed herein; x and y are each independently 0-6;
• B* and C* are each independently a connecting group
• P 1 and P 2 are independently r-armed polymers of 1-40 kDa average molecular weight, wherein r is an integer from 2 to 8. In preferred embodiments, P 1 and P 2 are r-armed poly(ethylene glycols).
• At least one of R la and R 2a is -CN. In some embodiments, at least one of R la and R 2a is -NO2. In some embodiments, at least one of R la and R 2a is optionally substituted aryl containing 6-10 carbons. For instance, in some embodiments, at least one of R la and R 2a is phenyl, naphthyl, or anthracenyl, each of which is optionally substituted. In some embodiments, at least one of R 1a and R 2a is optionally substituted heteroaryl comprising 3-7 carbons and containing at least one N, O, or S atom.
• At least one of R 1a and R 2a is pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl, each of which is optionally substituted.
• at least one of R 1a and R 2a is optionally substituted alkenyl containing 2-20 carbon atoms.
• at least one of R 1a and R 2a is optionally substituted alkynyl containing 2-20 carbon atoms.
• R 1a and R 2a is -COR 3a , -SOR 3a , or -SO2R 3a , wherein R 3a is H, optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 5a or –NR 5a 2, wherein each R 5a is independently H or optionally substituted alkyl containing 1-20 carbon atoms, or both R 5a groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring.
• At least one of R 1a and R 2a is -CN, -SOR 3a or -SO2R 3a . In some embodiments, at least one of R 1a and R 2a is –CN or -SO2R 3a . In some embodiments, at least one of R 1a and R 2a is –CN or -SO 2 R 3a , wherein R 3a is optionally substituted alkyl, optionally substituted aryl, or –NR 5a 2 .
• At least one of R 1a and R 2a 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 - SO 2 N(CH 2 CH 2 OCH 3 ) 2 .
• one of R 1a and R 2a is –CN or -SO 2 R 3a , wherein R 3a is optionally substituted alkyl, optionally substituted aryl, or –NR 5a 2; and the other is H.
• one of R 1a and R 2a is –CN, -SO2N(CH3)2, -SO2CH3, - SO 2 Phenyl, -SO 2 (chlorophenyl), -SO 2 (4-methylphenyl), -SO 2 N(CH 2 CH 2 ) 2 O, - SO2N(CH2CH2)2S, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2; and the other is H.
• each R 4a is independently C 1 -C 3 alkyl. In some embodiments, both R 4a are methyl.
• 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.
• 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.
• Hydrogels of Formula (III) where in P 1 and P 2 are r-armed poly(ethylene glycols) are prepared as described in, 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:el2213. These hydrogels provide for tuned rates of drug release and subsequent hydrogel dissolution.
• Typical connecting groups of B* and C* include, without limitation, triazole, carboxamide, carbamate, oxime, and thioether.
• the connecting group of B* and/or C* is triazole as described above.
• B* is triazole and C* is carboxamide.
• P 1 and P 2 are synthetic or natural polymers such as poly(ethylene glycols), dextrans, hyaluronic acids, and the like.
• the polymer chains are crosslinked to form an insoluble 3-dimensional matrix ( Figure 6), where each crosslink has an attachment point for a linker-CNP of Formula (II).
• the crosslinks slowly cleave by non-hydrolytic beta- elimination at rates governed primarily by groups R la and R 2a to give ultimately soluble polymer fragments.
• hydrogels allow for attachment of the linker-drugs via connecting group Z*, formed by reaction of cognate groups Z and Z’ on Formulas (II) and (III) as illustrated in Figure 2, thus producing a conjugate of Formula (I) wherein M is an insoluble hydrogel comprising a conjugated linker-peptide at each crosslink and having the more specific Formula (IV), wherein R 1 , R 2 , R 4 , E, n, Z*, y, P 1 , P 2 , B*, q, r, C*, x, R la , R 2a , and R 4a are as disclosed herein.
• Figure 2 further illustrates a method for prepared a conjugate of Formula (IV) comprising contacting a hydrogel of Formula (III) comprising connecting group Z’ with a linker-peptide of formula (II) comprising cognate connecting group Z, under conditions such that Z and Z’ react to form a residual group Z* that connects the linker-peptide to the hydrogel.
• Z and Z’ are azide/cyclooctyne, such conditions are at a temperature between 0 and 100 °C, preferably between 0 and 50 °C, and more preferably between 25 and 50 °C.
• the solvent in which the linker-peptide and hydrogel are suspended or dissolved may be aqueous, organic, or mixed depending on the solubility of the linker-peptide.
• Typical solvents are an aqueous buffer having a pH between 2 and 7, preferably between 2 and 5, optionally mixed with a water-miscible cosolvent such as methanol, ethanol, 2-propanol, tert- butanol, acetonitrile, dimethylformamide, acetonitrile, or tetrahydrofuran.
• the conjugates of formula (I) when M is an insoluble matrix are optionally isolated by washing to remove unreacted linker-peptide and reaction byproducts. Procedures for the reaction of (II) and (III) are analogous to those reported by Henise et al. (2020) Engineering Reports 2020;2:el2213.
• the linker-CNP of formula (II) may be prepared by reaction of a CNP peptide or protected version thereof with a linker reagent of formula (V) wherein X is a leaving group such as chloride, O-succinimidyl, O-nitrophenyl, and the like, and the remaining groups are as disclosed herein for formula (II).
• the linker may be attached to either the N-terminal alpha- amine or a lysine epsilon-amine group of the CNP peptide, using chemistry such as that described in US Patent No. 8,680,315.
• the linker is attached to the N-terminal alpha-amine, this is preferably done during synthesis of the peptide on solid support as illustrated in Figure 3.
• the linker is attached by reaction with an active carbonate such as a succinimidyl carbonate as detailed in the Examples below, and the resulting intermediate is deblocked, cleaved from the resin, and the disulfide is formed to provide the linker-CNP of formula (II).
• the invention is directed to protocols for formulating and administering the conjugates of formula (I).
• the conjugates are prepared as hydrogel microspheres suitable for subcutaneous injection using a narrow-gauge needle. These microspheres may be formulated in any solution suitable for injection and may comprise any excipients required to maintain the stability of the conjugate, for example buffers, antibacterial agents, antioxidant agents, agents for adjustment of density and osmolarity, and agents to promote suspension and prevent clumping of the microspheres.
• a low-pH buffer preferably a buffer at pH 2-7, more preferably pH 3-7, and most preferably pH 4-5.
• Suitable buffers are those known in the art for pharmaceutical applications, and include acetate, citrate, malate, maleate, phosphate, and others effective in these pH ranges.
• Administration can be by any route, including subcutaneous, intramuscular, or intra- articular. It is expected that the conjugates of the invention will be useful for the treatment of diseases and conditions in both humans and animals responsive to CNP, for example achondroplasia, with dosing frequencies of weekly, monthly, or longer.
• the mixture was allowed to warm to +10 °C over 2 h, then quenched with 20 mL of 6 N HC1.
• the mixture was diluted with methyl t-butyl ether (MTBE, 200 mL), washed 2x 100 mL of water and lx 100 mL of brine, dried over MgSO 4 , filtered, and concentrated to yield 14.05 g of crude ketone product.
• MTBE methyl t-butyl ether
• Example 1 Stability Study of (Gln 6,14 )CNP38 and Vosoritide [0075] In 1.5 mL glass vials, 100 ⁇ M [Gln 6,14 ]-CNP38 or 100 ⁇ M vosoritide was treated with 10 mM sodium phosphate containing 0.13 M NaCl, pH 7.4.
• Linker-CNPs of Formula (II) were prepared by solid-phase peptide synthesis. Methods for preparing CNP with suppression of C-terminal cysteine racemization have been described in Fujiwara et al., Chem. Pharm. Bull. (1996) 44(7): 1326-31.
• the linker was attached as the final residue using either 4-azido-3, 3-dimethyl- l-(isopropylsulfonyl)-2-butyl succinimidyl carbonate or 4- azido-3, 3-dimethyl- l-((N,N-dimethylamino)sulfonyl)-2-butyl succinimidyl carbonate.
• the linker-CNP was isolated by reversed-phase HPFC.
• the loaded microspheres were washed 4 times with 12 mF of the reaction solvent (until the OD 280 of the final wash was below detection) followed by 4 washes with 12 mF isotonic acetate buffer (10 mM Na acetate, 143 mM NaCl, 0.05% polysorbate 20 (w/v) pH 5.0.
• the CNP concentration and fraction loaded was determined by solubilizing ⁇ 30 ⁇ L of the packed slurry ( ⁇ 30 mg) in 9 volumes (-270 ⁇ L, 1 ⁇ L:l mg slurry) of 125 mM Borate pH 9.4 for 24 hours at 37 °C in duplicate.
• the PEG content in each slurry was determined using the previously described PEG assay.
• the percent loading of the microsphere slurry was determined as the ratio of peptide concentration to the theoretical PEG reactive end groups based on the PEG assay.
• the A 280 of the supernatant timepoints were plotted and fit to a single exponential using Prism 8.0 software to determine the release rate for each peptide.
• R 1 isopropyl-S0 2
• a release half-life of 6.1 h was observed, corresponding to a release half-life of 610 h at pH 7.4, 37 °C.
• CD-I mice male, 7 week old, -30 g weight
• CD-I mice Male, 7 week old, -30 g weight
• a slurry of the hydrogel microspheres of Example 3 suspended in 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 60kDa (w/v).
• the hydrogel microsphere conjugate wherein R 1 (N,N-dimethylamino)S0 2 was dosed at 0.7 ⁇ mol per mouse.
• Serum samples were collected with the addition of HALT protease inhibitors at the following timepoints: 0, 8, 24, 48, 96, 168, 240, 336, 408, 04, 576, 672, 840, 1008, 1176, 344, 1512, 1680, 1848, and 2016 hr.
• the complete time course was collected using 2 groups of 4 mice each and collecting serum samples at alternating timepoints from each group.
• the hydrogel microsphere conjugate wherein R 1 isopropyl-SO 2 was dosed at 0.22 ⁇ mol per mouse.
• the 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.
• Syringes 0.3 mL U-100 insulin syringe with fixed 29g x 1 ⁇ 2” needle, BD #34702 were filled under sterile conditions with 1 mL of formulated microspheres.
• Blood samples (approximately 1 mF) were collected from the peripheral vein of monkeys administered 4A and 4B at predose, 8, 24, 48, 96, 168, 240, 336, 408, 504, 576, 672, 744, and 840 hours after dose administration.
• blood was also collected at 1008, 1176, 1344, 1680, and 2160 hours after dose administration.
• Blood samples were processed to plasma by collection in tubes containing K 2 EDTA and protease inhibitor (Halt protease inhibitor, ThermoFisher Scientific) and split into two equal volumes for storage at - 80C.
• the animals were examined for general signs of toxicity, including fecal and urine quality, at least once weekly at the injection site, head, neck, limbs, trunk, tail, body orifices and genitalia. While handling, each animal was observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, lacrimation, piloerection, pupil size, and unusual respiratory pattern. The animals’ cages were also inspected for abnormal feces, urine, vomitus or other excretions or secretions.
• CNP-22 fluorescent EIA Kit from Phoenix Pharmaceuticals, Inc (cat # FEK-012-03) read on a Molecular Devices Spectramax i3 plate reader. Plasma samples were diluted in blank cynomolgus monkey plasma to obtain concentrations in the range of a standard curve (2 pM to 2 nM) generated using [Gln 6,14 ]CNP-38 in place of CNP-22. Analysis was repeated by two separate operators on separate days using the two different sample aliquots.
• the conjugate of Example 3, wherein R 1 isopropylsulfonyl, provided continuous exposure > 100 pM of [Gln 6,14 ]CNP-38 for approximately 1 month.
• the conjugate of Example 3, wherein R 1 (N,N-dimethylamino)- sulfonyl, provided continuous exposure > 100 pM of [Gln 6,14 ]CNP-38 for greater than 3 months.
• conjugate 4A was administered at either 1.5 or 2.2 umol/kg; conjugate 4B was administered at 6.1 umol/kg. Doses were calculated based on the body weight just prior to dosing and performed at approximately the same time of day.
• Comparator animals were dosed with either [Gln 6,14 ]CNP-38 or vosoritide at 70 nmol/kg formulated in 30 mM acetic acid pH 4.0 containing 10% (w/v) sucrose and 1% (v/v) benzyl alcohol (Wendt ;Breinholt), and control animals were dosed with 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 the body weight just prior to dosing and performed at approximately the same time each day.
• mice were anesthetized using isoflurane and placed on a table in the supine position, the tail was pulled straight, and a metal ruler was used to gently press down on the mouse to straighten any curvature of the spine so that the mouse was fully elongated.
• the naso-anal (distance from the tip of the nose to the anus) and the 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 conducted and recorded in centimeters.
• Total length (TF) is derived from the addition of the measured tail length and naso-anal length.
• mice were anesthetized and in addition to the nasal-anal and tail length measurements, bone length measurements were made by digital X-ray (spine, right femur, right tibia, humerus and ulna).
• mice were placed in the prone position and the length of the spine (lateral view, distance from the most cranial end of the Cl vertebra to the most caudal end of the S4 vertebra and accounting for curvature manually), the right femur (distance from the most proximal femoral head ossification center to the most distal ossification center in dorso-ventral view), the right tibia (distance from the most proximal ossification center to the most distal ossification center in dorso-ventral view), the right humerus (distance from the most proximal ossification center to the most distal ossification center in dorso-ventral view), and the right ulna (distance from the most proximal ossification center to the most distal ossification center in dorso-ventral view) of each mouse were measured by x
• FIG. 10 The total tail length measurements for the weight- adjusted dosing are shown in Figures 10 and 11, respectively.
• Figure 12 shows photograph of the anesthetized mice treated with A) vehicle control; B) QD [Gln 6,14 ]CNP-38 peptide at 70 nmol; C) biweekly (Q2Wk) conjugate 4A at 2.2 ⁇ mol/kg; D) weekly (QWk) conjugate 4A at 2.2 ⁇ mol/kg; and E) weekly (QWk) conjugate 4A at 1.5 ⁇ mol/kg, after five weeks.
• Biodegradable hydrogel microspheres loaded with (Gln 6,14 )CNP38 prepared according to Example 3 (R 1 isopropylsulfonyl or (N,N-dimethylamino)sulfonyl) were analyzed for release of free (Gln 6,14 )CNP38 and dissolved PEG under accelerated conditions of pH 9.4, 37 °C, according to the published procedures (Henise et al. (2020) Engineering Reports 2020;2:el2213).
• Solubilized peptide was quantitated by OD 280 while solubilized PEG was quantitated using a BaC1 2 /I 2 /Nal colorometric assay using PEG8000 as standard.
• the total solubilized peptide measured by OD280 may be the sum of free peptide released from the microspheres plus solubilized PEGylated peptide.
• the data were thus analyzed by correcting the amount of soluble peptide, assuming a constant first-order rate of cleavage of the drug-linker. Assuming that the solubilized PEG is representative of the bulk hydrogel, then the amount of soluble PEG-peptide contributing to the total OD280 is simply given as
• (PEG-peptide)soi PEG(t) ⁇ exp(-kt)
• PEG(t) the amount of solubilized PEG present at time t
• k the first-order rate constant for linker-peptide cleavage. This amount is subtracted from the total peptide measured by OD280 to give the amount of free peptide released from the hydrogel at time t.
• the experimental data for OD280 and PEG were normalized according to the totals in the assay as determined by the plateau values, and then the normalized data were fit using an iterative process to determine the value of k giving the best fit according to the sum of squared residuals.

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