Use of long-acting growth hormone to treat inflammation-induced diseases

Abstract

The present invention relates to long-acting growth hormone (GH) for use in the treatment of inflammation-induced diseases.

Description

[Technical field]

[0001] The present invention relates to long-acting growth hormone (GH) for use in the treatment of inflammation-induced diseases. [Background technology]

[0002] Nonalcoholic fatty liver disease (NAFLD) is a disorder that affects as many as one in three to five adults and one in ten children in the United States, and refers to the accumulation of excess fat in the liver in people who drink little or no alcohol. Some people with NAFLD can develop a more severe condition called nonalcoholic steatohepatitis (NASH): about 2-5% of adult Americans and up to 20% of obese people can suffer from NASH. In NASH, the accumulation of fat in the liver is associated with inflammation and varying degrees of scarring / fibrosis. NASH is a potentially serious condition with a significant risk of progressing to end-stage liver disease, cirrhosis, and hepatocellular carcinoma. Some patients who develop cirrhosis are at risk of liver failure and may eventually require a liver transplant.

[0003] The liver is rich in macrophages, which, when activated, accelerate the development of NASH through a wide range of inflammatory pathways. The majority of macrophages present in liver tissue are self-renewing resident phagocytic Kupffer cells, which can be divided into M1 (pro-inflammatory) and M2 (immunomodulatory) phenotypes. In a healthy liver, M1 and M2 functions are balanced to control inflammation. In NASH, an imbalance towards M1 is involved in causing excessive inflammation.

[0004] Historically, several pharmacological interventions have been attempted in NAFLD / NASH, but with limited overall benefit. Antioxidants may prevent lipid peroxidation, and cytoprotectants may stabilize phospholipid membranes, but previously attempted drugs such as ursodeoxycholic acid, vitamin E (α-tocopherol) and vitamin C, and pentoxifylline have shown no or only modest benefit. Most weight loss studies in NAFLD / NASH have been short-term, pilot studies of limited success, reporting no or only modest improvement in necroinflammation or fibrosis. A randomized, double-blind, placebo-controlled, 6-month trial of pioglitazone, a thiazolidinedione peroxisome proliferator-activated receptor (PPAR)-γ agonist and insulin sensitizer, versus weight loss alone demonstrated no improvement with weight loss alone, but treatment with pioglitazone improved glycemic control, insulin sensitivity, indices of systemic inflammation (including high-sensitivity C-reactive protein, tumor necrosis factor-α, and transforming growth factor-β), and liver histology in patients with NASH and impaired glucose tolerance or type 2 diabetes. Unfortunately, pioglitazone has also been associated with a marked increase in the risk of weight gain, edema, congestive heart failure, and osteoporotic fractures in both women and men. At the time of writing, phase III trials for NASH are ongoing for the thyroid hormone receptor-β (THR-β) agonist resmetirom, the CC chemokine receptor type 2 / 5 (CCR2 / CCR5) inhibitor cenicriviroc, the stearoyl-CoA desaturase-1 (SCD1) modulator aramchol, the galectin-3 inhibitor belapectin, and the selective sodium glucose cotransporter-2 (SGLT-2) inhibitor dapagliflozin.Obeticholic acid, a farnesoid X receptor (FXR) agonist, completed a Phase III trial for NASH by achieving one of the two FDA-proposed primary endpoints: "improvement of liver fibrosis by at least one stage using the NASH Clinical Research Network (CRN) fibrosis score and no worsening of NASH" or "resolution of NASH and no worsening of liver fibrosis using the NASH CRN fibrosis score." However, the results were not sufficient to obtain regulatory approval, indicating that the predicted benefit based on surrogate histopathology endpoints remains uncertain and does not sufficiently outweigh the potential risks for treating patients with liver fibrosis due to NASH. There were three Phase III trials that failed to achieve one of the two required primary endpoints for NASH. Selonsertib, an apoptosis signal-regulating kinase 1 ASK1 inhibitor, did not meet the primary endpoint of improvement of fibrosis by at least one stage without worsening of NASH in the STELLAR3 and STELLAR4 trials. Elafibranor, a PPARα / δ agonist, also failed to meet its primary endpoint of resolving NASH without worsening fibrosis.

[0005] There are other drugs currently in early clinical development that show potential for treating NAFLD / NASH. These include, among others, the fibroblast growth factor (FGF)-21 agonists Efruxifermin and Pegbelfermin, the FGF-19 agonist Aldafermin, the fibroblast growth factor receptor 1-beta Klotho (FGFR1-KLB) antibodies BFKB8488A and NGM313, the glucagon-like peptide 1 (GLP-1) receptor agonist Semaglutide, the dual receptor agonists with GLP-1 and glucagon activity Cotadutide and Efinopegdutide, the dual receptor agonists with GLP-1 and glucagon activity Tirzepatide, the dual receptor agonist with gastric inhibitory polypeptide (GIP) and GLP-1, and the PPAR alpha / delta / gamma agonist Lanifibranor. Summary of the Invention [Problem to be solved by the invention]

[0006] In summary, there is a need for more effective treatment of inflammation-induced diseases, particularly NAFLD / NASH. It is an object of the present invention to at least partially overcome the limitations of current treatment options. [Means for solving the problem]

[0007] In a first aspect, the present invention relates to long-acting growth hormone (GH) for use in treating inflammation-induced disease.In certain embodiments, inflammation-induced disease is inflammation-induced liver disease.In certain embodiments, inflammation-induced disease is NAFLD.In certain embodiments, inflammation-induced disease is NASH.

[0008] Surprisingly, it has been found that a stable level of growth hormone, such as that obtained from administering long-acting growth hormone to a patient, causes a rebalancing of macrophage phenotype between M1 and M2. In certain embodiments, such a rebalancing of macrophage phenotype is achieved through a reduction in M1. In certain embodiments, such a rebalancing of macrophage phenotype is achieved through an induction of M2.

[0009] The use of long-acting growth hormone may reduce dosing frequency, thereby increasing patient compliance and resulting in improved treatment outcomes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] In the present invention, the terms are used in the following sense:

[0011] As used herein, the term "growth hormone" or "GH" refers to all growth hormone protein sequences, preferably from mammalian species, more preferably from human and mammalian species, more preferably from human and mouse species, and in certain embodiments also includes variants, analogs, orthologs, homologs, and derivatives and fragments thereof. Growth hormone is characterized by promoting growth during anagen and maintaining normal body composition, anabolism, and lipid metabolism. In certain embodiments, the term "human growth hormone" or "hGH" refers to the hGH polypeptide of SEQ ID NO: 1, including its variants, homologs, and derivatives that exhibit essentially the same biological activity, i.e. promoting growth during anagen and maintaining normal body composition, anabolism, and lipid metabolism. In certain embodiments, the term "hGH" refers to the sequence of SEQ ID NO: 1.

[0012] SEQ ID NO:1 has the following sequence:

[0013] TIFF2024514095000001.tif9161TIFF2024514095000002.tif18170

[0014] As used herein, the term "GH variant" refers to a GH protein derived from the same species that is different from a reference GH protein, such as hGH of SEQ ID NO:1. In certain embodiments, such GH variants are at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference GH, such as hGH of SEQ ID NO:1. By a protein having an amino acid sequence that is at least, for example, 95% "identical" to a query amino acid sequence, it is intended that the amino acid sequence of the subject protein is identical to the query sequence, except that the subject protein sequence may contain up to 5 amino acid changes per each 100 amino acids of the query amino acid sequence. These changes in the reference sequence may occur at the amino (N-terminal) or carboxy-terminal (C-terminal) positions of the reference amino acid sequence, or at any position between these terminal positions, or any combination thereof. These changes in the reference sequence may be interspersed among the residues in the reference sequence, or may be in one or more contiguous groups within the reference sequence. Such GH variants may be naturally occurring variants, such as naturally occurring allelic variants encoded by one of several alternative forms of GH occupying a given locus on a chromosome or organism, or isoforms encoded by naturally occurring splice variants derived from a single primary transcript. Alternatively, the GH variant may be a variant that is not known to occur naturally and may be generated by mutagenesis techniques known in the art.

[0015] As used herein, the term "GH analog" refers to GH from different unrelated organisms that perform the same function in each organism but are not derived from a common ancestral structure shared by the ancestors of the organisms.Instead, similar GHs have arisen separately and have subsequently evolved to perform the same or similar functions.In other words, similar GH proteins are proteins that have completely different amino acid sequences but perform the same biological activity, namely, promoting growth during the growth period and maintaining normal body composition, anabolism, and lipid metabolism.

[0016] As used herein, the term "GH orthologs" refers to GHs in two different species whose sequences are related to each other through a common homologous GH in an ancestral species, but which have evolved divergently from each other.

[0017] As used herein, the term "GH homolog" refers to GH from different organisms that perform the same function in each organism and originate from an ancestral structure that the ancestors of the organisms shared. In other words, homologous GH proteins are proteins with highly similar amino acid sequences that perform the same biological activity, i.e., promote growth during puberty and maintain normal body composition, anabolism, and lipid metabolism. In certain embodiments, such GH homologs can be defined as proteins that show at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to a reference GH sequence, e.g., hGH of SEQ ID NO:1.

[0018] Thus, a GH according to the present invention may be, for example, (i) a GH in which at least one of the amino acid residues has been replaced with a conservative or non-conservative amino acid residue, in certain embodiments a conservative amino acid residue, where such a substituted amino acid residue may or may not be one encoded by the genetic code, and / or (ii) a GH in which at least one of the amino acid residues includes a substituent group, and / or (iii) a GH in which the GH has been fused to another compound, for example a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol), and / or (iv) a GH in which additional amino acids have been fused to the hGH polypeptide, such as an IgG Fc fusion region peptide or a leader or secretory sequence, or a sequence used for purification of the above forms of protein or preprotein sequences.

[0019] The GH protein may be a monomer or a multimer. The multimer may be a dimer, a trimer, a tetramer, or a multimer comprising at least 5 monomeric polypeptide units. The multimer may also be a homodimer or a heterodimer. The multimers of the present invention may be the result of hydrophobic, hydrophilic, ionic and / or covalent associations and / or may be indirectly linked, for example by liposome formation. In certain embodiments, the GH is a monomer, in particular an hGH monomer, for example an hGH monomer of SEQ ID NO: 1.

[0020] As used herein, the term "GH fragment" refers to any peptide or protein that comprises a contiguous span of a portion of the amino acid sequence of a GH protein, e.g., hGH of SEQ ID NO: 1. More specifically, a GH fragment comprises at least 6, preferably at least 8 or 10, more preferably at least 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 125, 150, 175, 191 contiguous amino acids of GH, e.g., hGH of SEQ ID NO: 1.

[0021] As used herein, the term "long-acting growth hormone" refers to a compound that contains GH in crystallized form or that contains GH embedded, fused, or covalently conjugated to at least one other chemical compound or moiety, such as a polymer, a fatty acid, or a fatty acid variant moiety, and has an increased clearance half-life in the patient's body compared to unmodified GH, for example, a clearance half-life that is at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, or at least 200 times higher than the clearance half-life of the corresponding unmodified GH. In certain embodiments, the GH is hGH, such as hGH of SEQ ID NO: 1. As used herein, the term "clearance half-life" refers to the time it takes for half of the total molecule administered to a patient to be cleared from the body.

[0022] As used herein, the terms "reversible", "reversibly", "degradable" or "degradably" with respect to a bond of a first moiety to a second moiety means that the bond linking the first and second moieties is cleavable under physiological conditions of an aqueous buffer at pH 7.4 at 37° C. with a half-life ranging from 1 hour to 3 months, e.g., 12 hours to 2 months, 1 day to 1 month. Cleavage may be enzymatic or non-enzymatic, and in certain embodiments is non-enzymatic. Thus, the terms "stable" or "permanent" with respect to a bond of a first moiety to a second moiety means that the bond linking the first and second moieties is cleavable under physiological conditions with a half-life of greater than 3 months.

[0023] As used herein, the term "reagent" refers to a chemical compound that contains at least one functional group for reaction with a functional group of another chemical compound or drug. Drugs that contain a functional group (e.g., a primary or secondary amine, or a hydroxyl functional group) are also understood to be reagents.

[0024] As used herein, the term "moiety" refers to a part of a molecule that is missing one or more atoms compared to the corresponding reagent. For example, when a reagent of the formula "HXH" reacts with another reagent to become part of a reaction product, the corresponding moiety of the reaction product has the structure "HX-" or "-X-", where each "-" indicates a bond to another moiety. Thus, the drug moiety is released as a drug from the reversible bond.

[0025] When a sequence of atoms or a chemical structure is provided that is connected to or interrupts two moieties, it is understood that the sequence or chemical structure can be connected to the two moieties in either orientation, unless expressly stated otherwise. For example, the moiety "-C(O)N(R 1 )-" is "-C(O)N(R 1 )-" or "-N(R 1 )C(O)-" or can be an interruption between the two moieties.

[0026] [ka] teeth,

[0027] [ka] Or

[0028] [ka] It can be joined into two parts either as a part or it can be interrupted into a part.

[0029] As used herein, the term "substituted" means that one or more --H atoms of a molecule or moiety have been replaced with a different atom or group of atoms, referred to as "substituents."

[0030] As used herein, the term "substituent" includes, in certain embodiments, halogen, -CN, -COOR, x1 , -OR x1 , -C(O)R x1 , -C(O)N(R x1 R x1a ), -S(O)2N(R x1 R x1a ), -S(O)N(R x1 R x1a ), -S(O)2R x1 , -S(O)R x1 , -N(R x1 )S(O)2N(R x1a R x1b ), -SR x1 , -N(R x1 R x1a ), -NO2, -OC(O)R x1 , -N(R x1 )C(O)R x1a , -N(R x1 )S(O)2R x1a , -N(R x1 )S(O)R x1a , -N(R x1 )C(O)OR x1a , -N(Rx1 )C(O)N(R x1a R x1b ), -OC(O)N(R x1 R x1a ), -T 0 , C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 alkynyl, where -T 0 , C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 Alkynyl may be one or more -R x2 Optionally replaced by C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 Alkynyl is -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R x3 )-, -S(O)2N(R x3 )-, -S(O)N(R x3 )-, -S(O)2-, -S(O)-, -N(R x3 )S(O)2N(R x3a )-, -S-, -N(R x3 )-, -OC(OR x3 )(R x3a )-, -N(R x3 )C(O)N(R x3a )- and -OC(O)N(R x3 )-, -R x1 , -R x1a , -R x1b -H, -T 0 , C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 alkynyl, wherein -T 0 , C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 Alkynyl may be one or more -R x2 Optionally replaced by C 1-50 Alkyl, C2-50 Alkenyl, and C 2-50 Alkynyl is -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R x3 )-, -S(O)2N(R x3 )-, -S(O)N(R x3 )-;-S(O)2-, -S(O)-, -N(R x3 )S(O)2N(R x3a )-, -S-, -N(R x3 )-, -OC(OR x3 )(R x3a )-, -N(R x3 )C(O)N(R x3a )- and -OC(O)N(R x3 )-, Each T 0 is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 each T is independently selected from the group consisting of cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; 0 is one or more -R x2 and optionally independently replaced by Each -R x2 is halogen, -CN, oxo(=O), -COOR x4 , -OR x4 , -C(O)R x4 , -C(O)N(R x4 R x4a ), -S(O)2N(R x4 R x4a ), -S(O)N(R x4 R x4a ), -S(O)2R x4 , -S(O)R x4 , -N(R x4 )S(O)2N(R x4a R x4b ), -SR x4 , -N(R x4 R x4a ), -NO2, -OC(O)R x4 , -N(R x4 )C(O)R x4a , -N(R x4)S(O)2R x4a , -N(R x4 )S(O)R x4a , -N(R x4 )C(O)OR x4a , -N(R x4 )C(O)N(R x4a R x4b ), -OC(O)N(R x4 R x4a ), and C 1-6 alkyl, wherein C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each -R x3 , -R x3a , -R x4 , -R x4a , -R x4b -H and C 1-6 alkyl, wherein C 1-6 The alkyl is optionally substituted with one or more halogens, which may be the same or different.

[0031] In certain embodiments, up to six -H atoms of an optionally substituted molecule are independently replaced by substituents, e.g., five -H atoms are independently replaced by substituents, four -H atoms are independently replaced by substituents, three -H atoms are independently replaced by substituents, two -H atoms are independently replaced by substituents, or one -H atom is replaced by a substituent.

[0032] As used herein, the term "fatty acid" refers to a saturated or unsaturated monocarboxylic acid with an aliphatic tail, which may contain from 4 to 28 carbon atoms. Fatty acids may be saturated or unsaturated, straight chain or branched. The term "fatty acid variant" refers to modified fatty acids, which may be substituted and in which certain carbon atoms may be replaced by other atoms or groups of atoms.

[0033] As used herein, the term "peptide" refers to a chain of at least two and up to 50 amino acid monomeric moieties linked by peptide (amide) bonds. The term "peptide" also includes peptidomimetics, such as D-peptides, peptoids, or beta-peptides, and encompasses such peptidomimetic chains having up to 50 monomeric moieties. Cyclic peptides, such as lasso peptides, are also included.

[0034] As used herein, the term "protein" refers to a chain of more than 50 amino acid monomer moieties (which may also be referred to as "amino acid residues") linked by peptide bonds, and in certain embodiments, up to 12,000 amino acid monomers, e.g., up to 10,000 amino acid monomer moieties, up to 8,000 amino acid monomer moieties, up to 5,000 amino acid monomer moieties, or up to 2,000 amino acid monomer moieties, are linked by peptide bonds.

[0035] As used herein, the term "about" in conjunction with a numerical value is used to indicate a range of the numerical value plus / minus 25% or less of the numerical value, in certain embodiments plus / minus 20% or less of the numerical value, and in certain embodiments plus / minus 10% or less of the numerical value, inclusive. For example, the phrase "about 200" is used to mean a range of 200 + / - 25% (inclusive), i.e., a range of 150 to 250 (inclusive), in certain embodiments a range of 200 + / - 20% (inclusive), i.e., a range of 160 to 240 (inclusive), and in certain embodiments a range of 200 + / - 10% (inclusive), i.e., a range of 180 to 220 (inclusive). It is understood that a percentage indicated as "about 50%" does not mean "50% + / - 25%", i.e., a range of 25-75%, inclusive, but rather that "about 50%" means 37.5-62.5%, i.e., a range of plus / minus 25% of the numerical value 50, inclusive.

[0036] As used herein, the term "polymer" refers to a molecule that includes repeating structural units, i.e., monomers, linked by chemical bonds in a linear, cyclic, branched, cross-linked, or dendrimeric fashion, or a combination thereof, and may be of synthetic or biological origin, or a combination of both. It is understood that a polymer may also include one or more other chemical groups and / or moieties, such as, for example, one or more functional groups. Similarly, it is understood that a peptide or protein is also a polymer, although the side chains of the individual amino acid residues may differ. In certain embodiments, a soluble polymer has a molecular weight of at least 0.5 kDa, such as a molecular weight of at least 1 kDa, a molecular weight of at least 2 kDa, a molecular weight of at least 3 kDa, or a molecular weight of at least 5 kDa. When a polymer is soluble, in certain embodiments, the polymer has a molecular weight of at most 1000 kDa, such as at most 750 kDa, such as at most 500 kDa, such as at most 300 kDa, such as at most 200 kDa, such as at most 100 kDa. It is understood that for insoluble polymers such as hydrogels, a meaningful molecular weight range cannot be provided.

[0037] As used herein, the term "polymeric" refers to an agent or moiety that includes one or more polymers or polymeric moieties / moieties. A polymeric agent or moiety may also optionally include one or more other moieties / moieties, which in certain embodiments are selected from the group consisting of: C 1-50 Alkyl, C 2-50 Alkenyl, C 2-50 Alkynyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl, and A bond selected from the group comprising:

[0038] [ka] [In the formula, The dashed line indicates the bond to the remainder of the moiety or reagent; -R and -R a are independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl.

[0039] Those skilled in the art will appreciate that the polymerization products resulting from a polymerization reaction do not all have the same molecular weight, but rather exhibit a molecular weight distribution. Thus, as used herein, molecular weight ranges, molecular weights, ranges of the number of monomers in a polymer, and the number of monomers in a polymer refer to number average molecular weights and monomer number averages, i.e., the arithmetic average of the molecular weight of a polymer or polymer portion, and the arithmetic average of the number of monomers of a polymer or polymer portion.

[0040] Thus, in a polymer portion that contains "x" monomer units, any integer assigned to "x" corresponds to the arithmetic average number of monomers. Any range of integers assigned to "x" provides a range of integers within which the arithmetic average number of monomers lies. An integer "x" designated as "about x" means that the arithmetic average number of monomers is within the integer range of x + / - 25%, preferably x + / - 20%, and more preferably x + / - 10%.

[0041] As used herein, the term "number average molecular weight" means the ordinary arithmetic average of the molecular weights of the individual polymers.

[0042] As used herein, the term "PEG-based" in reference to a moiety or reagent means that said moiety or reagent comprises PEG. In certain embodiments, the PEG-based moiety or reagent comprises at least 10% (w / w) PEG, such as at least 20% (w / w) PEG, such as at least 30% (w / w) PEG, such as at least 40% (w / w) PEG, such as at least 50% (w / w) PEG, such as at least 60% (w / w) PEG, such as at least 70% (w / w) PEG, such as at least 80% (w / w) PEG, such as at least 90% (w / w) PEG, such as at least 95% (w / w) PEG. The remaining weight percentage of the PEG-based moiety or reagent are other moieties, which in certain embodiments are selected from the following moieties and linkages: C 1-50 Alkyl, C 2-50 Alkenyl, C 2-50 Alkynyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl, and tetralinyl, and A bond selected from the group comprising:

[0043] [ka] [In the formula, The dashed line indicates the bond to the remainder of the moiety or reagent; -R and -R a are independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl.

[0044] The term "hyaluronic acid-based" is used accordingly.

[0045] As used herein, the term "PEG-based comprising at least X% PEG" in reference to a moiety or reagent means that said moiety or reagent comprises at least X% (w / w) ethylene glycol units (-CHCHO-), which may be arranged in alternating blocks or may be randomly distributed within said moiety or reagent, and in certain embodiments, all of the ethylene glycol units of said moiety or reagent are present in one block, with the remaining weight percentage of the PEG-based moiety or reagent being other moieties, which in certain embodiments are selected from the following moieties and linkages: C 1-50 Alkyl, C 2-50 Alkenyl, C 2-50 Alkynyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl, and tetralinyl, and A bond selected from the group comprising:

[0046] [ka] [In the formula, The dashed line indicates the bond to the remainder of the moiety or reagent; -R and -R a are independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl.

[0047] The term "a hyaluronic acid system comprising at least X% hyaluronic acid" is used accordingly.

[0048] As used herein, the term "hydrogel" refers to a hydrophilic or amphiphilic polymer network composed of homopolymers or copolymers, which is insoluble due to the presence of hydrophobic interactions, hydrogen bonds, ionic interactions, covalent chemical crosslinks, or any combination thereof. In certain embodiments, the hydrogel is insoluble due to the presence of covalent chemical crosslinks. In general, the crosslinks provide the network structure and physical integrity.

[0049] The term "interrupted" means that a moiety is inserted between two carbon atoms, or, if the insertion is at one of the ends of the moiety, between a carbon or heteroatom and a hydrogen atom.

[0050] As used herein, the term "C 1-4 "Alkyl," alone or in combination, means a straight or branched alkyl moiety having 1 to 4 carbon atoms. When present at the terminal end of a molecule, it is a straight or branched C 1-4 Examples of alkyl are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. 1-4 When bonded by an alkyl, such C 1-4 Examples of alkyl groups are -CH-, -CH-CH-, -CH(CH)-, -CH-CH-CH-, -CH(CH)-, and -C(CH)-. 1-4 Each hydrogen of an alkyl carbon may be optionally replaced by a substituent as defined above. 1-4 The alkyl may be interrupted by one or more moieties as defined below.

[0051] As used herein, the term "C 1-6 "Alkyl," alone or in combination, means a straight-chain or branched alkyl moiety having 1 to 6 carbon atoms. When present at the terminal end of a molecule, straight-chain and branched C 1-6Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl. 1-6 When the alkyl group is bonded to the C 1-6 Examples of alkyl groups are -CH-, -CH-CH-, -CH(CH)-, -CH-CH-CH-, -CH(CH)-, and -C(CH)-. 1-6 Each hydrogen atom of the carbon may be optionally replaced by a substituent as defined above. 1-6 The alkyl may be interrupted by one or more moieties as defined below.

[0052] Therefore, "C 1-10 Alkyl, C 1-20 Alkyl" or "C 1-50 "Alkyl" means an alkyl chain having 1 to 10, 1 to 20, or 1 to 50 carbon atoms, respectively; C 1-10 , C 1-20 Or C 1-50 Each hydrogen atom of the carbon may be optionally replaced by a substituent as defined above. 1-10 Or C 1-50 The alkyl may be interrupted by one or more moieties as defined below.

[0053] As used herein, the term "C 2-6 "Alkenyl", alone or in combination, means a straight or branched chain hydrocarbon moiety containing at least one carbon-carbon double bond, having 2 to 6 carbon atoms. When present at the end of the molecule, examples are -CH=CH2, -CH=CH-CH3, -CH2-CH=CH2, -CH=CHCH2-CH3 and -CH=CH-CH=CH2. When two portions of the molecule are C 2-6 When bonded by an alkenyl group, such C 2-6An example of an alkenyl is -CH=CH-. 2-6 Each hydrogen atom of the alkenyl moiety may be optionally replaced by a substituent as defined above. 2-6 The alkenyl may be interrupted by one or more moieties as defined below.

[0054] Hence the term "C 2-10 alkenyl", "C 2-20 alkenyl" or "C 2-50 "Alkenyl," alone or in combination, means a straight-chain or branched hydrocarbon moiety containing at least one carbon-carbon double bond, having 2 to 10, 2 to 20, or 2 to 50 carbon atoms. 2-10 Alkenyl, C 2-20 Alkenyl or C 2-50 Each hydrogen atom of the alkenyl group may be optionally replaced by a substituent as defined above. 2-10 Alkenyl, C 2-20 Alkenyl or C 2-50 The alkenyl may be interrupted by one or more moieties as defined below.

[0055] As used herein, the term "C 2-6 "Alkynyl", alone or in combination, means a straight or branched hydrocarbon moiety containing at least one carbon-carbon triple bond having 2 to 6 carbon atoms. When present at the end of the molecule, examples are -C≡CH, -CH2-C≡CH, -CH2-CH2-C≡CH and CH2-C≡C-CH3. When two parts of the molecule are linked by an alkynyl group, examples are -C≡C-. 2-6 Each hydrogen atom of the alkynyl group may be optionally replaced by a substituent as defined above. Optionally, one or more double bonds may be present. Optionally, C 2-6 The alkynyl may be interrupted by one or more moieties as defined below.

[0056] Thus, as used herein, the term "C 2-10 alkynyl", "C2-20 alkynyl" and "C 2-50 The term "alkynyl," alone or in combination, means a straight-chain or branched hydrocarbon moiety containing at least one carbon-carbon triple bond, having 2 to 10, 2 to 20, or 2 to 50 carbon atoms, respectively. 2-10 Alkynyl, C 2-20 Alkynyl or C 2-50 Each hydrogen atom of the alkynyl group may be optionally replaced by a substituent as defined above. Optionally, one or more double bonds may be present. Optionally, C 2-10 Alkynyl, C 2-20 Alkynyl or C 2-50 The alkynyl may be interrupted by one or more moieties as defined below.

[0057] As mentioned above, C 1-4 Alkyl, C 1-6 Alkyl, C 1-10 Alkyl, C 1-20 Alkyl, C 1-50 Alkyl, C 2-6 Alkenyl, C 2-10 Alkenyl, C 2-20 Alkenyl, C 2-50 Alkenyl, C 2-6 Alkynyl, C 2-10 Alkynyl, C 2-20 Alkenyl or C 2-50 The alkynyl may be optionally interrupted by one or more moieties, said one or more moieties preferably being

[0058] [ka] [In the formula, The dashed line indicates the bond to the remainder of the moiety or reagent; -R and -R a are independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl and hexyl. is selected from the group consisting of:

[0059] As used herein, the term "C3-10 "Cycloalkyl" means a cyclic alkyl chain having 3 to 10 carbon atoms, which may be saturated or unsaturated, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. 3-10 Each hydrogen atom of a cycloalkyl carbon may be replaced by a substituent as defined above. 3-10 "Cycloalkyl" also includes bridged bicycles such as norbornane or norbornene.

[0060] The term "8-30 membered carbopolycyclyl" or "8-30 membered carbopolycycle" refers to a cyclic moiety having 8-30 ring atoms, with two or more rings, two adjacent rings sharing at least one ring atom, and which may contain up to a maximum number of double bonds (fully saturated, partially saturated, or unsaturated, aromatic or non-aromatic rings). In certain embodiments, 8-30 membered carbopolycyclyl refers to a cyclic moiety having 2, 3, 4, or 5 rings, more preferably 2, 3, or 4 rings.

[0061] As used herein, the term "3- to 10-membered heterocyclyl" or "3- to 10-membered heterocycle" means a ring (fully saturated, partially saturated, or unsaturated, aromatic or non-aromatic ring) having 3, 4, 5, 6, 7, 8, 9, or 10 ring atoms and which may contain up to a maximum number of double bonds, in which at least one ring atom, and up to a maximum of four ring atoms, are replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen, and nitrogen (including =N(O)-), and the ring is attached to the remainder of the molecule by a carbon or nitrogen atom. Examples of 3- to 10-membered heterocycles include, but are not limited to, aziridine, oxirane, thiirane, azirine, oxirene, thiylene, azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazolidine, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepane, azepine, and homopiperazine. Each hydrogen atom of the 3- to 10-membered heterocyclyl or 3- to 10-membered heterocyclic group may be replaced by a substituent as defined below.

[0062] As used herein, the term "8- to 11-membered heterobicyclyl" or "8- to 11-membered heterobicycle" means a bicyclic heterocyclic moiety (fully saturated, partially saturated, or unsaturated, aromatic or non-aromatic ring) having 8 to 11 ring atoms and which may contain up to a maximum number of double bonds, in which at least one ring atom is shared by both rings, and at least one ring atom, up to a maximum of six ring atoms, is replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen, and nitrogen (including =N(O)-), and the ring is bonded to the remainder of the molecule by a carbon or nitrogen atom. Examples of 8-11 membered heterobicycles are indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine and pteridine. The term 8-11 membered heterobicycle also includes two ring spiro structures such as 1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocycles such as 8-aza-bicyclo[3.2.1]octane. Each hydrogen atom of the 8-11 membered heterobicyclyl or 8-11 membered heterobicycle carbon may be replaced by a substituent as defined below.

[0063] Similarly, the term "8- to 30-membered heteropolycyclyl" or "8- to 30-membered heteropolycycle" refers to a heterocyclic moiety (fully saturated, partially saturated or unsaturated, aromatic or non-aromatic rings) having 8 to 30 ring atoms and which may contain up to a maximum number of double bonds, with more than two rings, preferably 3, 4 or 5 rings, in which two adjacent rings share at least one ring atom, and at least one ring atom, up to a maximum of 10 ring atoms, is replaced by a heteroatom selected from the group of sulfur (including -S(O)-, -S(O)2-), oxygen, and nitrogen (including =N(O)-), and said rings are bonded to the remainder of the molecule by a carbon or nitrogen atom.

[0064] structure:

[0065] [ka] Regarding the part, "Pair R x / R y together with the atoms to which they are attached, C 3-10 The phrase "form a cycloalkyl or 3- to 10-membered heterocyclyl" is intended to mean x and R y is understood to be meant to form the following structure:

[0066] [ka] [Wherein, R is C 3-10 cycloalkyl or 3- to 10-membered heterocyclyl.

[0067] structure:

[0068] [ka] Regarding the part, "Pair R x / R y together with the atoms to which they are attached form a ring A" is intended to mean x and Ry is also understood to be meant to form the structure:

[0069] [ka]

[0070] As used herein, "halogen" means fluoro, chloro, bromo or iodo. It is generally preferred that halogen is fluoro or chloro.

[0071] As used herein, the term "functional group" refers to a group of atoms that can react with other groups of atoms. Exemplary functional groups are, for example, carboxylic acid (-(C=O)OH), primary or secondary amine (-NH2, -NH-), maleimide, thiol (-SH), sulfonic acid (-(O=S=O)OH), carbonate, carbamate (-O(C=O)N<), hydroxyl (-OH), aldehyde (-(C=O)H), ketone (-(C=O)-), hydrazine (>NN<), isocyanate, isothiocyanate, phosphate (-O(P=O)OHOH), phosphonate (-O(P=O)OHH), haloacetyl, alkyl halide, acryloyl, aryl fluoride, hydroxylamine, disulfide, sulfonamide, sulfate, vinyl sulfone, vinyl ketone, diazoalkane, oxirane, and aziridine.

[0072] When the long-acting GH contains one or more acidic or basic groups, the present invention also includes its corresponding pharma- ceutically or toxicologically acceptable salts, in particular its pharma- ceutically available salts.Thus, the long-acting GH containing an acidic group may be used according to the present invention, for example, as an alkali metal salt, an alkaline earth metal salt, or an ammonium salt.More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts, or salts with ammonia or organic amines, such as ethylamine, ethanolamine, triethanolamine, or amino acids.The long-acting GH containing one or more basic groups, i.e. groups that can be protonated, may be present and used according to the present invention in the form of its addition salts with inorganic or organic acids. Examples of suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to those skilled in the art. Those skilled in the art are aware of further methods of converting basic groups to cations, such as alkylation of amine groups, which results in a positively charged ammonium group and a suitable counterion of the salt. When the long-acting GH contains both acidic and basic groups, the present invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions). The respective salts can be obtained by the usual methods known to those skilled in the art, for example by contacting these compounds in a solvent or dispersant with an organic or inorganic acid or base, or by anion or cation exchange with other salts. The invention also includes all salts of long-acting GH which, due to their low physiological compatibility, are not directly suitable for use in medicines, but which can be used, for example, as intermediates in chemical reactions or for preparing pharmaceutically acceptable salts.

[0073] The term "pharmaceutical acceptable" means a substance that does not cause harm when administered to a patient, and preferably means approved for use in animals, e.g. for use in humans, by a regulatory authority, e.g. the EMA (Europe) and / or the FDA (US) and / or a regulatory authority of any other country.

[0074] As used herein, the term "excipient" refers to a diluent, adjuvant, or vehicle with which a therapeutic agent, such as a drug or prodrug, is administered. Such pharmaceutical excipients can be sterile liquids, such as water and oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is the preferred excipient when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are the preferred excipients when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably used as liquid excipients for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. If desired, the pharmaceutical compositions may also contain minor amounts of wetting or emulsifying agents, pH buffering agents, and the like, such as acetates, succinates, Tris, carbonates, phosphates, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), or surfactants, such as Tween, poloxamers, poloxamines, CHAPS, Igepal, or amino acids, such as glycine, lysine, or histidine. These pharmaceutical compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The pharmaceutical compositions may be formulated as suppositories with conventional binders and excipients, such as triglycerides. Oral formulations may contain standard excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Such compositions contain a therapeutically effective amount of the drug or biologically active moiety, together with a suitable amount of excipient so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0075] In general, the terms "comprise" or "comprising" also encompass "consist of" or "consisting of."

[0076] In certain embodiments, long-acting growth hormone inhibits the recruitment of inflammatory monocytes to the site of inflammation.When the inflammation-induced disease is NAFLD, particularly NASH, the site of such inflammation is the liver.In certain embodiments, when the inflammation-induced disease is NAFLD, particularly NASH, administration of long-acting growth hormone leads to an increase in HLA-G in the liver.In certain embodiments, administration of long-acting growth hormone leads to an increase in IGF-1 levels.When animal studies are performed, it is understood that the corresponding gene from such animal species is used, for example, the mouse ortholog of HLA-G is H2 / B1.

[0077] In certain embodiments, administration of long-acting growth hormone leads to the change of one or more markers of liver inflammation selected from the group consisting of cytokines, chemokines and other transcriptional and histological markers.In certain embodiments, long-acting growth hormone for use in treating inflammation-induced diseases, such as NAFLD, particularly NASH, leads to the improvement of transcriptional or histological markers of fibrosis.

[0078] In certain embodiments, administration of long-acting growth hormone results in a reduction in steatosis. In certain embodiments, when the inflammation-induced disease is an inflammation-induced liver disease, long-acting growth hormone results in liver regeneration.

[0079] In certain embodiments, the long-acting growth hormone is administered to the patient once a week. In certain embodiments, the long-acting growth hormone is administered to the patient once every two weeks. In certain embodiments, the long-acting growth hormone is administered to the patient once every four weeks. In certain embodiments, the long-acting growth hormone is administered to the patient once a month.

[0080] In another aspect, the present invention provides a long-acting GH for use in the treatment of an inflammation-induced disease, the treatment comprising: (a) administering to a patient having an inflammation-induced disease at least an initial dose of long-acting GH; (b) measuring insulin-like growth factor-1 (IGF-1) levels, and (c) decreasing the dose of long-acting GH by at least 5% if the IGF-1 level is above a standard deviation score of +3 and increasing the dose of long-acting GH by at least 5% if the IGF-1 level is below a standard deviation score of 0. This relates to long-acting GH, including:

[0081] It is understood that no dose adjustment is required in step (c) if the IGF-1 level falls within the range of 0 to +3 standard deviation scores. In certain embodiments, steps (a)-(c) are repeated until the IGF-1 level stabilizes within the range of 0 to +3 standard deviation scores.

[0082] In certain embodiments, the dose of long-acting GH is decreased by at least 5% in step (c) if the IGF-1 level is above a standard deviation score of +2, and increased by at least 5% if the IGF-1 level is below a standard deviation score of +0.5. Thus, in such embodiments, no dose adjustment is required in step (c) if the IGF-1 level falls within the range of standard deviation scores of +0.5 to +2. In certain embodiments, steps (a) to (c) are repeated until the IGF-1 level stabilizes within the range of standard deviation scores of +0.5 to +2.

[0083] Embodiments for inflammation-induced disorders, frequency of administration and long-acting growth hormone are as described elsewhere herein.

[0084] In certain embodiments, the dose reduction in step (c) is 5%. In certain embodiments, the dose reduction in step (c) is 6%. In certain embodiments, the dose reduction in step (c) is 7%. In certain embodiments, the dose reduction in step (c) is 8%. In certain embodiments, the dose reduction in step (c) is 9%. In certain embodiments, the dose reduction in step (c) is 10%. In certain embodiments, the dose reduction in step (c) is 11%. In certain embodiments, the dose reduction in step (c) is 12%. In certain embodiments, the dose reduction in step (c) is 13%. In certain embodiments, the dose reduction in step (c) is 14%. In certain embodiments, the dose reduction in step (c) is 15%. In certain embodiments, the dose reduction in step (c) is 16%. In certain embodiments, the dose reduction in step (c) is 17%. In certain embodiments, the dose reduction in step (c) is 18%. In certain embodiments, the dose reduction in step (c) is 19%. In certain embodiments, the dose reduction in step (c) is 20%. In certain embodiments, the dose increase in step (c) is 5%. In certain embodiments, the dose increase in step (c) is 6%. In certain embodiments, the dose increase in step (c) is 7%. In certain embodiments, the dose increase in step (c) is 8%. In certain embodiments, the dose increase in step (c) is 9%. In certain embodiments, the dose increase in step (c) is 10%. In certain embodiments, the dose increase in step (c) is 11%. In certain embodiments, the dose increase in step (c) is 12%. In certain embodiments, the dose increase in step (c) is 13%. In certain embodiments, the dose increase in step (c) is 14%. In certain embodiments, the dose increase in step (c) is 15%. In certain embodiments, the dose increase in step (c) is 16%.In certain embodiments, the dose increase in step (c) is 17%. In certain embodiments, the dose increase in step (c) is 18%. In certain embodiments, the dose increase in step (c) is 19%. In certain embodiments, the dose increase in step (c) is 20%.

[0085] In another aspect, the present invention provides a long-acting GH for use in the treatment of an inflammation-induced disease, the treatment comprising: (a) administering to a patient having an inflammation-induced disease at least an initial dose of long-acting GH; (b) measuring biomarkers indicative of M1 and M2 macrophages; (c) adjusting the dose of long-acting GH based on the change in macrophage phenotype due to M1 reduction or M2 induction as indicated by the biomarkers. This relates to long-acting GH, including:

[0086] Embodiments for inflammation-induced disorders, frequency of administration and long-acting growth hormone are as described elsewhere herein.

[0087] In certain embodiments, biomarkers indicative of M1 and M2 macrophages are measured prior to the initiation of treatment with long-acting GH.

[0088] In certain embodiments, the biomarkers indicative of M1 macrophages are selected from the group consisting of interleukin (IL)-1β, IL-6, IL-12, IL-23, IL-27, tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-y), monocyte chemoattractant protein (MCP)-1, CCL2, CCL3, CCL5, CXCL8, CXCL9, CXCL10, CXCL1, CXCL16, CCR2, CCR7, IL1R1, TLR2, TLR4, MARCO, CD11c, CD38, and iNOS. In certain embodiments, the biomarkers indicative of M1 macrophages are selected from the group consisting of IL-6, TNF-α, CD11c, and iNOS.

[0089] In certain embodiments, the biomarkers indicative of M2 macrophages are selected from the group consisting of IL-2, IL-4, IL-10, IL-13, CCL17, CCL18, CCL22, CCL24, CCL13, CCL16, CXCR1, CXCR2, CD14, CD23, CD36, CD163, mannose receptor (CD206), scavenger receptor A, Chi3l3 / Ym1, Retnla / Fizz-1, and Arginase-1. In certain embodiments, the biomarkers indicative of M2 macrophages are selected from the group consisting of IL-10, CD36, CD206, Retnla / Fizz-1, and Arginase-1.

[0090] In certain embodiments, if the ratio of M1 to M2 macrophages is unbalanced, e.g., if biomarkers indicative of M1 type macrophages are still elevated or predominant compared to biomarkers indicative of M2 macrophages, the dose of long-acting growth hormone is increased in step (c). In certain embodiments, such dose increase is a 5% increase. In certain embodiments, such dose increase is a 6% increase. In certain embodiments, such dose increase is a 7% increase. In certain embodiments, such dose increase is an 8% increase. In certain embodiments, such dose increase is a 9% increase. In certain embodiments, such dose increase is a 10% increase. In certain embodiments, such dose increase is an 11% increase. In certain embodiments, such dose increase is a 12% increase. In certain embodiments, such dose increase is a 13% increase. In certain embodiments, such dose increase is a 14% increase. In certain embodiments, such dose increase is a 15% increase. In certain embodiments, such dose increase is a 16% increase. In certain embodiments, such dose increase is a 17% increase. In certain embodiments, such dose increase is an 18% increase. In certain embodiments, such dose increase is a 19% increase. In certain embodiments, such dose increase is a 20% increase. If in step (c) the ratio of M1 to M2 macrophages is balanced, e.g., biomarkers indicative of M1 and M2 have reached a steady state, the dose is, in certain embodiments, not adjusted but remains constant.

[0091] In certain embodiments, the dose adjustment in step (c) involves measuring IGF-1 levels and adjusting the dose of the long-acting GH such that the IGF-1 levels are in the range of 0 to +3 standard deviation scores. In certain embodiments, the dose adjustment in step (c) involves measuring IGF-1 levels and adjusting the dose of the long-acting GH such that the IGF-1 levels are in the range of +0.5 to +2 standard deviation scores.

[0092] In certain embodiments, steps (b) and (c) are repeated until macrophage rebalance is achieved.

[0093] In another aspect, the invention relates to a method of treating an inflammation-induced disease comprising administering a pharmacologic effective amount of a long-acting growth hormone. Embodiments of long-acting growth hormone and inflammation-induced diseases are described elsewhere herein.

[0094] In another aspect, the present invention provides a method for producing a composition comprising: (a) administering to a patient having an inflammation-induced disease at least an initial dose of long-acting GH; (b) measuring insulin-like growth factor-1 (IGF-1) levels, and (c) decreasing the dose of long-acting GH by at least 5% if the IGF-1 level is above a standard deviation score of +3 and increasing the dose of long-acting GH by at least 5% if the IGF-1 level is below a standard deviation score of 0. The present invention relates to a method comprising the steps of:

[0095] It is understood that no dose adjustment is required in step (c) if the IGF-1 level falls within the range of 0 to +3 standard deviation scores. In certain embodiments, steps (a)-(c) are repeated until the IGF-1 level stabilizes within the range of 0 to +3 standard deviation scores.

[0096] In certain embodiments, the dose of long-acting GH is decreased by at least 5% in step (c) if the IGF-1 level is above a standard deviation score of +2, and increased by at least 5% if the IGF-1 level is below a standard deviation score of +0.5. Thus, in such embodiments, no dose adjustment is required in step (c) if the IGF-1 level falls within the range of standard deviation scores of +0.5 to +2. In certain embodiments, steps (a) to (c) are repeated until the IGF-1 level stabilizes within the range of standard deviation scores of +0.5 to +2.

[0097] Embodiments for the dose adjustment in step (c) are as described elsewhere herein.

[0098] In another aspect, the present invention provides a method for producing a composition comprising: (a) administering to a patient having an inflammation-induced disease at least an initial dose of long-acting GH; (b) measuring biomarkers indicative of M1 and M2 macrophages; (c) adjusting the dose of long-acting GH based on the change in macrophage phenotype due to M1 reduction or M2 induction as indicated by the biomarkers. The present invention relates to a method comprising the steps of:

[0099] In certain embodiments, steps (b) and (c) are repeated until macrophage rebalance is achieved.

[0100] Embodiments of the biomarkers in step (b) and dose adjustments in step (c) are described elsewhere herein.

[0101] In certain embodiments, the long-acting growth hormone comprises at least one human growth hormone (hGH). In certain embodiments, the hGH has a sequence of SEQ ID NO:1. In certain embodiments, the hGH has a sequence having at least 90% identity to the sequence of SEQ ID NO:1. In certain embodiments, the hGH has a sequence having at least 92% identity to the sequence of SEQ ID NO:1. In certain embodiments, the hGH has a sequence having at least 94% identity to the sequence of SEQ ID NO:1. In certain embodiments, the hGH has a sequence having at least 95% identity to the sequence of SEQ ID NO:1. In certain embodiments, the hGH has a sequence having at least 96% identity to the sequence of SEQ ID NO:1. In certain embodiments, the hGH has a sequence having at least 97% identity to the sequence of SEQ ID NO:1. In certain embodiments, the hGH has a sequence having at least 98% identity to the sequence of SEQ ID NO:1. In certain embodiments, the hGH has a sequence having at least 99% identity to the sequence of SEQ ID NO:1.

[0102] In one embodiment, the long-acting GH comprises growth hormone non-covalently embedded or encapsulated in a polymer or lipid-containing matrix, hi certain embodiments, the long-acting GH comprises growth hormone non-covalently embedded or encapsulated in a polymer. Preferred polymer matrices are 2-methacryloyl-oxyethyl phosphorylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymers, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxypropyl methacrylate). , poly(hydroxypropyl oxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyl oxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinyl amine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, cellulose, carbomethyl cellulose, hydroxypropyl methyl cellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch, and other carbohydrate based polymers, xylan, and copolymers thereof.

[0103] In certain embodiments, the polymer is selected from the group consisting of PEG, polylactide-co-glycolide (PLGA) and hyaluronic acid. In certain embodiments, the polymer is PEG. In certain embodiments, the polymer is PLGA. In certain embodiments, the polymer is hyaluronic acid.

[0104] In certain embodiments, the polymer matrix is ​​selected from the group consisting of 2-methacryloyl-oxyethyl phosphorylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymer, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxypropyl methacrylate), A hydrogel comprising a polymer selected from the group consisting of poly(hydroxypropyloxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic acid-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, cellulose, carbomethylcellulose, hydroxypropylmethylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch, and other carbohydrate-based polymers, xylan, and copolymers thereof. In certain embodiments, the hydrogel comprises a polymer selected from the group consisting of PEG, polylactide-co-glycolide (PLGA) and hyaluronic acid. In certain embodiments, the hydrogel is a PEG-based hydrogel. In certain embodiments, the hydrogel comprises PLGA.In certain embodiments, the hydrogel comprises hyaluronic acid.

[0105] In certain embodiments, the long-acting growth hormone is crystalline growth hormone.

[0106] In certain embodiments, the long-acting growth hormone comprises a growth hormone moiety fused to at least one natural or non-natural amino acid sequence. Such an amino acid sequence is understood to comprise one or more amino acid residues. In certain embodiments, such a growth hormone fusion protein comprises a recognition sequence for enzymatic cleavage between the growth hormone moiety and the natural or non-natural amino acid sequence. In certain embodiments, the growth hormone fusion protein comprises a chemical cleavage site between the growth hormone moiety and the natural or non-natural amino acid sequence. In certain embodiments, the amino acid sequence is selected from the group consisting of a carboxyl terminal peptide of chorionic gonadotropin described in US 2012 / 0035101, albumin, an XTEN sequence described in WO2011123813A2, a proline / alanine random coil sequence described in WO2011 / 144756A1, a proline / alanine / serine random coil sequence described in WO2008 / 155134, and an Fc fusion protein. In certain embodiments, the long-acting growth hormone comprises an hGH-CTP fusion protein. In certain embodiments, the long-acting growth hormone comprises an hGH-XTEN fusion protein. In certain embodiments, the long-acting growth hormone comprises an hGH-HSA fusion protein. In certain embodiments, the long-acting growth hormone comprises an hGH-Fc fusion protein.

[0107] In certain embodiments, the long-acting growth hormone comprises a growth hormone moiety covalently conjugated to one or more chemical moieties. In certain embodiments, the chemical moiety is a polymer moiety, e.g., 2-methacryloyl-oxyethyl phosphoylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymer, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxypropyl ... and the polymeric portion comprises a polymer selected from the group consisting of poly(hydroxypropyl oxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, cellulose, carbomethylcellulose, hydroxypropyl methylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch, and other carbohydrate based polymers, xylan, and copolymers thereof. In certain embodiments, the polymer portion comprises a PEG-based polymer, hi certain embodiments, the polymer portion comprises a hyaluronic acid-based polymer.

[0108] In certain embodiments, the polymeric portion comprises a linear, branched, dendrimeric, or cyclic polymer, or any combination thereof. In certain embodiments, the polymeric portion comprises a linear polymer. In certain embodiments, the polymeric portion comprises a branched polymer, e.g., a polymer having 1, 2, 3, 4, or 5 branch points, which in certain embodiments are -N<, -CR b1 < and >C<, -R b1 -H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 alkynyl, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl may be one or more -R b2 Optionally replaced by C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl is -C(O)O-, -O-, -C(O)-, -C(O)N(R b3 )-, -S(O)2N( b3 )-, -S(O)N(R b3 )-, -S(O)2-, -S(O)-, -N(R b3 )S(O)2N(R b3a )-, -S-, -N(R b3 )-, -OC(OR b3 )(R b3a )-, -N(R b3 )C(O)N(R b3a )- and -OC(O)N(R b3 )-, optionally interrupted by -R b2 , -R b3 and -R b3a -H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 alkynyl. In certain embodiments, the polymeric portion comprises a dendrimeric polymer. In certain embodiments, the polymeric portion comprises a cyclic polymer.

[0109] In certain embodiments, the chemical moiety is a fatty acid or fatty acid variant moiety, which may be optionally substituted. In certain embodiments, such fatty acid or fatty acid variant moiety has a structure disclosed in WO2005 / 027978A2 and WO2014 / 060512A1. In certain embodiments, the long-acting growth hormone is a growth hormone-fatty acid conjugate. In certain embodiments, the long-acting growth hormone is a growth hormone-fatty acid variant conjugate.

[0110] In certain embodiments, the long-acting growth hormone has the formula (F):

[0111] [ka] [In the formula, The dashed line indicates the binding to the remainder of the long-acting growth hormone.] Includes parts of.

[0112] In certain embodiments, the dashed line in formula (F) indicates a bond to the growth hormone moiety. In certain embodiments, the dashed line in formula (F) indicates a bond to the sulfur of the thiol of the cysteine ​​side chain of the growth hormone moiety. In certain embodiments, the growth hormone moiety is of SEQ ID NO: 1, in which the leucine at position 101 is mutated to cysteine, and the dashed line in formula (F) indicates a bond to this cysteine ​​at position 101. It is understood that "a bond to this cysteine" means a bond to the sulfur of the thiol of the cysteine.

[0113] In certain embodiments, the fatty acid or fatty acid variant moiety has the formula (Fi):

[0114] [ka] [In the formula, The dashed line indicates the binding to the remainder of the long-acting growth hormone.] It has the structure:

[0115] In certain embodiments, the dashed line in formula (Fi) indicates a bond to the growth hormone moiety. In certain embodiments, the dashed line in formula (Fi) indicates a bond to the sulfur of the thiol of the cysteine ​​side chain of the growth hormone moiety. In certain embodiments, the growth hormone moiety is of SEQ ID NO: 1, in which the leucine at position 101 is mutated to cysteine, and the dashed line in formula (Fi) indicates a bond to this cysteine ​​at position 101. It is understood that "bond to this cysteine" means a bond to the sulfur of the thiol of cysteine.

[0116] When the long-acting GH is of formula (Fi), where the growth hormone portion has the sequence of SEQ ID NO:1, in which the leucine at position 101 is replaced by a cysteine ​​and the dashed line indicates the bond of the thiol to the cysteine ​​via the sulfur, the long-acting GH is somapacitan, sold as Sogroya®.

[0117] In certain embodiments, the bond between the growth hormone moiety and the chemical moiety is a stable covalent bond. In certain embodiments, the bond between the growth hormone moiety and the chemical moiety is a reversible covalent bond. When the growth hormone moiety is reversibly conjugated to one or more chemical moieties, such a conjugate may also be referred to as a prodrug.

[0118] In certain embodiments, the long-acting growth hormone is represented by formula (Ia) or (Ib):

[0119] [ka] [In the formula, each -D is independently a growth hormone moiety, Each-L 1 - is independently a linker moiety covalently and reversibly attached to -D; Each-L 2 - is independently a chemical bond or a spacer moiety; each -Z is independently an optionally substituted polymer moiety or a fatty acid moiety; x is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16; y is an integer selected from the group consisting of 2, 3, 4 and 5. or a pharma- ceutically acceptable salt thereof.

[0120] In certain embodiments, the growth hormone conjugate is of formula (Ia). In certain embodiments, x is 1. In certain embodiments, the growth hormone conjugate is of formula (Ib). In certain embodiments, y is 2. In certain embodiments, y is 3. In certain embodiments, y is 4.

[0121] In certain embodiments, all moieties-D of the conjugate are the same. In certain embodiments, the conjugates of the invention comprise more than one type of -D, e.g., 2, 3, 4 or 5 different types of moieties-D, provided that more than one moiety-D is present in the conjugate.

[0122] In certain embodiments, -D of formula (Ia) and (Ib) has a sequence of SEQ ID NO: 1. In certain embodiments, -D of formula (Ia) and (Ib) has a sequence having at least 90% identity to the sequence of SEQ ID NO: 1. In certain embodiments, -D of formula (Ia) and (Ib) has a sequence having at least 92% identity to the sequence of SEQ ID NO: 1. In certain embodiments, -D of formula (Ia) and (Ib) has a sequence having at least 94% identity to the sequence of SEQ ID NO: 1. In certain embodiments, -D of formula (Ia) and (Ib) has a sequence having at least 95% identity to the sequence of SEQ ID NO: 1. In certain embodiments, -D of formula (Ia) and (Ib) has a sequence having at least 96% identity to the sequence of SEQ ID NO: 1. In certain embodiments, -D of formula (Ia) and (Ib) has a sequence having at least 97% identity to the sequence of SEQ ID NO: 1. In certain embodiments, -D of formula (Ia) and (Ib) has a sequence having at least 98% identity to the sequence of SEQ ID NO: 1. In certain embodiments, -D of formula (Ia) and (Ib) has a sequence having at least 99% identity to the sequence of SEQ ID NO: 1.

[0123] In certain embodiments, all moieties -L of the conjugate 1 - are identical, provided that there are two or more moieties -L in the conjugate. 1 In certain embodiments, the conjugate of the invention comprises two or more types of -L 1 -, for example, 2, 3, 4 or 5 different moieties -L 1 - Including.

[0124] Part-L 1 - may be any moiety that allows for reversible binding of -D. In certain embodiments, -L 1 - is a traceless linker, i.e. the linker from the drug DH is released in its unmodified form, which may also be called the free form.

[0125] Part-L 1- may be attached to a proteinogenic or non-proteinogenic amino acid residue of -D. In certain embodiments, -L 1 - is attached to a non-proteinogenic amino acid residue. 1 The bond of - is to a proteinogenic amino acid residue. When the bond occurs at a proteinogenic amino acid residue, said proteinogenic amino acid residue is, in certain embodiments, selected from the group consisting of cysteine, methionine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, glutamine, and arginine. In certain embodiments, such a proteinogenic amino acid residue is selected from the group consisting of cysteine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, and arginine. In certain embodiments, such a proteinogenic amino acid is cysteine. In certain embodiments, such a proteinogenic amino acid is histidine. In certain embodiments, such a proteinogenic amino acid is lysine. In certain embodiments, such a proteinogenic amino acid is tryptophan. In certain embodiments, such a proteinogenic amino acid is serine. In certain embodiments, such a proteinogenic amino acid is threonine. In certain embodiments, such a proteinogenic amino acid is aspartic acid. In certain embodiments, such a proteinogenic amino acid is glutamic acid. In certain embodiments, such a proteinogenic amino acid is arginine.

[0126] Part-L 1 - may be linked to -D through any type of bond, so long as it is reversible, and the functional group of -D selected for attachment is -L 1 It is understood that the bond between - and -D is affected. In certain embodiments, -L 1- is linked to -D through a bond selected from the group consisting of amide, ester, carbamate, acetal, aminal, imine, oxime, hydrazone, disulfide, and acylguanidine. 1 - is linked to -D through a bond selected from the group consisting of amide, ester, carbamate, and acylguanidine. These bonds do not have to be reversible themselves, but reversibility can be achieved by -L 1 It is understood that this may be a function of the specific atomic groups or moieties present in -L. 1 - is linked to -D through an ester bond. In certain embodiments, -L 1 - is linked to -D through a carbamate bond. In certain embodiments, -L 1 - is linked to -D through an acylguanidine. In certain embodiments, -L 1 - is linked to -D through an amide bond.

[0127] In certain embodiments, -D is linked to -L via the nitrogen of the amine functional group of -D. 1 Such an amine functionality may be the N-terminal amine functionality of -D or an amine functionality from the side chain of a lysine residue. In certain embodiments, -D is conjugated to -L through the nitrogen of the N-terminal amine functionality. 1 In certain embodiments, -D is conjugated to -L through the nitrogen of the amine functional group from the side chain of a lysine residue. 1 In certain embodiments, -L 1 - is linked to -D through the nitrogen of the amine functional group of the side chain of the lysine residue of -D, and -D and -L 1 In certain embodiments, the bond formed between -L is a carbamate. 1 - is linked to -D through the nitrogen of the amine functional group of the side chain of the lysine residue of -D, and -D and -L 1 The bond formed between - is an amide.

[0128] In certain embodiments, -L1 - has the structure disclosed in WO 2009 / 095479 A2. Thus, in certain embodiments, the moiety -L 1 - is a compound of formula (II):

[0129] [ka] [In the formula, The dashed line indicates the attachment of -D to the nitrogen by forming an amide bond; -X- is -C(R 4 R 4a )-, -N(R 4 )-, -O-, -C(R 4 R 4a )-C(R 5 R 5a )-, -C(R 5 R 5a )-C(R 4 R 4a )-, -C(R 4 R 4a )-N(R 6 )-, -N(R 6 )-C(R 4 R 4a )-, -C(R 4 R 4a )-O-, -OC(R 4 R 4a )- and -C(R 7 R 7a )-, X 1 is selected from the group consisting of C and S(O); -X 2 - is -C(R 8 R 8a )- and -C(R 8 R 8a )-C(R 9 R 9a )-, =X 3 is selected from the group consisting of =O, =S and =N-CN; -R 1 , -R 1a , -R 2 , -R 2a , -R4 , -R 4a , -R 5 , -R 5a , -R 6 , -R 8 , -R 8a , -R 9 , -R 9a -H and C 1-6 independently selected from the group consisting of alkyl, -R 3 , -R 3a -H and C 1-6 alkyl, with the proviso that -R 3 , -R 3a When one or both of are other than -H, they may be joined to the N to which they are attached by SP 3 linked through a hybridized carbon atom, -R 7 is -N(R 10 R 10a ) and -NR 10 -(C=O)-R 11 is selected from the group consisting of -R 7a , -R 10 , -R 10a , -R 11 are each independently -H and C 1-6 is selected from the group consisting of alkyl, Optionally, Pair-R 1a / -R 4a , -R 1a / -R 5a , -R 1a / -R 7a , -R 4a / -R 5a , -R 8a / -R 9a one or more of the following forms a chemical bond; Optionally, Pair-R 1 / -R 1a , -R 2 / -R 2a , -R 4 / -R 4a , -R 5 / -R 5a , -R 8 / -R 8a , -R 9 / -R9a One or more of the following, together with the atoms to which they are attached, are C 3-10 forming a cycloalkyl or 3- to 10-membered heterocyclyl; Optionally, Pair-R 1 / -R 4 , -R 1 / -R 5 , -R 1 / -R 6 , -R 1 / -R 7a , -R 4 / -R 5 , -R 4 / -R 6 , -R 8 / -R 9 , -R 2 / -R 3 one or more of, together with the atoms to which they are attached, form a ring A; Optionally, R 3 / R 3a form, together with the nitrogen atom to which they are attached, a 3- to 10-membered heterocyclic ring; A is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl. The -L 1 - At least one -L 2 Replaced by -Z and, optionally, -L 1 - is further substituted, with the proviso that the hydrogen marked with an asterisk in formula (II) is -L 2 is not replaced by -Z or a substituent.

[0130] In certain embodiments, -L of formula (II) 1 - is one part - L 2 Replaced by -Z.

[0131] In certain embodiments, -L of formula (II) 1 - is not further substituted.

[0132] -R in formula (II) 3 / -R 3a However, when they are combined with the nitrogen atom to which they are bonded to form a 3- to 10-membered heterocycle, the atom directly bonded to the nitrogen atom is sp 3 It is understood that only such 3- to 10-membered heterocyclic rings can be formed that are hybridized carbon atoms. 3 / -R 3a and the nitrogen atom to which they are attached have the following structure:

[0133] [ka] [In the formula, The dashed line is -L 1 - represents a bond to the remainder of the ring contains 3 to 10 atoms, including at least one nitrogen atom; and R # and R ## sp 3 represents a hybridized carbon atom].

[0134] It is also understood that the 3- to 10-membered heterocycle may be further substituted.

[0135] -R in formula (II) 3 / -R 3a and the nitrogen atom to which they are attached are the following:

[0136] [ka] [In the formula, The dashed line indicates the bond to the rest of the molecule; -R is -H and C 1-6 alkyl].

[0137] Moiety-L of formula (II) 1- may be optionally further substituted. In general, any substituent may be used as long as it does not affect the principle of cleavage, i.e., the hydrogen marked with an asterisk in formula (II) is not replaced and the moiety of formula (II)

[0138] [ka] The nitrogen in remains part of a primary, secondary or tertiary amine, i.e., -R 3 and -R 3a are each independently -H or sp 3 It is linked to -N< through a hybridized carbon atom.

[0139] In one embodiment, the -R 1 or -R 1a -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 2 or -R 2a -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 3 or -R 3a -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 4 -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 5 or -R 5a -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 6 -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 7 or -R 7a -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 8 or -R8a -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 9 or -R 9a -L 2 -Z or -L 2 -Z' is substituted.

[0140] In certain embodiments, -L 1 - has the structure disclosed in WO 2016 / 020373 A1. Thus, in certain embodiments, the moiety -L 1 - is a compound of formula (III):

[0141] [ka] [In the formula, The dashed lines indicate attachment of -D by forming an amide or ester bond to a primary or secondary amine or hydroxyl, respectively; -R 1 , -R 1a , -R 2 , -R 2a , -R 3 and -R 3a -H, -C(R 8 R 8a R 8b ), -C(=O)R 8 , -C≡N, -C(=NR 8 )R 8a , -CR 8 (=CR 8a R 8b ), -C≡CR 8 and -T, -R 4 , -R 5 and -R 5a -H, -C(R 9 R 9a R 9b -T; a1 and a2 are each independently 0 or 1; Each-R 6 , -R 6a , -R7 , -R 7a , -R 8 , -R 8a , -R 8b , -R 9 , -R 9a , -R 9b -H, halogen, -CN, -COOR 10 , -OR 10 , -C(O)R 10 , -C(O)N(R 10 R 10a ), -S(O)2N(R 10 R 10a ), -S(O)N(R 10 R 10a ), -S(O)2R 10 , -S(O)R 10 , -N(R 10 )S(O)2N(R 10a R 10b ), -SR 10 , -N(R 10 R 10a ), -NO2, -OC(O)R 10 , -N(R 10 )C(O)R 10a , -N(R 10 )S(O)2R 10a , -N(R 10 )S(O)R 10a , -N(R 10 )C(O)OR 10a , -N(R 10 )C(O)N(R 10a R 10b ), -OC(O)N(R 10 R 10a ), -T, C 1-20 Alkyl, C 2-20 Alkenyl, and C 2-20 alkynyl, wherein -T, -C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkynyl may be one or more -R 11 Optionally replaced by C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20Alkynyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 12 )-, -S(O)2N(R 12 )-, -S(O)N(R 12 )-, -S(O)2-, -S(O)-, -N(R 12 )S(O)2N(R 12a )-, -S-, -N(R 12 )-, -OC(OR 12 )(R 12a )-, -N(R 12 )C(O)N(R 12a )- and -OC(O)N(R 12 )-, Each-R 10 , -R 10a , -R 10b -H, -T, C 1-20 Alkyl, C 2-20 Alkenyl, and C 2-20 alkynyl; 1-20 Alkyl, C 2-20 Alkenyl, and C 2-20 Alkynyl may be one or more -R 11 Optionally replaced by C 1-20 Alkyl, C 2-20 Alkenyl, and C 2-20 Alkynyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 12 )-, -S(O)2N(R 12 )-, -S(O)N(R 12 )-, -S(O)2-, -S(O)-, -N(R 12 )S(O)2N(R 12a )-, -S-, -N(R 12 )-, -OC(OR 12 )(R 12a )-, -N(R 12 )C(O)N(R 12a )- and -OC(O)N(R 12 )-, Each T is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 each T is independently selected from the group consisting of cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl, and each T is independently selected from the group consisting of one or more of the same or different -R 11 is optionally replaced by Each-R 11 is halogen, -CN, oxo(=O), -COOR 13 , -OR 13 , -C(O)R 13 , -C(O)N(R 13 R 13a ), -S(O)2N(R 13 R 13a ), -S(O)N(R 13 R 13a ), -S(O)2R 13 , -S(O)R 13 , -N(R 13 )S(O)2N(R 13a R 13b ), -SR 13 , -N(R 13 R 13a ), -NO2, -OC(O)R 13 , -N(R 13 )C(O)R 13a , -N(R 13 )S(O)2R 13a , -N(R 13 )S(O)R 13a , -N(R 13 )C(O)OR 13a , -N(R 13 )C(O)N(R 13a R 13b ), -OC(O)N(R 13 R 13a ), and C 1-6 alkyl, C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R 12 , -R 12a , -R 13 , -R 13a , -R 13b -H and C 1-6 alkyl, C 1-6the alkyl is optionally substituted with one or more halogens which may be the same or different; Optionally, Pair-R 1 / -R 1a , -R 2 / -R 2a , -R 3 / -R 3a , -R 6 / -R 6a , -R 7 / -R 7a One or more of the following, together with the atoms to which they are attached, are C 3-10 forming a cycloalkyl or 3- to 10-membered heterocyclyl; Optionally, Pair-R 1 / -R 2 , -R 1 / -R 3 , -R 1 / -R 4 , -R 1 / -R 5 , -R 1 / -R 6 , -R 1 / -R 7 , -R 2 / -R 3 , -R 2 / -R 4 , -R 2 / -R 5 , -R 2 / -R 6 , -R 2 / -R 7 , -R 3 / -R 4 , -R 3 / -R 5 , -R 3 / -R 6 , -R 3 / -R 7 , -R 4 / -R 5 , -R 4 / -R 6 , -R 4 / -R 7 , -R 5 / -R 6 , -R 5 / -R 7 , -R 6 / -R 7one or more of, together with the atoms to which they are attached, form a ring A; A is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; -L 1 - At least one -L 2 Replaced by -Z and, optionally, -L 1 - is further substituted.

[0142] -L in formula (III) 1 The optional further substituents of - are preferably as described above.

[0143] Preferably, -L of formula (III) 1 - 1 part - L 2 Replaced by -Z.

[0144] In one embodiment, -L of formula (III) 1 - is not further substituted.

[0145] In another embodiment, -L 1 has the structure disclosed in EP 1536334 B1, WO 2009 / 009712 A1, WO 2008 / 034122 A1, WO 2009 / 143412 A2, WO 2011 / 082368 A2, and U.S. Pat. No. 8,618,124 B2, which are incorporated herein by reference.

[0146] In certain embodiments, -L 1 - has the structure disclosed in U.S. Pat. No. 8,946,405 B2 and U.S. Pat. No. 8,754,190 B2. Thus, in certain embodiments, -L 1 - is a compound of formula (IV):

[0147] [ka] [In the formula, The dashed line indicates a bond to -D through a functional group of -D selected from the group consisting of -OH, -SH, and -NH; m is 0 or 1; -R 1 and -R 2 At least one or both of are -CN, -NO2, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -C(O)R 3 , -S(O)R 3 , -S(O)2R 3 , and -SR 4 are independently selected from the group consisting of -R 1 and -R 2 is selected from the group consisting of: -H, optionally substituted alkyl, optionally substituted arylalkyl, and optionally substituted heteroarylalkyl; -R 3 is -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 9 and -N(R 9 ) 2 selected from the group consisting of -R 4 is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; Each-R 5 is independently selected from the group consisting of: -H, optionally substituted alkyl, optionally substituted alkenylalkyl, optionally substituted alkynylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; -R 9is selected from the group consisting of -H and optionally substituted alkyl; -Y- is absent and -X- is selected from the group consisting of -O- and -S-; or -Y- is -N(Q)CH2- and -X- is -O-; Q is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; Optionally, -R 1 and-R 2 may be taken together to form a 3- to 8-membered ring, Optionally, both -R 9 together with the nitrogen to which they are attached form a heterocyclic ring. The -L 1 - is -L 2 Replaced by -Z and, optionally, -L 1 - is further substituted.

[0148] The terms used in relation to formula (IV) only have the following meanings:

[0149] The term "alkyl" as used herein includes straight chain, branched or cyclic saturated hydrocarbon groups of 1 to 8 carbons, or, in some embodiments, 1 to 6 or 1 to 4 carbon atoms.

[0150] The term "alkoxy" includes an alkyl group linked to an oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and the like.

[0151] The term "alkenyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon double bond.

[0152] The term "alkynyl" includes non-aromatic unsaturated hydrocarbon groups having a carbon-carbon triple bond.

[0153] The term "aryl" includes aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. The term "heteroaryl" includes aromatic rings containing 3 to 15 carbons and containing at least one N, O, or S atom, preferably 3 to 7 carbons and 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.

[0154] In some cases, the alkenyl, alkynyl, aryl, or heteroaryl moiety may be coupled to the remainder of the molecule through an alkylene bond. In these situations, 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.

[0155] The term "halogen" includes bromo, fluoro, chloro and iodo.

[0156] The term "heterocyclic ring" refers to a 4-8 membered aromatic or non-aromatic ring containing from 3 to 7 carbon atoms and at least one N, O or S atom. Examples are piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine and tetrahydrofuranyl, as well as the exemplary groups provided above for the term "heteroaryl".

[0157] When the ring system is optionally substituted, suitable substituents are selected from the group consisting of alkyl, alkenyl, alkynyl, or further rings, each of which is optionally further substituted. Optional substituents for any of the groups, including those listed above, include halo, nitro, cyano, -OR, -SR, -NR2, -OCOR, -NRCOR, -COOR, -CONR2, -SOR, -SOR, -SONR2, -SON2NR2, where each R is independently alkyl, alkenyl, alkynyl, aryl, or heteroaryl, or two R groups, together with the atoms to which they are attached, form a ring.

[0158] In certain embodiments, -L of formula (IV) 1 - 1 part - L 2 Replaced with -Z.

[0159] In certain embodiments, -L of formula (IV) 1 - is not further substituted.

[0160] In certain embodiments, -L 1 - has the structure disclosed in WO2013 / 036857A1. Thus, in certain embodiments, -L 1 - is a compound of formula (V):

[0161] [ka] [In the formula, The dashed line indicates the bond to -D through the amine functionality of -D; -R 1 is an optionally substituted C1-C6 straight chain, branched or cyclic alkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an alkoxy, and -NR 5 2, -R 2 is selected from the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; -R 3 is selected from the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; -R 4 is selected from the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; Each-R 5 are independently selected from the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl, or when acting together, two -R 5 can be cycloalkyl or cycloheteroalkyl. The -L 1 - is -L 2 Replaced by -Z and, optionally, -L 1 - is further substituted.

[0162] The terms used in relation to formula (V) only have the following meanings:

[0163] "Alkyl", "alkenyl" and "alkynyl" include straight chain, branched or cyclic hydrocarbon groups of 1 to 8 carbons, or 1 to 6 carbons, or 1 to 4 carbons, where alkyl is a saturated hydrocarbon, alkenyl contains one or more carbon-carbon double bonds, and alkynyl contains one or more carbon-carbon triple bonds. Unless otherwise specified, they contain 1 to 6 C.

[0164] "Aryl" includes aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. "Heteroaryl" includes aromatic rings containing 3 to 15 carbons and at least one N, O, or S atom, preferably 3 to 7 carbons and at least one N, O, or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiszolyl, isothiazolyl, quinolyl, indolyl, indenyl, and the like.

[0165] The term "substituted" refers to an alkyl, alkenyl, alkynyl, aryl, or heteroaryl group that contains one or more substituents in place of one or more hydrogen atoms. Substituents generally include halogen (including F, Cl, Br, and I), lower alkyl (including linear, branched, and cyclic), lower haloalkyl (including fluoroalkyl, chloroalkyl, bromoalkyl, and iodoalkyl), OH, lower alkoxy (including linear, branched, and cyclic), SH, lower alkylthio (including linear, branched, and cyclic), amino, alkylamino, dialkylamino, silyl (including alkylsilyl, alkoxysilyl, and arylsilyl), nitro, cyano, carbonyl, carboxylic acid, carboxylic acid ester, carboxylic acid amide, aminocarbonyl, aminoacyl, carbamate, urea, thiocarbamate ... mate, thiourea, ketone, sulfone, sulfonamide, aryl (including phenyl, naphthyl and anthracenyl), heteroaryl (including 5-membered heteroaryl such as pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole and tetrazole, 6-membered heteroaryl such as pyridine, pyrimidine, pyrazine, and fused heteroaryl such as benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole and benzisothiazole).

[0166] In certain embodiments, -L of formula (V) 1 - 1 part - L 2 Replaced by -Z.

[0167] In certain embodiments, -L of formula (V) 1 - is not further substituted.

[0168] In certain embodiments, -L 1 - has the structure disclosed in U.S. Patent No. 7,585,837 B2. Thus, in certain embodiments, -L 1 - is a compound of formula (VI):

[0169] [ka] [In the formula, The dashed line indicates the bond to -D through the amine functionality of -D; R 1 and R 2 is hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro, -SO3H, -SO2NHR 5 , amino, ammonium, carboxyl, PO3H2, and OPO3H2; R 3 , R 4 and R 5 are independently selected from the group consisting of hydrogen, alkyl and aryl. The -L 1 - is -L 2 Replaced by -Z and, optionally, -L 1 - is further substituted.

[0170] Suitable substituents for formula (VI) include alkyl (e.g., C 1-6 Alkyl), alkenyl (e.g., C 2-6 alkenyl), alkynyl (e.g., C 2-6alkynyl), aryl (e.g., phenyl), heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl (e.g., aromatic 4- to 7-membered heterocycle) or halogen moiety.

[0171] The terms used in relation to formula (VI) only have the following meanings:

[0172] The terms "alkyl", "alkoxy", "alkoxyalkyl", "aryl", "alkaryl" and "aralkyl" refer to alkyl radicals of 1 to 8, preferably 1 to 4, carbon atoms, such as methyl, ethyl, propyl, isopropyl and butyl, and aryl radicals of 6 to 10 carbon atoms, such as phenyl and naphthyl. The term "halogen" includes bromo, fluoro, chloro and iodo.

[0173] In certain embodiments, -L of formula (VI) 1 - 1 part - L 2 Replaced with -Z.

[0174] In certain embodiments, -L of formula (VI) 1 - is not further substituted.

[0175] In certain embodiments, -L 1 - has the structure disclosed in WO2002 / 089789A1. Thus, in certain embodiments, -L 1 - is a group represented by the formula (VII):

[0176] [ka] [In the formula, The dashed line indicates the bond to -D through the amine functionality of -D; Y1 and Y2 are independently O, S or NR 7 and R 2 , R 3 , R 4 , R 5 , R 6 and R 7is hydrogen, C 1-6 Alkyl, C 3-12 Branched alkyl, C 3-8 Cycloalkyl, C 1-6 Substituted alkyl, C 3-8 Substituted cycloalkyl, aryl, substituted aryl, aralkyl, C 1-6 Heteroalkyl, substituted C 1-6 Heteroalkyl, C 1-6 Alkoxy, phenoxy and C 1-6 heteroalkoxy; Ar is a moiety which, when included in formula (VII), forms a polysubstituted aromatic hydrocarbon or a polysubstituted heterocyclic group; X is a chemical bond or a moiety that is actively transported into a target cell, a hydrophobic moiety, or a combination thereof; y is 0 or 1. The -L 1 - is -L 2 Replaced by -Z and, optionally, -L 1 - is further substituted.

[0177] The terms used in relation to formula (VII) only have the following meanings:

[0178] The term "alkyl" includes, for example, alkoxy, C 3-8 Linear, branched, substituted C, including cycloalkyl or substituted cycloalkyl 1-12 It is understood to include alkyl.

[0179] The term "substituted" shall be understood to include the addition of one or more different atoms or the replacement of one or more atoms contained in a functional group or compound with one or more different atoms.

[0180] Substituted alkyl includes carboxyalkyl, aminoalkyl, dialkylamino, hydroxyalkyl, and mercaptoalkyl, substituted cycloalkyl includes moieties such as 4-chlorocyclohexyl, aryl includes moieties such as naphthyl, substituted aryl includes moieties such as 3-bromo-phenyl, aralkyl includes moieties such as toluyl, heteroalkyl includes moieties such as ethylthiophene, substituted heteroalkyl includes moieties such as 3-methoxythiophone, alkoxy includes moieties such as methoxy, and phenoxy includes moieties such as 3-nitrophenoxy. Halo- is understood to include fluoro, chloro, iodo, and bromo.

[0181] In certain embodiments, -L of formula (VII) 1 - 1 part - L 2 Replaced with -Z.

[0182] In certain embodiments, -L of formula (VII) 1 - is not further substituted.

[0183] In certain embodiments, -L 1 - is a substructure of formula (VIII)

[0184] [ka] [In the formula, The dashed line with an asterisk indicates the attachment of -D to the nitrogen by forming an amide bond, and the unmarked dashed line indicates the attachment of -L 1 - indicates the bond to the remainder of Including, -L 1 - is -L 2 Replaced by -Z and, optionally, -L 1 - is further substituted.

[0185] In certain embodiments, -L of formula (VIII) 1 - 1 part - L 2 Replaced by -Z.

[0186] In certain embodiments, -L of formula (VIII) 1 - is not further substituted.

[0187] In certain embodiments, -L 1 - is a substructure of formula (IX)

[0188] [ka] [In the formula, The dashed line with an asterisk indicates attachment by forming a carbamate bond to the nitrogen of -D; The unmarked dashed line is -L 1 - indicates the bond to the remainder of Including, -L 1 - is -L 2 Replaced by -Z and, optionally, -L 1 - is further substituted.

[0189] In certain embodiments, -L of formula (IX) 1 - 1 part - L 2 Replaced by -Z.

[0190] In certain embodiments, -L of formula (IX) 1 - is not further substituted.

[0191] In certain embodiments, -L 1 - is a compound of formula (IX-a):

[0192] [ka] [In the formula, The dashed line with an asterisk indicates the bond of -D to the nitrogen, and the unmarked dashed line indicates the bond of -L. 2 - indicates the bond to Z, n is 0, 1, 2, 3, or 4; =Y1 and =Y5 are independently selected from the group consisting of =O and =S; -Y2- and -Y3- are independently selected from the group consisting of -O- and -S-; -Y4- is -O-, -NR 5 - and -C(R 6 R 6a )-, -R 3 , -R 5 , -R 6 , and -R 6a are independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl; -R 4 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl; -W- is C 3-10 Cycloalkyl, 8-30 membered carbopolycyclyl, 3-10 membered heterocyclyl, -C(O)-, -C(O)N(R 7 )-, -O-, -S-, and -N(R 7 C optionally interrupted by one or more groups selected from the group consisting of 1-20 is selected from the group consisting of alkyl, -Nu is -N(R 7 R 7a ), -N(R 7 OH), -N(R 7 )-N(R 7a R 7b ), -S(R 7 ), -COOH,

[0193] [ka] is a nucleophile selected from the group consisting of -Ar- is

[0194] [ka] [ka] (In the formula, The dashed line is -L 1 - represents a bond to the remainder of -Z 1 - is -O-, -S-, and -N(R 7 )-, -Z 2 - is -N(R 7 )-is) is selected from the group consisting of -R 7 , -R 7a , and -R 7b -H, C 1-6 Alkyl, C 2-6 Alkenyl, and C 2-6 alkynyl] The -L 1 - is optionally further substituted.

[0195] In certain embodiments, -D is linked to -L of formula (IX-a) through the nitrogen of the amine functional group of -D, e.g., through the nitrogen of the amine functional group of the lysine side chain of -D. 1 - is bonded to.

[0196] In certain embodiments, -L of formula (IX-a) 1 - 1 part - L 2 Replaced by -Z.

[0197] In certain embodiments, -L of formula (IX-a) 1 - is not further substituted.

[0198] In certain embodiments, -L 1 - is a compound of formula (IX-b):

[0199] [ka] [In the formula, The dashed line with an asterisk indicates the bond of -D to the nitrogen, and the unmarked dashed line indicates the bond of -L. 2 - indicates the bond to Z, n is 0, 1, 2, 3, or 4; =Y1 and =Y5 are independently selected from the group consisting of =O and =S; -Y2- and -Y3- are independently selected from the group consisting of -O- and -S-; -Y4- is -O-, -NR 5 - and -C(R 6 R 6a )-, -R 2 , -R 3 , -R 5 , -R 6 , -R 6a are independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl; -R 4 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl; -W- is C 3-10 Cycloalkyl, 8-30 membered carbopolycyclyl, 3-10 membered heterocyclyl, -C(O)-, -C(O)N(R 7 )-, -O-, -S-, and -N(R 7 C optionally interrupted by one or more groups selected from the group consisting of1-20 is selected from the group consisting of alkyl, -Nu is -N(R 7 R 7a ), -N(R 7 OH), -N(R 7 )-N(R 7a R 7b ), -S(R 7 ), -COOH,

[0200] [ka] is a nucleophile selected from the group consisting of -Ar- is

[0201] [ka] (In the formula, The dashed line is -L 1 - represents a bond to the remainder of -Z 1 - is -O-, -S-, and -N(R 7 )-, -Z 2 - is -N(R 7 )-is) is selected from the group consisting of -R 7 , -R 7a , -R 7b -H, C 1-6 Alkyl, C 2-6 Alkenyl, and C 2-6 alkynyl] The -L 1 - is optionally further substituted.

[0202] In certain embodiments, -D is linked to -L of formula (IX-b) through the nitrogen of the amine functional group of -D, e.g., through the nitrogen of the amine functional group of the lysine side chain of -D. 1 - is bonded to.

[0203] In certain embodiments, -L of formula (IX-b) 1 - 1 part - L 2 Replaced by -Z.

[0204] In certain embodiments, -L of formula (IX-b) 1 - is not further substituted.

[0205] In certain embodiments, the =Y 1 In certain embodiments, -Y in formula (IX-a) and (IX-b) is ═O. 2 In certain embodiments, -Y in formula (IX-a) and (IX-b) is -O-. 3 In certain embodiments, -Y in formula (IX-a) and (IX-b) is -O-. 4 -NR 5 In certain embodiments, =Y in formula (IX-a) and (IX-b) is 5 is =O.

[0206] In certain embodiments, n in formulas (IX-a) and (IX-b) is 0 or 1. In certain embodiments, n in formulas (IX-a) and (IX-b) is 0. In certain embodiments, n in formulas (IX-a) and (IX-b) is 1.

[0207] In certain embodiments, -R of formula (IX-b) 2 is selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. 2 is selected from the group consisting of -H, methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, -R 2 is selected from -H, methyl, and ethyl. In certain embodiments, -R 2 is -H.

[0208] In certain embodiments, -R3 is selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, -R 3 is selected from the group consisting of -H, methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, -R 3 is selected from -H, methyl, and ethyl. In certain embodiments, -R 3 is -H.

[0209] In certain embodiments, -R in each of formulas (IX-a) and (IX-b) 4 is independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, -R 4 is selected from the group consisting of methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, -R 4 is selected from methyl and ethyl.

[0210] In certain embodiments, -R 5 is selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, -R 5 is selected from the group consisting of -H, methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, -R 5 In certain embodiments, -R in formula (IX-a) and (IX-b) is selected from methyl and ethyl. 5 is methyl.

[0211] In certain embodiments, -R6 and -R 6a is independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, -R 6 and -R 6a is independently selected from the group consisting of -H, methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, -R 6 and -R 6a is independently selected from -H, methyl, and ethyl. In certain embodiments, -R 6 and -R 6a are both -H.

[0212] In certain embodiments, Ar in formula (IX-a) and (IX-b) is phenyl. In certain embodiments, Ar in formula (IX-a) and (IX-b) is

[0213] [ka] wherein the dashed lines indicate the bonds of the moieties of formulae (IX-a) and (IX-b) to the remainder. It is.

[0214] In certain embodiments, W in formula (IX-a) and (IX-b) is C 3-10 Cycloalkyl, -C(O)-, -C(O)N(R 7 )-, -O-, -S-, and -N(R 7 Optionally interrupted by )-C 1-20 In certain embodiments, W in formula (IX-a) and (IX-b) is C 3-10 Cycloalkyl, -C(O)-, -C(O)N(R 7 )-, -O-, -S-, and -N(R 7 Optionally interrupted by )-C 1-10 In certain embodiments, W in formula (IX-a) and (IX-b) is C3-10 Cycloalkyl, -C(O)-, -C(O)N(R 7 )-, -O-, -S-, and -N(R 7 Optionally interrupted by )-C 1-6 In certain embodiments, W in formulas (IX-a) and (IX-b) is

[0215] [ka] [In the formula, The dashed line indicates the bond of the moiety of formula (IX-a) or (IX-b), respectively, to the remainder. It is.

[0216] In certain embodiments, -Nu in formulas (IX-a) and (IX-b) is -N(R 7 R 7a ).

[0217] In certain embodiments, -R 7 , -R 7a , and -R 7b are each independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, -R 7 , -R 7a , and -R 7b are each independently selected from -H, methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, -R 7 , -R 7a , and -R 7b are each independently selected from methyl or ethyl. In certain embodiments, -R 7 , -R 7a , and -R 7b are all methyl.

[0218] In certain embodiments, -L 1- is represented by the formula (IX-c)

[0219] [ka] [In the formula, The dashed line with an asterisk indicates the bond of -D to the nitrogen; The unmarked dashed lines are -L 2 - indicates the bond to Z, s1 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. It is of the following.

[0220] In certain embodiments, -D is linked to -L of formula (IX-c) through the nitrogen of the amine functional group of -D, e.g., through the nitrogen of the amine functional group of the lysine side chain of -D. 1 - is bonded to.

[0221] In certain embodiments, s1 in formula (IX-c) is an integer selected from the group consisting of 1, 2, 3, 4, and 5. In certain embodiments, s1 in formula (IX-c) is 1. In certain embodiments, s1 in formula (IX-c) is 2. In certain embodiments, s1 in formula (IX-c) is 3. In certain embodiments, s1 in formula (IX-c) is 4. In certain embodiments, s1 in formula (IX-c) is 5.

[0222] In certain embodiments, -L 1 - is represented by the formula (IX-d)

[0223] [ka] [In the formula, The dashed line with an asterisk indicates the bond of -D to the nitrogen; The unmarked dashed lines are -L 2 -Z] It is of the following.

[0224] In certain embodiments, -D is linked to -L of formula (IX-d) through the nitrogen of the amine functional group of -D, e.g., through the nitrogen of the amine functional group of the lysine side chain of -D. 1 - is bonded to.

[0225] In certain embodiments, -L 1 - has the structure disclosed in WO 2020 / 206358 A1. Thus, in certain embodiments, the moiety -L 1 - is a compound of the formula (X):

[0226] [ka] [In the formula, The unmarked dashed line indicates a bond to -D; The dashed line with an asterisk indicates -L 2 -Z or -L 2 - indicates the bond to Z', n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6; -R 1 and -R 2 are independently an electron withdrawing group, an alkyl, or -H; -R 1 or -R 2 at least one of is an electron withdrawing group, Each -R 4 are independently C1-C3 alkyl or two -R 4 form a 3- to 6-membered ring together with the carbon atom to which they are attached, -Y- is absent when -D is a drug moiety linked through an amine, or -Y- is not absent when -D is a drug moiety linked through a phenol, alcohol, thiol, thiophenol, imidazole, or a non-basic amine, and 6 )CH2-, where -R 6 is an optionally substituted C1-C6 alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl. It is of the following.

[0227] In certain embodiments, -L of formula (X) 1 - 1 part - L 2 Replaced by -Z.

[0228] In certain embodiments, -L of formula (X) 1 - is not further substituted.

[0229] In certain embodiments, n in formula (X) is an integer selected from 1, 2, 3, 4, 5, and 6. In certain embodiments, n in formula (X) is an integer selected from 1, 2, and 3. In certain embodiments, n in formula (X) is an integer from 0, 1, 2, and 3. In certain embodiments, n in formula (X) is 1. In certain embodiments, n in formula (X) is 2. In certain embodiments, n in formula (X) is 3.

[0230] In certain embodiments, -R of formula (X) 1 and -R 2 The electron withdrawing group is -CN, -NO2, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR 3 , -SOR 3 , or -SO2R 3 (where -R 3 is -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl), -OR 8 or -NR 8 2 (where each -R 8 are independently -H or optionally substituted alkyl, or both -R 8 groups taken together with the nitrogen to which they are attached form a heterocyclic ring), or -SR 9 (where -R 9is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

[0231] In certain embodiments, -R of formula (X) 1 and -R 2 In certain embodiments, the electron withdrawing group of formula (X) is -CN. 1 and -R 2 In certain embodiments, the electron withdrawing group of formula (X) is -NO. 1 and -R 2 The electron withdrawing group of formula (X) is an optionally substituted aryl containing 6 to 10 carbons. 1 and -R 2 The electron withdrawing group of formula (X) is optionally substituted phenyl, naphthyl, or anthracenyl. 1 and -R 2 The electron withdrawing group of formula (X) is an optionally substituted heteroaryl containing 3 to 7 carbons and at least one N, O, or S atom. 1 and -R 2 The electron withdrawing group of formula (X) is optionally substituted pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl. 1 and -R 2 is an optionally substituted alkenyl having 2 to 20 carbon atoms. 1 and -R 2 is an optionally substituted alkynyl having 2 to 20 carbon atoms. 1 and -R 2 The electron-withdrawing group is -COR 3 , -SOR 3, or -SO2R 3 (where -R 3 is -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl containing 1 to 20 carbon atoms, -OR 8 or -NR 8 2 (where each -R 8 are independently -H or optionally substituted alkyl containing 1 to 20 carbon atoms, or both -R 8 In certain embodiments, the -R of formula (X) is a heterocyclic ring, wherein the -R groups together with the nitrogen to which they are attached form a heterocyclic ring. 1 and -R 2 The electron-withdrawing group is -SR 9 where -R 9 is an optionally substituted alkyl, an optionally substituted aryl, an optionally substituted arylalkyl, an optionally substituted heteroaryl, or an optionally substituted is heteroarylalkyl.

[0232] In certain embodiments, -R of formula (X) 1 or -R 2 At least one of the following is -CN, -SOR 3 , or -SO2R 3 In certain embodiments, -R of formula (X) 1 and -R 2 At least one of is -CN or -SO2R 3 In certain embodiments, -R of formula (X) 1 and -R 2 At least one of is -CN or -SO2R 3 where -R 3 is an optionally substituted alkyl, an optionally substituted aryl, or -NR 8 In certain embodiments, -R of formula (X) is 2. 1 and -R 2At least one of is -CN, -SO2N(CH3)2, -SO2CH3, phenyl substituted with -SO2, phenyl substituted with -SO2 and -Cl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0233] In certain embodiments, each -R of formula (X) 4 is independently C1-C3 alkyl. In certain embodiments, both -R 4 is methyl.

[0234] In certain embodiments, -Y- in formula (X) is absent. In certain embodiments, -Y- in formula (X) is -N(R 6 )CH2-.

[0235] In certain embodiments, -L 1 - is of formula (X), where n is 1, -R 1 is -CN and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 1, -R 1 is -SO2N(CH3)2, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 1, -R 1 is SO2CH3, -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 1, -R 1 is -SO2N(CH2CH2)2CHCH3, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 1, -R 1is phenyl substituted by -SO2, -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 1, -R 1 is phenyl substituted by -SO2 and -Cl, -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 1, -R 1 is -SO2N(CH2CH2)2O, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 1, -R 1 is -SO2CH(CH3)2, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 1, -R 1 is -SO2N(CH3)(CH2CH3), and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 1, -R 1 is -SO2N(CH2CH2OCH3)2, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 1, -R 1 is phenyl substituted by -SO2 and -CH3, -R 2 is -H and -R 4 is -CH3.

[0236] In certain embodiments, -L 1 - is of formula (X), where n is 2; -R 1 is -CN and -R 2is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 2, -R 1 is -SO2N(CH3)2, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 2, -R 1 is SO2CH3, -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 2, -R 1 is -SO2N(CH2CH2)2CHCH3, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 2, -R 1 is phenyl substituted by -SO2, -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 2, -R 1 is phenyl substituted by -SO2 and -Cl, -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 2, -R 1 is -SO2N(CH2CH2)2O, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 2, -R 1 is -SO2CH(CH3)2, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 2, -R1 is -SO2N(CH3)(CH2CH3), and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 2, -R 1 is -SO2N(CH2CH2OCH3)2, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 2, -R 1 is phenyl substituted by -SO2 and -CH3, -R 2 is -H and -R 4 is -CH3.

[0237] In certain embodiments, -L 1 - is of formula (X), where n is 3; -R 1 is -CN and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 3, -R 1 is -SO2N(CH3)2, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 3, -R 1 is SO2CH3, -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 3, -R 1 is -SO2N(CH2CH2)2CHCH3, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 3, -R 1 is phenyl substituted by -SO2, -R 2is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 3, -R 1 is phenyl substituted by -SO2 and -Cl, -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 3, -R 1 is -SO2N(CH2CH2)2O, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 3, -R 1 is -SO2CH(CH3)2, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 3, -R 1 is -SO2N(CH3)(CH2CH3), and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 3, -R 1 is -SO2N(CH2CH2OCH3)2, and -R 2 is -H and -R 4 is -CH3. In certain embodiments, -L 1 - is of formula (X), where n is 3, -R 1 is phenyl substituted by -SO2 and -CH3, -R 2 is -H and -R 4 is -CH3.

[0238] The terms used in relation to formula (X) only have the following meanings:

[0239] The term "alkyl" refers to a linear, branched, or cyclic saturated hydrocarbon group of 1 to 20, 1 to 12, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. In certain embodiments, the alkyl is linear or branched. Examples of linear or branched alkyl groups include 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 certain embodiments, the alkyl is cyclic. Examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, and cyclohexyl.

[0240] The term "alkoxy" refers to an alkyl group attached to an oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, and cyclobutoxy.

[0241] The term "alkenyl" refers to a non-aromatic unsaturated hydrocarbon having a carbon-carbon double bond and 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0242] The term "alkynyl" refers to a non-aromatic unsaturated hydrocarbon having a carbon-carbon triple bond and 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0243] The term "aryl" refers to aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. The term "heteroaryl" refers to aromatic rings containing 3 to 15 carbons and at least one N, O, or S atom, preferably 3 to 7 carbons and at least one N, O, or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, and indenyl.

[0244] In certain embodiments, the alkenyl, alkynyl, aryl, or heteroaryl moiety may be attached to the remainder of the molecule through an alkyl bond. In these situations, the substituent is referred to as alkenylalkyl, alkynylalkyl, arylalkyl, or heteroarylalkyl, indicating that an alkylene moiety is between the alkenyl, alkynyl, aryl, or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl, or heteroaryl is attached.

[0245] The term "halogen" or "halo" refers to bromo, fluoro, chloro and iodo.

[0246] The term "heterocyclic ring" or "heterocyclyl" refers to a 3- to 15-membered aromatic or non-aromatic ring containing at least one N, O, or S atom. Examples include piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, as well as the exemplary groups provided above for the term "heteroaryl." In certain embodiments, the heterocyclic ring or heterocyclyl is non-aromatic. In certain embodiments, the heterocyclic ring or heterocyclyl is aromatic.

[0247] The term "optionally substituted" refers to a group that may be unsubstituted or substituted with one or more (e.g., 1, 2, 3, 4, or 5) substituents that may be the same or different. Examples of substituents include alkyl, alkenyl, alkynyl, halogen, -CN, -OR aa , -SR aa , -NR aa R bb , -NO2, -C=NH(OR aa ), -C(O)R aa , -OC(O)R aa , -C(O)OR aa , -C(O)NR aa R bb , -OC(O)NR aa R bb , -NR aa C(O)R bb , -NR aaC(O)OR bb , -S(O)R aa , -S(O)2R aa , -NR aa S(O)R bb , -C(O)NR aa S(O)R bb , -NR aa S(O)2R bb , -C(O)NR aa S(O)2R bb , -S(O)NR aa R bb , -S(O)NR aa R bb , -P(O)(OR aa )(OR bb ), heterocyclyl, heteroaryl, or aryl, where alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl are each independently -R cc is optionally replaced by -R aa and -R bb are each independently -H, alkyl, alkenyl, alkynyl, heterocyclyl, heteroaryl, or aryl, or -R aa and -R bb together with the nitrogen atom to which they are attached form a heterocyclyl, which is optionally substituted by alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, or -CN; cc is independently alkyl, alkenyl, alkynyl, halogen, heterocyclyl, heteroaryl, aryl, -CN, or -NO2.

[0248] In certain embodiments, all moieties -L of the conjugate 2 - are identical, provided that there are two or more moieties -L in the conjugate. 2 In certain embodiments, the conjugate of the invention comprises two or more types of -L 2 -, for example, 2, 3, 4 or 5 different moieties -L 2 -, including two or more such types of -L 2 -There is only one type of -L1 -, or two or more types of -L 1 - may be linked to

[0249] In certain embodiments, -L 2 - is a chemical bond. In certain embodiments, -L 2 - is a spacer moiety.

[0250] In certain embodiments, -L 2 - stands for -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-, -S(O)2N(R y1 )-, -S(O)N(R y1 )-, -S(O)2-, -S(O)-, -N(R y1 )S(O)2N(R y1a )-, -S-, -N(R y1 )-, -OC(OR y1 )(R y1a )-, -N(R y1 )C(O)N(R y1a )-, -OC(O)N(R y1 )-, C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 alkynyl, where -T'-, C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 Alkynyl may be one or more -R y2 Optionally replaced by C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 Alkynyl is -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y3 )-, -S(O)2N(R y3 )-, -S(O)N(R y3 )-, -S(O)2-, -S(O)-, -N(R y3 )S(O)2N(R y3a )-, -S-, -N(R y3 )-, -OC(OR y3 )(R y3a )-, -N(Ry3 )C(O)N(R y3a )- and -OC(O)N(R y3 )-, -R y1 and -R y1a -H, -T', C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 alkynyl, wherein -T', C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 Alkynyl may contain one or more of the same or different -R y2 Optionally replaced by C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 Alkynyl is -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y4 )-, -S(O)2N(R y4 )-, -S(O)N(R y4 )-, -S(O)2-, -S(O)-, -N(R y4 )S(O)2N(R y4a )-, -S-, -N(R y4 )-, -OC(OR y4 )(R y4a )-, -N(R y4 )C(O)N(R y4a )- and -OC(O)N(R y4 )-, Each T' is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl, each T' being the same or different and selected from the group consisting of one or more -R y2 and optionally independently replaced by Each -R y2 is halogen, -CN, oxo(=O), -C(O)OR y5 , -OR y5 , -C(O)Ry5 , -C(O)N(R y5 )(R y5a ), -S(O)2N(R y5 )(R y5a ), -S(O)N(R y5 )(R y5a ), -S(O)2R y5 , -S(O)R y5 , -N(R y5 )S(O)2N(R y5 )(R y5a ), -SR y5 , -N(R y5 )(R y5a ), -NO2, -OC(O)R y5 , -N(R y5 )C(O)R y5a , -N(R y5 )S(O)2R y5a , -N(R y5 )S(O)R y5a , -N(R y5 )C(O)OR y5a , -N(R y5 )C(O)N(R y5 )(R y5a ), -OC(O)N(R y5 )(R y5a ), and C 1-6 alkyl, wherein C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each -R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b -H, and C 1-6 alkyl, wherein C 1-6 The alkyl is optionally substituted with one or more halogens, which may be the same or different.

[0251] In certain embodiments, -L 2 - stands for -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-, -S(O)2N(Ry1 )-, -S(O)N(R y1 )-, -S(O)2-, -S(O)-, -N(R y1 )S(O)2N(R y1a )-, -S-, -N(R y1 )-, -OC(OR y1 )(R y1a )-, -N(R y1 )C(O)N(R y1a )-, -OC(O)N(R y1 )-, C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 alkynyl, wherein -T'-, C 1-20 Alkyl, C 2-20 Alkenyl, and C 2-20 Alkynyl may be one or more -R y2 Optionally replaced by C 1-20 Alkyl, C 2-20 Alkenyl, and C 2-20 Alkynyl is -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y3 )-, -S(O)2N(R y3 )-, -S(O)N(R y3 )-, -S(O)2-, -S(O)-, -N(R y3 )S(O)2N(R y3a )-, -S-, -N(R y3 )-, -OC(OR y3 )(R y3a )-, -N(R y3 )C(O)N(R y3a )- and -OC(O)N(R y3 )-, -R y1 and -R y1a -H, -T', C 1-10 Alkyl, C 2-10 Alkenyl, and C 2-10 alkynyl, wherein -T', C 1-10 Alkyl, C 2-10 Alkenyl, and C 2-10 Alkynyl may be one or more -Ry2 Optionally replaced by C 1-10 Alkyl, C 2-10 Alkenyl, and C 2-10 Alkynyl is -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y4 )-, -S(O)2N(R y4 )-, -S(O)N(R y4 )-, -S(O)2-, -S(O)-, -N(R y4 )S(O)2N(R y4a )-, -S-, -N(R y4 )-, -OC(OR y4 )(R y4a )-, -N(R y4 )C(O)N(R y4a )- and -OC(O)N(R y4 )-, Each T' is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl, each T' being the same or different and selected from the group consisting of one or more -R y2 and optionally independently replaced by -R y2 is halogen, -CN, oxo(=O), -C(O)OR y5 , -OR y5 , -C(O)R y5 , -C(O)N(R y5 )(R y5a ), -S(O)2N(R y5 )(R y5a ), -S(O)N(R y5 )(R y5a ), -S(O)2R y5 , -S(O)R y5 , -N(R y5 )S(O)2N(R y5a )(R y5b ), -SR y5 , -N(R y5 )(R y5a ), -NO2, -OC(O)R y5, -N(R y5 )C(O)R y5a , -N(R y5 )S(O)2R y5a , -N(R y5 )S(O)R y5a , -N(R y5 )C(O)OR y5a , -N(R y5 )C(O)N(R y5a )(R y5b ), -OC(O)N(R y5 )(R y5a ), and C 1-6 alkyl, wherein C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each -R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b -H, and C 1-6 alkyl, wherein C 1-6 The alkyl is optionally substituted with one or more halogens which may be the same or different.

[0252] In certain embodiments, -L 2 - stands for -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-, -S(O)2N(R y1 )-, -S(O)N(R y1 )-, -S(O)2-, -S(O)-, -N(R y1 )S(O)2N(R y1a )-, -S-, -N(R y1 )-, -OC(OR y1 )(R y1a )-, -N(R y1 )C(O)N(R y1a )-, -OC(O)N(R y1 )-, C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 alkynyl, wherein -T'-, C 1-50Alkyl, C 2-50 Alkenyl, and C 2-50 Alkynyl may be one or more -R y2 Optionally replaced by C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 Alkynyl is -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y3 )-, -S(O)2N(R y3 )-, -S(O)N(R y3 )-, -S(O)2-, -S(O)-, -N(R y3 )S(O)2N(R y3a )-, -S-, -N(R y3 )-, -OC(OR y3 )(R y3a )-, -N(R y3 )C(O)N(R y3a )- and -OC(O)N(R y3 )-, -R y1 and -R y1a -H, -T', C 1-10 Alkyl, C 2-10 Alkenyl, and C 2-10 alkynyl, Each T' is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 independently selected from the group consisting of cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; Each-R y2 is halogen and C 1-6 alkyl, each -R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b -H and C 1-6 alkyl, wherein C 1-6The alkyl is optionally substituted with one or more halogens, which may be the same or different.

[0253] In certain embodiments, -L 2 - is C 1-20 Alkyl chains, which are -O-, -T'- and -C(O)N(R y1 )-, wherein C is optionally interrupted by one or more groups independently selected from the group consisting of 1-20 The alkyl chains are -OH, -T' and -C(O)N(R y6 R y6a Optionally substituted with one or more groups independently selected from the group consisting of -R y1 , -R y6 , -R y6a H and C 1-4 alkyl, T' is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 It is selected from the group consisting of cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl.

[0254] In certain embodiments, -L 2 - has a molecular weight in the range of 14 g / mol to 750 g / mol.

[0255] In certain embodiments, -L 2 -teeth,

[0256] [ka] [In the formula, The dashed lines are -L 1 -, -L 2 - represents the bond to the remainder of - or to Z; -R and -R aare independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl. The moiety is selected from the group consisting of:

[0257] In certain embodiments, all moieties Z of the conjugate are identical, provided there is more than one moiety Z in the conjugate. In certain embodiments, the conjugates of the invention contain more than one type of Z, e.g., 2, 3, 4 or 5 different moieties Z. Such more than one type of Z may be represented by only one type of -L. 2 -, or two or more types of -L 2 - may be linked to

[0258] In certain embodiments, Z is an optionally substituted fatty acid moiety. In certain embodiments, Z is an optionally substituted fatty acid moiety as disclosed in WO 2005 / 027978 A2 and WO 2014 / 060512 A1.

[0259] In certain embodiments, Z is a polymer moiety, i.e., at least one polymer, such as, for example, 2-methacryloyl-oxyethylphosphoylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymer, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide ... and the moiety comprises a polymer selected from the group consisting of poly(propyl methacrylate), poly(hydroxypropyl oxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic acid-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinyl amine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, cellulose, carbomethylcellulose, hydroxypropyl methylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch, and other carbohydrate based polymers, xylan, and copolymers thereof. In certain embodiments, the polymer portion comprises a PEG-based polymer, hi certain embodiments, the polymer portion comprises a hyaluronic acid-based polymer.

[0260] In certain embodiments, Z is water-insoluble.

[0261] In certain embodiments, Z comprises a hydrogel. Such hydrogels may be degradable or non-degradable, i.e., stable. In certain embodiments, such hydrogels are degradable. In certain embodiments, such hydrogels are non-degradable. In certain embodiments, such hydrogels include 2-methacryloyl-oxyethyl phosphorylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymers, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxypropyl ... The polymers may comprise one or more polymers selected from the group consisting of poly(hydroxypropyl oxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicones, cellulose, carbomethylcellulose, hydroxypropyl methylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch, and other carbohydrate-based polymers, xylan, and copolymers thereof.In certain embodiments, the hydrogel comprises a PEG-based or hyaluronic acid-based hydrogel. In certain embodiments, Z comprises a PEG-based hydrogel. In certain embodiments, Z comprises a hyaluronic acid-based hydrogel.

[0262] In certain embodiments, Z is a hydrogel disclosed in WO2006 / 003014, WO2011 / 012715A1, WO2014 / 056926A1, or WO2020 / 064846A1.

[0263] In certain embodiments, Z is a hydrogel as disclosed in WO2013 / 036847 A1. In particular, in certain embodiments, Z is a hydrogel produced by a method comprising reacting at least a first reactive polymer with a cleavable crosslinker compound, said cleavable crosslinker compound comprising a first functional group -Y that reacts with the first reactive polymer. 1 and further comprising a moiety that is cleaved by elimination under physiological conditions, said moiety comprising a second functional group -Y 2 In certain embodiments, the cleavable crosslinker compound comprises the formula (PL-1):

[0264] [ka] [In the formula, m is 0 or 1; -X is a functional group capable of linking to a reactive polymer that is subject to cleavage under physiological conditions, and the second functional group -Y 2 Including, -R 1 , -R 2 and -R 5 At least one of the first functional group -Y 1 Including, -R 1 and -R 2 one, and only one, of is selected from the group consisting of -H, alkyl, arylalkyl, and heteroarylalkyl; Optionally, -R1 and -R 2 may be bonded to form a 3- to 8-membered ring, -R 1 and -R 2 At least one or both of the groups are -CN, -NO2, aryl, heteroaryl, alkenyl, alkynyl, -COR 3 , -SOR 3 , -SO2R 3 , and -SR 4 are independently selected from the group consisting of: -R 3 is -H, alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -OR 9 , and -NR 9 2, -R 4 is selected from the group consisting of alkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; Each -R 5 is -H, alkyl, alkenylalkyl, alkynylalkyl, p is an integer ranging from 1 to 1000 (OCH2CH2) p is independently selected from the group consisting of O-alkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; Each -R 9 is independently selected from the group consisting of -H and alkyl, or both -R 9 together with the nitrogen to which they are attached form a heterocyclic ring, The moiety of formula (PL-1) may be further substituted. It is of the following.

[0265] In certain embodiments, -X in formula (PL-1) is selected from succinimidyl carbonate, sulfosuccinimidyl carbonate halide, thioether, ester, nitrophenyl carbonate, chloroformate, fluoroformate, optionally substituted phenol, and formula (PL-2):

[0266] [ka] [In the formula, The dashed line indicates the bond to the remainder of formula (PL-1); -T*- is -O-, -S-, and -NR 6 - selected from the group consisting of z is an integer selected from the group consisting of 1, 2, 3, 4, 5, and 6; -X'- is absent or -OR 7 -and-SR 7 - selected from the group consisting of -Y 2 is a functional group that can be linked to a reactive polymer, -R 6 is selected from the group consisting of -H, alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; -R 7 is alkylene, phenylene, and p is an integer ranging from 1 to 1000 (OCH2CH2) p selected from the group consisting of is selected from the group consisting of:

[0267] In certain embodiments, -X of formula (PL-1) comprises an activated carbonate, such as succinimidyl carbonate, sulfosuccinimidyl carbonate, or nitrophenyl carbonate. In certain embodiments, -X of formula (PL-1) comprises a carbonyl halide, such as O(C=O)Cl or O(C=O)F. In certain embodiments, -X of formula (PL-1) has the formula (PL-2). In certain embodiments, -X of formula (PL-1) has the formula OR 7 or SR 7 where R 7 is an optionally substituted alkylene, an optionally substituted phenylene or (OCH2CH2) p and p is 1 to 1000.

[0268] In certain embodiments, p in formula (PL-2) is an integer in the range of 1 to 100. In certain embodiments, p in formula (PL-2) is an integer in the range of 1 to 10.

[0269] In certain embodiments, -Y in formula (PL-1) 1 and -Y in formula (PL-2) 2 are, independently, N3, NH2, and NH-CO2 t Bu, S.H., S. t Bu, Maleimide, CO2H, CO2 t Bu, 1,3-diene, cyclopentadiene, furan, alkyne, cyclooctyne, acrylate, or acrylamide, t Bu is tert-butyl, -Y 1 or -Y 2 contains N3, the other does not contain an alkyne or cyclooctyne; -Y 1 or -Y 2 contains SH, the other does not contain maleimide, acrylate, or acrylamide; -Y 1 or -Y 2 If one of them contains NH2, the other does not contain CO2H, and -Y 1 or -Y 2 When one of them contains a 1,3-diene or a cyclopentadiene, the other does not contain furan.

[0270] In certain embodiments, the cleavable crosslinker compound has the formula (PL-3):

[0271] [ka] [In the formula, m is 0 or 1; n is an integer selected from 1 to 1000; s is 0, 1 or 2; t is selected from the group consisting of 2, 4, 8, 16, and 32; -W- stands for -O(C=O)O-, -O(C=O)NH-, -O(C=O)S-, -O(C=O)NR 6 CH2O- and -O(C=O)NR 6 S-, -Q is a core group having a valence = t and links the arms of the cleavable crosslinking compound; t is an integer selected from 2, 4, 8, 16, and 32; -R 1 , -R 2 , and -R 5 is defined as in formula (PL-1). It is of the following.

[0272] In certain embodiments, t in formula (PL-3) is 2. In certain embodiments, t in formula (PL-3) is 4. In certain embodiments, t in formula (PL-3) is 8. In certain embodiments, t in formula (PL-3) is 16. In certain embodiments, t in formula (PL-3) is 32.

[0273] In certain embodiments, -Q in formula (PL-3) is

[0274] [ka] having a structure selected from the group consisting of: where the dashed line indicates the bond to the remainder of the cleavable crosslinker compound.

[0275] In certain embodiments, -Q of formula (PL-3) has the structure (PL-3-i). In certain embodiments, -Q of formula (PL-3) has the structure (PL-3-ii). In certain embodiments, -Q of formula (PL-3) has the structure (PL-3-iii).

[0276] In certain embodiments, the cleavable crosslinker compound is of formula (PL-3), where m is 0, n is approximately 100, s is 0, t is 4, -W- is -O(C=O)NH-, -Q has the structure of (PL-3i), and -R 2 is H, while -R 5 is -H and the other is -R 5 is (CH2)5N3, -R 1is (4-chlorophenyl)SO2, phenyl substituted with -SO2, morpholino-SO2, or -CN.

[0277] In certain embodiments, -Y in formula (PL-3) 1 are N3, NH2, and NH-CO2 t Bu, S.H., S. t Bu, Maleimide, CO2H, CO2 t Bu, 1,3-diene, cyclopentadiene, furan, alkyne, cyclooctyne, acrylate, or acrylamide, t Bu is tert-butyl.

[0278] In certain embodiments, -Y 1 and -Y in formula (PL-2) 2 are, independently, N3, NH2, and NH-CO2 t Bu, S.H., S. t Bu, Maleimide, CO2H, CO2 t Bu, 1,3-diene, cyclopentadiene, furan, alkyne, cyclooctyne, acrylate, or acrylamide.

[0279] In certain embodiments, -Y 1 and -Y 2 One of -Y is an azide and the other is a reactive functional group selected from the group consisting of acetylene, cyclooctyne, and maleimide. 1 and -Y 2 One of -Y is a thiol and the other is a reactive functional group selected from the group consisting of maleimide, acrylate, acrylamide, vinylsulfone, vinylsulfonamide, and halocarbonyl. 1 and -Y 2 One of -Y is an amine and the other is a selectively reactive functional group selected from a carboxylic acid and an activated carboxylic acid. 1 and -Y 2One of the groups is a maleimide and the other is a selectively reactive functional group selected from the group consisting of 1,3-dienes, cyclopentadienes, and furans.

[0280] In certain embodiments, the first polymer and any second polymer may include suitable reactive functional groups or may be represented by the formula [Y 3 -(CH2) s (CH2CH2O) n ] t Q, where -Y 3 is a reactive functional group, s is 0, 1, or 2, n is an integer selected from the group ranging from 10 to 1000, -Q is a core group having a valence t, where t is an integer selected from the group consisting of 2, 4, 8, 16, and 32), homopolymeric or copolymeric polyethylene glycol, polypropylene glycol, poly(N-vinylpyrrolidone), polymethacrylate, polyphosphazene, polylactide, polyacrylamide, polyglycolate, polyethyleneimine, agarose, dextran, gelatin, collagen, polylysine, chitosan, alginate, hyaluronan, pectin, and carrageenan.

[0281] In certain embodiments, the first polymer comprises a multi-arm polymer. In certain embodiments, the first polymer comprises at least three arms. In certain embodiments, the first polymer comprises at least four arms. In certain embodiments, the first polymer comprises at least five arms. In certain embodiments, the first polymer comprises at least six arms. In certain embodiments, the first polymer comprises at least seven arms. In certain embodiments, the first polymer comprises at least eight arms.

[0282] In certain embodiments, the second polymer comprises a multi-arm polymer. In certain embodiments, the second polymer comprises at least three arms. In certain embodiments, the second polymer comprises at least four arms. In certain embodiments, the second polymer comprises at least five arms. In certain embodiments, the second polymer comprises at least six arms. In certain embodiments, the second polymer comprises at least seven arms. In certain embodiments, the second polymer comprises at least eight arms.

[0283] In certain embodiments, the first polymer comprises a 2-arm polyethylene glycol polymer. In certain embodiments, the first polymer comprises a 4-arm polyethylene glycol polymer. In certain embodiments, the first polymer comprises an 8-arm polyethylene glycol polymer. In certain embodiments, the first polymer comprises a 16-arm polyethylene glycol polymer. In certain embodiments, the first polymer comprises a 32-arm polyethylene glycol polymer.

[0284] In certain embodiments, the second polymer comprises a 2-arm polyethylene glycol polymer. In certain embodiments, the second polymer comprises a 4-arm polyethylene glycol polymer. In certain embodiments, the second polymer comprises an 8-arm polyethylene glycol polymer. In certain embodiments, the second polymer comprises a 16-arm polyethylene glycol polymer. In certain embodiments, the second polymer comprises a 32-arm polyethylene glycol polymer.

[0285] In certain embodiments, the first and second reactive polymers are reacted sequentially or simultaneously with the cleavable crosslinker compound.

[0286] In certain embodiments, the first functional group and the second functional group are the same.

[0287] The terms used solely in connection with formulae (PL-1), (PL-2) and (PL-3) have the following meanings:

[0288] The term "moiety cleavable by elimination under physiological conditions" refers to the group HC-(CH=CH) m -C-X' (where m is 0 or 1 and X' is a leaving group) and the above elimination reaction removing the HX' element can occur at a rate such that the half-life of the reaction is 1-10,000 hours under physiological conditions of pH and temperature. Preferably, the half-life of the reaction is 1-5,000 hours, more preferably 1-1,000 hours under physiological conditions of pH and temperature. Physiological conditions of pH and temperature refer to a pH of 7-8 and a temperature of 30-40°C.

[0289] The term "reactive polymers and oligomers" refers to polymers or oligomers that contain functional groups that are reactive towards other functional groups, most preferably under mild conditions compatible with the stability requirements of peptides, proteins, and other biomolecules. Suitable functional groups found in reactive polymers include maleimides, thiols or protected thiols, alcohols, acrylates, acrylamides, amines or protected amines, carboxylic acids or protected carboxylic acids, azides, alkynes including cycloalkynes, 1,3-dienes including cyclopentadiene and furan, α-halocarbonyls, and N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, or nitrophenyl esters or carbonates.

[0290] The term "functional group that can be linked to a reactive polymer" refers to a functional group that reacts with a corresponding functional group of a reactive polymer to form a covalent bond to the polymer. Suitable functional groups that can be linked to a reactive polymer include maleimide, thiol or protected thiol, acrylate, acrylamide, amine or protected amine, carboxylic acid or protected carboxylic acid, azide, alkyne including cycloalkyne, 1,3-dienes including cyclopentadiene and furan, α-halocarbonyl, and N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, or nitrophenyl ester or carbonate.

[0291] The term "substituted" refers to an alkyl, alkenyl, alkynyl, aryl, or heteroaryl group that contains one or more substituents in place of one or more hydrogen atoms. Substituents are generally halogen (including F, CI, Br, and I), lower alkyl (including linear, branched, and cyclic), lower haloalkyl (including fluoroalkyl, chloroalkyl, bromoalkyl, and iodoalkyl), OH, lower alkoxy (including linear, branched, and cyclic), SH, lower alkylthio (including linear, branched, and cyclic), amino, alkylamino, dialkylamino, silyl (including alkylsilyl, alkoxysilyl, and arylsilyl), nitro, cyano, carbonyl, carboxylic acid, carboxylic acid ester, carboxylic acid amide, aminocarbonyl, aminoacyl, carbamate, urea, thiocarbamate, may be selected from thiourea, ketone, sulfone, sulfonamide, aryl (including phenyl, naphthyl, and anthracenyl), heteroaryl (including 5-membered heteroaryl such as pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole, and tetrazole, 6-membered heteroaryl such as pyridine, pyrimidine, pyrazine, and fused heteroaryl such as benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole, and benzisothiazole).

[0292] R 1 and R 2 The properties of R can be adjusted by the optional addition of electron donating or electron withdrawing substituents. 1 R 2means a substituent that results in a decrease in the acidity of CH, where electron donating groups are typically associated with negative Hammett σ or Taft σ* constants and are well known in the art of physical organic chemistry (Hammett constants refer to aryl / heteroaryl substituents and Taft constants refer to substituents on non-aromatic moieties). Examples of suitable electron donating substituents include lower alkyl, lower alkoxy, lower alkylthio, amino, alkylamino, dialkylamino, and silyl.

[0293] The term "electron-withdrawing group" refers to R 1 R 2 Refers to a substituent that results in an increase in the acidity of the CH group; electron-withdrawing groups are typically associated with positive Hammett σ or Taft σ* constants and are well known in the art of physical organic chemistry. Examples of suitable electron-withdrawing substituents include halogen, difluoromethyl, trifluoromethyl, nitro, cyano, C(=O)-R x where -R x is H, lower alkyl, lower alkoxy, or amino, or S(O) m R Y m is 1 or 2; -R Y is lower alkyl, aryl, or heteroaryl. As is well known in the art, the electronic influence of a substituent can depend on the position of the substituent. For example, an alkoxy substituent at the ortho or para position of an aryl ring is electron donating and is characterized by a negative Hammett σ constant, while an alkoxy substituent at the meta position of an aryl ring is electron withdrawing and is characterized by a positive Hammett σ constant.

[0294] The terms "alkyl," "alkenyl," and "alkynyl" include straight-chain, branched, or cyclic hydrocarbon groups of 1 to 8 carbons, or 1 to 6 carbons, or 1 to 4 carbons, where alkyl is a saturated hydrocarbon, alkenyl contains one or more carbon-carbon double bonds, and alkynyl contains one or more carbon-carbon triple bonds. Unless otherwise specified, they contain 1 to 6 carbons.

[0295] The term "aryl" includes aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. "Heteroaryl" includes aromatic rings containing 3 to 15 carbons and at least one N, O, or S atom, preferably 3 to 7 carbons and 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.

[0296] The term "halogen" includes fluoro, chloro, bromo and iodo.

[0297] The term "maleimide" means a compound of the formula

[0298] [ka] is the basis.

[0299] In certain embodiments, Z is a hydrogel as disclosed in WO 2020 / 206358 A1. In particular, in certain embodiments, Z is (a) Multiarm polymer-P 2 providing a first prepolymer comprising the formula (PL-4):

[0300] [ka] [In the formula, n is an integer selected from 0, 1, 2, 3, 4, 5, and 6; r is an integer greater than 2, -Y is a reactive functional group for linking the first prepolymer to a second prepolymer; -R 1 and -R 2 are independently an electron withdrawing group, an alkyl, or -H, where -R 1 and -R2 at least one of is an electron-withdrawing group; Each -R 4 are independently C1-C3 alkyl, or two -R 4 form a 3- to 6-membered ring together with the carbon atom to which they are attached, -W- is absent or

[0301] [ka] (In the formula, The dashed line with an asterisk indicates a bond to -NH-, and the unmarked dashed line indicates a bond to -P 2 indicates a bond to each of x, y, and z is independently an integer selected from 0, 1, 2, 3, 4, 5, and 6; -B' is -NH, -ONH, a ketone, an aldehyde, -SH, -OH, -COH, a carboxamide group, or a group containing a cyclooctyne or bicyclononyne; -C* is a carboxamide, a thioether, a thiosuccinimidyl, a triazole, or an oxime. is] The steps are: (b) Multiarm polymer-P 1 wherein each arm is terminated by a reactive functional group -Y″ that reacts with -Y of step (a); (c) mixing the two prepolymers of steps (a) and (b) under conditions in which -Y and -Y″ react to form a bond -Y*-, and optionally (d) isolating the resulting hydrogel; and (b) providing a hydrogel comprising:

[0302] Thus, Z is a hydrogel obtainable from the above method. In certain embodiments, the hydrogels produced by the aforementioned methods are degradable.

[0303] In certain embodiments, -Y and -Y″ react under step (c) to give a compound of formula (PL-4′):

[0304] [ka] wherein n, r, -P 1 , -Y*-, -R 4 , -R 1 , -R 2 , -W-, and -P 2 is as defined above.

[0305] In certain embodiments, n in formula (PL-4) or (PL-4') is an integer selected from 1, 2, 3, 4, 5, and 6. In certain embodiments, n in formula (PL-4) or (PL-4') is an integer selected from 1, 2, and 3. In certain embodiments, n in formula (PL-4) or (PL-4') is an integer selected from 0, 1, 2, and 3. In certain embodiments, n in formula (PL-4) or (PL-4') is 1. In certain embodiments, n in formula (PL-4) is 2. In certain embodiments, n in formula (PL-4) or (PL-4') is 3.

[0306] In certain embodiments, the multi-arm-P of formula (PL-4) or (PL-4′) 2 is an r-arm polymer, where r is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In certain embodiments, r in formula (PL-4) or (PL-4') is an integer selected from 2, 3, 4, 5, 6, 7, and 8. In certain embodiments, r in formula (PL-4) or (PL-4') is an integer selected from 2, 4, 6, and 8. In certain embodiments, r in formula (PL-4) or (PL-4') is 2. In certain embodiments, r in formula (PL-4) or (PL-4') is 4. In certain embodiments, r in formula (PL-4) or (PL-4') is 6. In certain embodiments, r in formula (PL-4) or (PL-4') is 8.

[0307] In certain embodiments, -P of formula (PL-4) or (PL-4') 2 In certain embodiments, -P of formula (PL-4) or (PL-4') has a molecular weight of at least 1 kDa. 2 In certain embodiments, -P of formula (PL-4) or (PL-4') has a molecular weight of 1 to 100 kDa. 2 In certain embodiments, -P of formula (PL-4) or (PL-4') has a molecular weight of 1 to 80 kDa. 2 In certain embodiments, -P of formula (PL-4) or (PL-4') has a molecular weight of 1 to 60 kDa. 2 In certain embodiments, -P of formula (PL-4) or (PL-4') has a molecular weight of 1 to 40 kDa. 2 In certain embodiments, -P of formula (PL-4) or (PL-4') has a molecular weight of 1 to 20 kDa. 2 In certain embodiments, -P of formula (PL-4) or (PL-4') has a molecular weight of 1 to 10 kDa. 2 In certain embodiments, -P of formula (PL-4) or (PL-4') has a molecular weight of 1 to 5 kDa. 2 In certain embodiments, -P of formula (PL-4) or (PL-4') has a molecular weight of about 20 kDa. 2 In certain embodiments, -P of formula (PL-4) or (PL-4') has a molecular weight of about 40 kDa. 2 In certain embodiments, -P of formula (PL-4) or (PL-4') has a molecular weight of about 60 kDa. 2 has a molecular weight of approximately 80 kDa.

[0308] In certain embodiments, the multi-arm polymer-P in step (b) 1 is an r-arm polymer, where r is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In certain embodiments, the multi-arm-P 1is an r-arm polymer, where r is an integer selected from 2, 3, 4, 5, 6, 7, and 8. In certain embodiments, the multi-arm-P 1 is an r arm polymer, where r is an integer selected from 2, 4, 6, and 8. In certain embodiments, the multi-arm-P 1 is an r-arm polymer, where r is 2. In certain embodiments, the multi-arm-P 1 is an r-arm polymer, where r is 4. In certain embodiments, the multi-arm-P 1 is an r-arm polymer, where r is 6. In certain embodiments, the multi-arm-P 1 is an r-arm polymer, where r is 8.

[0309] In certain embodiments, in step (b)-P 1 has a molecular weight of at least 1 kDa. In certain embodiments, the multi-arm polymer-P 1 In certain embodiments, the multi-arm polymer-P in step (b) has a molecular weight of 1 to 100 kDa. 1 In certain embodiments, the multi-arm polymer-P in step (b) has a molecular weight of 1 to 80 kDa. 1 In certain embodiments, the multi-arm polymer-P in step (b) has a molecular weight of 1 to 60 kDa. 1 In certain embodiments, the multi-arm polymer-P in step (b) has a molecular weight of 1 to 40 kDa. 1 In certain embodiments, the multi-arm polymer-P in step (b) has a molecular weight of 1 to 20 kDa. 1 In certain embodiments, the multi-arm polymer-P in step (b) has a molecular weight of 1 to 10 kDa. 1 In certain embodiments, the multi-arm polymer-P in step (b) has a molecular weight of 1 to 5 kDa. 1In certain embodiments, the multi-arm polymer-P in step (b) has a molecular weight of about 20 kDa. 1 In certain embodiments, the multi-arm polymer-P in step (b) has a molecular weight of about 40 kDa. 1 In certain embodiments, the multi-arm polymer-P in step (b) has a molecular weight of about 60 kDa. 1 has a molecular weight of approximately 80 kDa.

[0310] In certain embodiments, in step (b)-P 1 and -P of formula (PL-4) or (PL-4') 2 comprises poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(ethyleneimine) (PEI), dextran, hyaluronic acid, or copolymers thereof. In certain embodiments, the -P in step (b) 1 and P of formula (PL-4) or (PL-4') 2 is a PEG-based polymer. In certain embodiments, -P in step (b) 1 and -P of formula (PL-4) or (PL-4') 2 is a hyaluronic acid based polymer.

[0311] In certain embodiments, -R of formula (PL-4) or (PL-4') 1 and -R 2 are independently an electron withdrawing group, an alkyl, or -H; -R 1 and -R 2 At least one of the groups is an electron withdrawing group.

[0312] In certain embodiments, -R of formula (PL-4) or (PL-4') 1 and -R 2 The electron withdrawing group is -CN, -NO2, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -COR 3 , -SOR 3 or -SO2R 3 (where -R3 is -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl), -OR 8 or -NR 8 2 (where each -R 8 are independently -H or optionally substituted alkyl, or both -R 8 groups taken together with the nitrogen to which they are attached form a heterocyclic ring), or -SR 9 (where -R 9 is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

[0313] In certain embodiments, -R of formula (PL-4) or (PL-4') 1 and -R 2 In certain embodiments, the electron withdrawing group of formula (PL-4) or (PL-4′) is —CN. 1 and -R 2 In certain embodiments, the electron withdrawing group of formula (PL-4) or (PL-4′) is —NO. 1 and -R 2 In certain embodiments, the electron withdrawing group of formula (PL-4) or (PL-4′) is an optionally substituted aryl having 6 to 10 carbons. 1 and -R 2 The electron withdrawing group of formula (PL-4) or (PL-4′) is optionally substituted phenyl, naphthyl, or anthracenyl. 1 and -R 2 The electron withdrawing group of formula (PL-4) or (PL-4') is an optionally substituted heteroaryl containing 3 to 7 carbons and at least one N, O, or S atom. 1 and -R 2The electron withdrawing group of formula (PL-4) or (PL-4') is optionally substituted pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl. 1 and -R 2 In certain embodiments, the electron withdrawing group of formula (PL-4) or (PL-4′) is an optionally substituted alkenyl containing 2 to 20 carbon atoms. 1 and -R 2 In certain embodiments, the electron withdrawing group of formula (PL-4) or (PL-4′) is an optionally substituted alkynyl containing 2 to 20 carbon atoms. 1 and -R 2 The electron-withdrawing group 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 8 or -NR 8 2, where each -R 8 are independently -H or optionally substituted alkyl containing 1 to 20 carbon atoms, or both -R 8 The -R groups together with the nitrogen to which they are attached form a heterocyclic ring. In certain embodiments, -R of formula (PL-4) or (PL-4') 1 and -R 2 The electron-withdrawing group is -SR 9 where -R 9 is an optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl containing 1 to 20 carbon atoms. 1 and -R2 At least one of the groups is -CN or -SO2R 3 It is.

[0314] In certain embodiments, -R of formula (PL-4) or (PL-4') 1 and -R 2 At least one of the following is -CN, -SOR 3 or -SO2R 3 In certain embodiments, -R of formula (PL-4) or (PL-4') is 1 and -R 2 At least one of the groups is -CN or -SO2R 3 In certain embodiments, -R of formula (PL-4) or (PL-4') is 1 and -R 2 At least one of the groups is -CN or -SO2R 3 where -R 3 is an optionally substituted alkyl, an optionally substituted aryl, or -NR 8 In certain embodiments, -R of formula (PL-4) or (PL-4') is 1 and -R 2 At least one of is -CN, -SO2N(CH3)2, -SO2CH3, phenyl substituted with -SO2, phenyl substituted with -SO2 and -Cl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0315] In certain embodiments, -R in each of formula (PL-4) or (PL-4') 4 are independently C1-C3 alkyl or may be joined together to form a 3-6 membered ring. In certain embodiments, each -R in formula (PL-4) or (PL-4') 4 is independently C1-C3 alkyl. In certain embodiments, -R of formula (PL-4) or (PL-4') 4 are both methyl.

[0316] In certain embodiments, -Y and -Y″ are independently selected from the group consisting of amine, aminooxy, ketone, aldehyde, maleimidyl, thiol, alcohol, azide, 1,2,4,6-tetrazinyl, trans-cyclooctenyl, bicyclononynyl, cyclooctynyl, and protected variants thereof.

[0317] In certain embodiments, Y and Y" may react with each other, e.g., selectively. For example, when -Y is an amine, -Y" is a carboxylic acid, an active ester, or an active carbonate, resulting in a residual linking functionality -Y*- that is an amide or a carbamate. As another example, when -Y is an azide, -Y" is an alkynyl, a bicyclononynyl, or a cyclooctynyl, resulting in a residual linking functionality -Y*- that is a 1,2,3-triazole. As another example, when -Y is NHO, -Y" is a ketone or an aldehyde, resulting in a residual linking functionality -Y*- that is an oxime. As another example, when -Y is SH, -Y" is a maleimide or a halocarbonyl, resulting in a residual linking functionality -Y*- that is a thiosuccinimidyl or a thioether. Similarly, these roles of -Y and -Y" can be reversed to result in -Y*- in the opposite orientation.

[0318] In certain embodiments, -Y*- comprises an amide, an oxime, a 1,2,3-triazole, a thioether, a thiosuccinimide, or an ether. 2 -It is.

[0319] These conjugation reactions can be carried out under conditions known in the art; for example, when -Y is an azide and -Y" is a cyclooctyne, the conjugation can occur in any solvent in which both components have suitable solubility, although aqueous solutions are known to exhibit more favorable reaction kinetics. When mixed in a suitable solvent, typically an aqueous buffer with a pH of 2-7 when -Y and -Y" are azides / cyclooctynes, or 6-9 when -Y and -Y" are activated esters and amines, the -Y and -Y" groups react to form an insoluble hydrogel matrix containing crosslinks of formula (PL-4'). This process can be carried out in bulk phase or under emulsification conditions in a mixed organic / aqueous system to form particulate suspensions, e.g., microspheres, suitable for injection.

[0320] In certain embodiments, the conjugate comprising hydrogel Z is (a) providing a first prepolymer of formula (PL-4); Formula (b) (PL-5)

[0321] [ka] [In the formula, n, -R 1 , -R 2 , -R 4 and -Y are as defined in formula (PL-4), -D is a drug moiety, -X- is absent when -D is a drug moiety linked through an amine, or -X- is replaced by -N(R 6 )CH2-, where -R 6 is an optionally substituted C1-C6 alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl. reacting the prepolymer of formula (PL-4) with a linker drug of formula (PL-5) such that -Y reacts with -B' of formula (PL-4); (c) Multiarm polymer-P 1 wherein each arm is terminated with a reactive functional group -Y″ that reacts with -Y of step (a), and -P 1 The embodiments are as described above, (d) mixing the two prepolymers of steps (a) and (b) under conditions in which -Y and -Y″ react to form a residual linking functional group -Y*-; and, optionally, (e) isolating the resulting hydrogel; The method includes:

[0322] In certain embodiments, the conjugate is obtained by a method comprising the step of reacting a hydrogel Z with a linker drug of formula (PL-5), where -B' on the hydrogel Z reacts with -Y of formula (PL-5).

[0323] The terms used solely in connection with formulae (PL-4), (PL-4') and (PL-5) have the following meanings:

[0324] The term "alkyl" refers to a linear, branched, or cyclic saturated hydrocarbon group of 1 to 20, 1 to 12, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. In certain embodiments, the alkyl is linear or branched. Examples of linear or branched alkyl groups include 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 certain embodiments, the alkyl is cyclic. Examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, and cyclohexyl.

[0325] The term "alkoxy" refers to an alkyl group attached to an oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, and cyclobutoxy.

[0326] The term "alkenyl" refers to a non-aromatic unsaturated hydrocarbon having a carbon-carbon double bond and 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0327] The term "alkynyl" refers to a non-aromatic unsaturated hydrocarbon having a carbon-carbon triple bond and 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0328] The term "aryl" refers to aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. The term "heteroaryl" refers to aromatic rings containing 3 to 15 carbons and at least one N, O, or S atom, preferably 3 to 7 carbons and at least one N, O, or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, and indenyl.

[0329] In certain embodiments, the alkenyl, alkynyl, aryl, or heteroaryl moiety may be attached to the remainder of the molecule through an alkyl bond. In these situations, the substituent is referred to as alkenylalkyl, alkynylalkyl, arylalkyl, or heteroarylalkyl, indicating that an alkylene moiety is between the alkenyl, alkynyl, aryl, or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl, or heteroaryl is attached.

[0330] The term "halogen" or "halo" refers to bromo, fluoro, chloro and iodo.

[0331] The term "heterocyclic ring" or "heterocyclyl" refers to a 3- to 15-membered aromatic or non-aromatic ring containing at least one N, O, or S atom. Examples include piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, as well as the exemplary groups provided above for the term "heteroaryl." In certain embodiments, the heterocyclic ring or heterocyclyl is non-aromatic. In certain embodiments, the heterocyclic ring or heterocyclyl is aromatic.

[0332] The term "optionally substituted" refers to a group that may be unsubstituted or substituted with one or more (e.g., 1, 2, 3, 4, or 5) substituents that may be the same or different. Examples of substituents include alkyl, alkenyl, alkynyl, halogen, -CN, -OR aa , -SR aa , -NR aa R bb , -NO2, -C=NH(OR aa ), -C(O)R aa , -OC(O)R aa , -C(O)OR aa , -C(O)NR aa R bb , -OC(O)NR aa R bb , -NR aa C(O)R bb , -NR aa C(O)OR bb , -S(O)R aa , -S(O)2R aa , -NR aa S(O)R bb , -C(O)NR aa S(O)R bb , -NR aa S(O)2R bb , -C(O)NR aa S(O)2R bb , -S(O)NR aa R bb , -S(O)NR aa R bb , -P(O)(OR aa )(OR bb), heterocyclyl, heteroaryl, or aryl, where alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl are each independently -R cc is optionally replaced by -R aa and -R bb are each independently -H, alkyl, alkenyl, alkynyl, heterocyclyl, heteroaryl, or aryl, or -R aa and -R bb together with the nitrogen atom to which they are attached form a heterocyclyl, which is optionally substituted by alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, or -CN; cc is independently alkyl, alkenyl, alkynyl, halogen, heterocyclyl, heteroaryl, aryl, -CN, or -NO2.

[0333] In certain embodiments, Z is a hyaluronic acid-based hydrogel as disclosed in WO2018 / 175788A1. In certain embodiments, Z is a hyaluronic acid-based hydrogel as disclosed in WO2020 / 064847A1.

[0334] In certain embodiments, Z is a water soluble polymer moiety.

[0335] In certain embodiments, Z comprises a polymer selected from the group consisting of linear, branched, dendritic, multi-armed or cyclic polymers or combinations thereof. In certain embodiments, Z comprises a linear polymer. In certain embodiments, Z is a branched PEG-based polymer. In certain embodiments, Z is a branched PEG-based polymer having 1, 2, 3, 4, 5 or 6 branch points. In certain embodiments, Z is a branched PEG-based polymer having 1, 2 or 3 branch points. In certain embodiments, Z is a branched PEG-based polymer having 1 branch point. In certain embodiments, Z is a branched PEG-based polymer having 2 branch points. In certain embodiments, Z is a branched PEG-based polymer having 3 branch points. In certain embodiments, Z is a branched PEG-based polymer having 4 branch points. In certain embodiments, the branch points are selected from the group consisting of -N<, -CH< and >C<. In certain embodiments, Z comprises a dendritic polymer. In certain embodiments, Z comprises a multi-arm polymer, for example, a polymer comprising 3, 4, 5, 6, 7, or 8 arms. In certain embodiments, Z comprises a multi-arm polymer comprising 3 arms. In certain embodiments, Z comprises a multi-arm polymer comprising 4 arms. In certain embodiments, Z comprises a multi-arm polymer comprising 5 arms. In certain embodiments, Z comprises a multi-arm polymer comprising 6 arms. In certain embodiments, Z comprises a multi-arm polymer comprising 7 arms. In certain embodiments, Z comprises a multi-arm polymer comprising 8 arms. In certain embodiments, Z comprises a cyclic polymer. In certain embodiments, Z comprises a polymer comprising any combination of polymers selected from the group consisting of linear, branched, dendritic, multi-arm, or cyclic polymers.

[0336] When Z is a water-soluble polymer moiety, such a polymer moiety may have a molecular weight in the range of 1 kDa to 1000 kDa, inclusive. In certain embodiments, Z has a molecular weight in the range of 5 kDa to 1000 kDa, inclusive. In certain embodiments, Z has a molecular weight in the range of 6 kDa to 500 kDa, inclusive. In certain embodiments, Z has a molecular weight in the range of 7 kDa to 250 kDa, inclusive. In certain embodiments, Z has a molecular weight in the range of 10 kDa to 150 kDa, inclusive. In certain embodiments, Z has a molecular weight in the range of 12 kDa to 100 kDa, inclusive. In certain embodiments, Z has a molecular weight in the range of 15 kDa to 80 kDa, inclusive. In certain embodiments, Z has a molecular weight in the range of 10 kDa to 80 kDa, inclusive. In certain embodiments, Z has a molecular weight of about 80 kDa. In certain embodiments, Z has a molecular weight of about 70 kDa. In certain embodiments, Z has a molecular weight of about 60 kDa. In certain embodiments, Z has a molecular weight of about 50 kDa. In certain embodiments, Z has a molecular weight of about 40 kDa. In certain embodiments, Z has a molecular weight of about 30 kDa. In certain embodiments, Z has a molecular weight of about 20 kDa. In certain embodiments, Z has a molecular weight of about 10 kDa. In certain embodiments, Z has a molecular weight of about 5 kDa.

[0337] In certain embodiments, Z is 2-methacryloyl-oxyethyl phosphoylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymer, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxypropyl methacrylate), poly(hydrogen). and the water soluble polymeric portion comprises a polymer selected from the group consisting of poly(hydroxypropyl oxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic acid-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, cellulose, carbomethylcellulose, hydroxypropyl methylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch, and other carbohydrate based polymers, xylan, and copolymers thereof.

[0338] In certain embodiments, Z is a water soluble polymer moiety that comprises a protein, such as a protein selected from the group consisting of the carboxyl terminal peptide of chorionic gonadotropin described in US 2012 / 0035101 A1, albumin, the XTEN sequences described in WO 2011123813 A2, the proline / alanine random coil sequences described in WO 2011 / 144756 A1, the proline / alanine / serine random coil sequences described in WO 2008 / 155134 A1 and WO 2013 / 024049 A1, and Fc-fusion proteins.

[0339] In certain embodiments, Z comprises a random coil protein portion in which at least 80%, in certain embodiments at least 85%, in certain embodiments at least 90%, in certain embodiments at least 95%, in certain embodiments at least 98%, and in certain embodiments at least 99% of the total number of amino acids forming the random coil protein portion are selected from alanine and proline. In certain embodiments, at least 10% but less than 75%, and in certain embodiments less than 65% of the total number of amino acid residues in such a random coil protein portion are proline residues. In certain embodiments, such a random coil protein portion is as described in WO 2011 / 144756 A1. In certain embodiments, Z comprises at least one portion selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:51, and SEQ ID NO:61, as disclosed in WO2011 / 144756, which is incorporated herein by reference. Such a random coil protein-containing moiety containing alanine and proline is referred to as “PA” or a “PA moiety.” Thus, in certain embodiments, Z comprises a PA moiety.

[0340] In certain embodiments, Z comprises a random coil protein moiety in which at least 80%, in certain embodiments at least 85%, in certain embodiments at least 90%, in certain embodiments at least 95%, in certain embodiments at least 98%, and in certain embodiments at least 99% of the total number of amino acids forming the random coil protein moiety are selected from alanine, serine, and proline. In certain embodiments, at least 4% but less than 40% of the total number of amino acid residues in such a random coil protein moiety are proline residues. In certain embodiments, such a random coil protein moiety is as described in WO 2008 / 155134 A1. In certain embodiments, Z comprises at least one moiety selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54 and SEQ ID NO:56, as disclosed in WO 2008 / 155134 A1, which is incorporated herein by reference. Moieties that include such random coil protein moieties that include alanine, serine and proline are referred to as "PAS" or "PAS moieties". Thus, in certain embodiments, Z comprises a PAS moiety.

[0341] In certain embodiments, Z comprises a random coil protein moiety in which at least 80%, in certain embodiments at least 85%, in certain embodiments at least 90%, in certain embodiments at least 95%, in certain embodiments at least 98%, and in certain embodiments 99% of the total number of amino acids forming the random coil protein moiety are selected from alanine, glycine, serine, threonine, glutamic acid, and proline. In certain embodiments, such a random coil protein moiety is as described in WO 2010 / 091122 A1. In certain embodiments, Z is selected from the group consisting of SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO 4, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:759, SEQ ID NO:760, SEQ ID NO:761, SEQ ID NO:762, SEQ ID NO:763, SEQ ID NO:764, SEQ ID NO:765, SEQ ID NO:766, SEQ ID NO:767, SEQ ID NO:768, SEQ ID NO:769, SEQ ID NO:770, SEQ ID NO:771, SEQ ID NO:772, SEQ ID NO:773, SEQ ID NO:774, SEQ ID NO:775, SEQ ID NO:776, SEQ ID NO:777, SEQ ID NO:778, SEQ ID NO:779, SEQ ID NO:1715, SEQ ID NO:1716, SEQ ID NO:1718, SEQ ID NO:1719, SEQ ID NO:1720, SEQ ID NO:1721, and SEQ ID NO:1722.A moiety comprising such a random coil protein moiety comprising alanine, glycine, serine, threonine, glutamic acid and proline is referred to as an "XTEN" or "XTEN moiety", consistent with its designation in WO 2010 / 091122 A1. Thus, in certain embodiments, Z comprises an XTEN moiety.

[0342] In certain embodiments, Z comprises at least 60, such as at least 90 or at least 120, elastin-like peptide structural units selected from any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 of WO2021 / 030196. In certain embodiments, Z comprises a protein of SEQ ID NO:34 of WO2021 / 030196. In certain embodiments, Z comprises a protein of SEQ ID NO:35 of WO2021 / 030196.

[0343] In certain embodiments, Z comprises a hyaluronic acid based polymer.

[0344] In certain embodiments, Z comprises a polymer moiety disclosed in WO 2013 / 024047 A1. In certain embodiments, Z comprises a polymer moiety disclosed in WO 2013 / 024048 A1.

[0345] In certain embodiments, Z comprises a multi-arm PEG-based polymer, hi certain embodiments, Z comprises a multi-arm PEG-based polymer having at least two PEG-based arms, such as 2, 3, 4, 5, 6, 7, or 8 PEG-based arms.

[0346] In certain embodiments, Z is of the formula (Zi):

[0347] [ka] [In the formula, -BP 1 <, -BP2 <, -BP 3 < is -N< and -C(R 8 )< R 8 But, H, C 1-6 Alkyl, C 2-6 Alkenyl, and C 2-6 alkynyl, -P 1 , -P 2 , -P 3 , -P 4 are, independently of one another, PEG-based chains containing at least 40% PEG and having a molecular weight in the range of 2 to 45 kDa; -C 1 -, -C 2 -But C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 alkynyl, C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 Alkynyl may be one or more of the same or different R 9 Optionally replaced by C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 Alkynyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 10 )-, -S(O)2N(R 10 )-, -S(O)N(R 10 )-, -S(O)2-, -S(O)-, -N(R 10 )S(O)2N(R 10a )-, -S-, -N(R 10 )-, -OC(OR 10 )(R 10a )-, -N(R 10 )C(O)N(R 10a )- and -OC(O)N(R 10 )-, Each T is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl, each T being the same or different and selected from the group consisting of one or more R 9 and optionally independently replaced by Each R 9 is halogen, -CN, oxo(=O), -COOR 11 , -OR 11 , -C(O)R 11 , -C(O)N(R 11 R 11a ), -S(O)2N(R 11 R 11a ), -S(O)N(R 11 R 11a ), -S(O)2R 11 , -S(O)R 11 , -N(R 11 )S(O)2N(R 11a R 11b ), -SR 11 , -N(R 11 R 11a ), -NO2, -OC(O)R 11 , -N(R 11 )C(O)R 11a , -N(R 11 )S(O)2R 11a , -N(R 11 )S(O)R 11a , -N(R 11 )C(O)OR 11a , -N(R 11 )C(O)N(R 11a R 11b ), -OC(O)N(R 11 R 11a ), and C 1-6 alkyl, wherein C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each R 10 , R 10a , R 11 , R 11a , and R 11b But -H and C 1-6 alkyl, wherein C 1-6The alkyl is optionally substituted with one or more halogens which may be the same or different. Including the branched portion of

[0348] In certain embodiments, the BP of formula (Zi) 1 In certain embodiments, BP of formula (Zi) is -N<. 2 and B.P. 2 Both are -CH<. The first branch point BP 1 And, -L 1 It is advantageous if the attachment site of Z to - is separated by no more than a certain number of atoms. Thus, in certain embodiments, the critical distance is less than 60 atoms, such as less than 50 atoms, such as less than 40 atoms, such as less than 30 atoms, such as less than 20 atoms or less than 10 atoms. The term "critical distance" refers to the distance between the first branch point BP 1 And, -L 1 - Refers to the shortest distance measured as the number of atoms between the binding site of Z to .

[0349] In certain embodiments, C of formula (Zi) 1 and C 2 -O-, -C(O)N(R 10 )- and 3- to 10-membered heterocyclyl 1-50 alkyl, where the 3- to 10-membered heterocyclyl is substituted by at least one oxo (=O).

[0350] In certain embodiments, P 1 , P 2 , P 3 and P 4 Each of the P independently has a molecular weight in the range of about 5 to about 40 kDa. 1 , P 2 , P 3 and P 4 Each of the P independently has a molecular weight in the range of about 8 to about 25 kDa. 1 , P 2 , P 3 and P4 Each of the P independently has a molecular weight in the range of about 9 to about 22 kDa. 1 , P 2 , P 3 and P 4 Each of the P independently has a molecular weight in the range of about 9 to about 15 kDa. 1 , P 2 , P 3 and P 4 Each of the P independently has a molecular weight in the range of 10 to 12 kDa. 1 , P 2 , P 3 and P 4 Each, independently of the other, has a molecular weight of about 10 kDa. 1 , P 2 , P 3 and P 4 Each of the P independently has a molecular weight in the range of 15 to 22 kDa. 1 , P 2 , P 3 and P 4 Each of the P independently has a molecular weight in the range of 18 to 21 kDa. 1 , P 2 , P 3 and P 4 each independently has a molecular weight of about 20 kDa.

[0351] In certain embodiments, C of formula (Zi) 1 and C 2 is the formula (Z-ia)

[0352] [ka] [In the formula, The dashed line with stars indicates BP 1 indicates a bond to Unmarked dashed lines represent BP 2 or BP 3 indicates a bond to q1 is 1, 2, 3, 4, 5, 6, 7, or 8; q2 is 1, 2, 3, 4, or 5; q3 is 1, 2, 3, 4, 5, 6, 7, or 8; q4 is 1, 2, or 3. It is of the following.

[0353] In certain embodiments, q1 of formula (Z-ia) is 4, 5, 6, 7, or 8. In certain embodiments, q1 of formula (Z-ia) is 5, 6, or 7. In certain embodiments, q1 of formula (Z-ia) is 6. In certain embodiments, q2 of formula (Z-ia) is 1, 2, or 3. In certain embodiments, q3 of formula (Z-ia) is 2, 3, 4, or 5. In certain embodiments, q3 is 2, 3, or 4. In certain embodiments, q3 of formula (Z-ia) is 3. In certain embodiments, q4 of formula (Z-ia) is 1.

[0354] In certain embodiments, P of formula (Zi) 1 , P 2 , P 3 , and P 4 are independently of each other the formula (Z-ib)

[0355] [ka] [In the formula, The dashed line indicates R 1 The remainder of each, i.e., BP 2 or BP 3 indicates a bond to m is 0 or 1; p is an integer ranging from 45 to 1000; q is selected from the group consisting of 1, 2, 3, 4, 5, and 6. It is of the following.

[0356] In certain embodiments, p in formula (Z-ib) is in the range of 115 to 900. In certain embodiments, p in formula (Z-ib) is in the range of 180 to 580. In certain embodiments, p in formula (Z-ib) is in the range of 200 to 340. In certain embodiments, p in formula (Z-ib) is in the range of 220 to 270. In certain embodiments, p in formula (Z-ib) is in the range of 220 to 240. In certain embodiments, p in formula (Z-ib) is about 225. In certain embodiments, p in formula (Z-ib) is in the range of 340 to 500. In certain embodiments, p in formula (Z-ib) is in the range of 410 to 475. In certain embodiments, p in formula (Z-ib) is about 450.

[0357] In certain embodiments, Z is of the formula (Z-ic):

[0358] [ka] [In the formula, p1, p2, p3, and p4 are independently integers ranging from 45 to 1000. Includes parts of.

[0359] In certain embodiments, p1, p2, p3, and p4 are independently an integer in the range of 115 to 900. In certain embodiments, p1, p2, p3, and p4 are independently an integer in the range of 180 to 580. In certain embodiments, p1, p2, p3, and p4 are independently an integer in the range of 200 to 340. In certain embodiments, p1, p2, p3, and p4 are independently an integer in the range of 220 to 270. In certain embodiments, p1, p2, p3, and p4 are independently an integer in the range of 220 to 240. In certain embodiments, p1, p2, p3, and p4 are independently an integer in the range of 210 to 240. In certain embodiments, p1, p2, p3, and p4 are independently about 225. In certain embodiments, p1, p2, p3, and p4 are independently integers in the range of 340 to 500. In certain embodiments, p1, p2, p3, and p4 are independently integers in the range of 410 to 475. In certain embodiments, p1, p2, p3, and p4 are independently integers in the range of 420 to 480. In certain embodiments, p1, p2, p3, and p4 are independently about 450.

[0360] In certain embodiments, the growth hormone conjugate has the formula (Ci), as disclosed, for example, in WO05099768 A2:

[0361] [ka] [In the formula, -D is an hGH moiety linked to the remainder of the molecule through the nitrogen of the amine functional group of -D; p1, p2, p3 and p4 are independently integers ranging from 400 to 500. It is a conjugate of.

[0362] In certain embodiments, -D of formula (Ci) is the hGH moiety of SEQ ID NO: 1. In certain embodiments, -D of formula (Ci) is linked to the remainder of the molecule through the nitrogen of the amine functionality provided by the lysine side chain of -D.

[0363] In certain embodiments, p1, p2, p3, and p4 of formula (Ci) are independently integers ranging from 420 to 480. In certain embodiments, p1, p2, p3, and p4 of formula (Ci) are independently integers ranging from 420 to 470. In certain embodiments, p1, p2, p3, and p4 of formula (Ci) are independently integers ranging from 440 to 460. In certain embodiments, p1, p2, p3, and p4 of formula (Ci) are about 450.

[0364] In certain embodiments, the growth hormone conjugate is of formula (C-ii), e.g., as disclosed in WO2016 / 079114A1:

[0365] [ka] [In the formula, -D is an hGH moiety linked to the remainder of the molecule through the nitrogen of the amine functional group of -D; p1, p2, p3 and p4 are independently integers ranging from 200 to 250. It is a conjugate of.

[0366] In certain embodiments, -D of formula (C-ii) is the hGH moiety of SEQ ID NO: 1. In certain embodiments, -D of formula (C-ii) is linked to the remainder of the molecule through the nitrogen of the amine functionality provided by the lysine side chain of -D.

[0367] In certain embodiments, p1, p2, p3, and p4 of formula (C-ii) are independently integers ranging from 210 to 240. In certain embodiments, p1, p2, p3, and p4 of formula (C-ii) are independently integers ranging from 220 to 240. In certain embodiments, p1, p2, p3, and p4 of formula (C-ii) are about 225.

[0368] When the long-acting GH is of formula (C-ii), -D is of SEQ ID NO: 1, -D is linked to the remainder of the molecule through the nitrogen of the amine functionality provided by the lysine side chain of -D, and p1, p2, p3 and p4 are independently integers in the range of 220 to 240, the long-acting GH is lonapegsomatropin, sold in the US as Skytrofa®.

[0369] In certain embodiments, the long-acting growth hormone is selected from the group consisting of Somatrogon, Albutropin, ARX201, ALTU-238, PHA-794428, hGH-OctoDex, Norditropin, Nutropin Depot, LB03002, Somatropin Biopartners, Efpegsomatropin, Somapacitan, Jintrolong, Eftansomatropin, and TV-1106. In certain embodiments, the long-acting growth hormone is selected from the group consisting of Somatrogen, Albutropin, Norditropin, Nutropin Depot, Somatropin Biopartners, Efpegsomatropin, Somapasitan, Eftansomatropin, and Jintrolong. In certain embodiments, the long-acting growth hormone is Somatrogen. In certain embodiments, the long-acting growth hormone is Albutropin. In certain embodiments, the long-acting growth hormone is ARX201. In certain embodiments, the long-acting growth hormone is ALTU-238. In certain embodiments, the long-acting growth hormone is PHA-794428. In certain embodiments, the long-acting growth hormone is hGH-OctoDex. In certain embodiments, the long-acting growth hormone is Norditropin. In certain embodiments, the long-acting growth hormone is Nutropin Depot. In certain embodiments, the long-acting growth hormone is LB03002. In certain embodiments, the long-acting growth hormone is Somatropin BioPartners. In certain embodiments, the long-acting growth hormone is ef-peg-somatropin. In certain embodiments, the long-acting growth hormone is somapacitan. Somapacitan is sold as Sogroya®. In certain embodiments, the long-acting growth hormone is TV-1106.In certain embodiments, the long-acting growth hormone is jintrolong. In certain embodiments, the long-acting growth hormone is efthanol somatropine.

[0370] In another aspect, the present invention relates to a pharmaceutical composition comprising at least one long-acting growth hormone and at least one excipient for use in treating inflammation-induced diseases, wherein the at least one long-acting growth hormone and the treatment are as described elsewhere herein. In certain embodiments, such pharmaceutical compositions have a pH in the range of pH 3 to pH 8, inclusive. In certain embodiments, such pharmaceutical compositions are suspension formulations. In certain embodiments, such pharmaceutical compositions are liquid formulations. In certain embodiments, such pharmaceutical compositions are dry formulations.

[0371] Such liquid, suspension or dry pharmaceutical compositions comprise at least one excipient. Excipients used in parenteral formulations can be classified as, for example, buffers, isotonicity adjusting agents, preservatives, stabilizers, anti-adsorption agents, oxidation protectants, viscosifiers / viscosity enhancing agents, or other auxiliaries. However, in some cases, an excipient may have a dual or triple function. In certain embodiments, at least one excipient of the pharmaceutical composition of the invention is selected from the group consisting of: (i) Buffers: physiologically acceptable buffers to maintain pH within a desired range, such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulfate, nitrate, chloride, pyruvate; antacids, such as Mg(OH)2 or ZnCO3, may also be used; (ii) Isotonicity adjusting agents: to minimize pain that may result from cell damage due to osmotic pressure differences at the injection depot; glycerin and sodium chloride are examples; effective concentrations can be determined by osmometry using an assumed osmolality of 285-315 mOsmol / kg for serum; (iii) Preservatives and / or antimicrobial agents: Preservatives are required for multiple dose parenteral formulations in sufficient concentrations to minimize the risk of patient infection upon injection, and corresponding regulatory requirements have been established; typical preservatives include m-cresol, phenol, methylparaben, ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosol, sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and benzalkonium chloride; (iv) Stabilizers: stabilization is achieved by enhancing the protein stabilizing power, by destabilizing the denatured state, or by direct binding of excipients to the protein; stabilizers can be amino acids (e.g., alanine, arginine, aspartic acid, glycine, histidine, lysine, proline), sugars (e.g., glucose, sucrose, trehalose), polyols (e.g., glycerol, mannitol, sorbitol), salts (e.g., potassium phosphate, sodium sulfate), chelating agents (e.g., EDTA, hexaphosphate), ligands (e.g., divalent metal ions (zinc, calcium, etc.)), other salts or organic molecules (e.g., phenolic derivatives); in addition, oligomers or polymers, such as cyclodextrin, dextran, dendrimers, PEG or PVP, or protamine or HSA, can be used; (v) Anti-adsorption agents: primarily ionic or non-ionic surfactants or other proteins or soluble polymers are used to coat or competitively adsorb to the inner surface of the formulation container; for example, poloxamer (Pluronic F-68), PEG dodecyl ether (Brij 35), polysorbate 20 and 80, dextran, polyethylene glycol, PEG-polyhistidine, BSA and HSA, and gelatin; the excipient concentration and type selected depends on the effect to be avoided, but typically a monolayer of surfactant is formed at the interface just above the CMC value; (vi) Oxidative protection agents: antioxidants such as ascorbic acid, ectoine, methionine, glutathione, monothioglycerol, morin, polyethyleneimine (PEI), propyl gallate, and vitamin E; chelating agents such as citric acid, EDTA, hexaphosphate, and thioglycolic acid may also be used; (vii) Thickening or Viscosity Enhancing Agents: These retard settling of particles in vials and syringes, and are used to aid in mixing and resuspension of the particles and to make the suspension easier to inject (i.e., less force on the syringe plunger); suitable thickening or viscosity enhancing agents include, for example, carbomer thickeners (e.g., Carbopol 940, Carbopol Ultrez 10), cellulose derivatives (e.g., hydroxypropyl methylcellulose (hypromellose, HPMC) or diethylaminoethylcellulose (DEAE or DEAE-C)), colloidal magnesium silicate (Veegum) or sodium silicate, hydroxyapatite gel, tricalcium phosphate gel, xanthan, carrageenan (e.g., Satia gum UTC 30), aliphatic poly(hydroxy acids) (e.g., poly(D,L- or L-lactic acid) (PLA) and poly(glycolic acid) (PGA) and copolymers thereof (PLGA), terpolymers of D,L-lactide, glycolide and caprolactone), poloxamers, hydrophilic poly(oxyethylene) blocks and hydrophobic poly(oxypropylene) blocks to construct poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) triblocks (e.g., Pluronic®), polyetherester copolymers (e.g., polyethylene glycol terephthalate / polybutylene terephthalate copolymers), sucrose acetate isobutyrate (SAIB), dextran, or derivatives thereof, combinations of dextran and PEG, polydimethylsiloxane, collagen, chitosan, polyvinyl alcohol (PVA) and derivatives, polyalkylimides, poly(acrylamide-co-diallyldimethylammonium (DADMA)), polyvinylpyrrolidone (PVP), glycosaminoglycans (GAGs) (e.g., dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronan), ABA triblock or AB block copolymers composed of a hydrophobic A block (e.g., polylactide (PLA) or poly(lactide-co-glycolide) (PLGA)) and a hydrophilic B block (e.g., polyethylene glycol (PEG) or polyvinylpyrrolidone);Such block copolymers and the poloxamers mentioned above may exhibit inverse thermogelation behavior (fluid state at room temperature to facilitate administration, gel state above the sol-gel transition temperature at body temperature after injection); (viii) spreading or diffusing agents: modulate the permeability of connective tissue by hydrolysis of components of the extracellular matrix in the interstitial spaces (e.g., but not limited to, hyaluronic acid, a polysaccharide found in the intercellular spaces of connective tissue); spreading agents, such as, but not limited to, hyaluronidase, temporarily reduce the viscosity of the extracellular matrix and facilitate the diffusion of injected drugs; and (ix) Other auxiliary agents: wetting agents, viscosity adjusting agents, antibiotics, hyaluronidase, etc.; acids and bases such as hydrochloric acid and sodium hydroxide are necessary auxiliary agents for adjusting pH during production.

[0372] In another aspect, the present invention relates to a long-acting growth hormone for use in treating inflammation-induced disease, and administration of the long-acting growth hormone inhibits the recruitment of inflammatory monocytes to the site of inflammation.In certain embodiments, the inflammation-induced disease is NAFLD, for example NASH.When the inflammation-induced disease is NAFLD, particularly NASH, the site of inflammation is the liver.In certain embodiments, when the inflammation-induced disease is NAFLD, particularly NASH, administration of the long-acting growth hormone leads to the increase of HLA-G in the liver.In certain embodiments, the long-acting growth hormone for use in treating inflammation-induced disease leads to the increase of IGF-1 level.

[0373] In certain embodiments, the long-acting growth hormone for use in the treatment of inflammation-induced diseases, such as NAFLD, particularly NASH, causes the change of one or more markers of liver inflammation selected from the group consisting of cytokines, chemokines, and other transcriptional and histological markers.In certain embodiments, the long-acting growth hormone for use in the treatment of inflammation-induced diseases, such as NAFLD, particularly NASH, causes the improvement of transcriptional or histological markers of fibrosis.

[0374] In certain embodiments, the long-acting growth hormone for use in the treatment of inflammation-induced diseases, such as NAFLD, particularly NASH, causes steatosis reduction.In certain embodiments, the long-acting growth hormone for use in the treatment of inflammation-induced liver diseases, such as NAFLD, particularly NASH, causes liver regeneration.

[0375] The long-acting growth hormone and frequency of administration are as described elsewhere herein. EXAMPLES

[0376] Example 1 Synthesis of compound 1

[0377] [ka] n=200~250

[0378] Compound 1 can be synthesized as described in WO2016 / 079114A1 for compound 2 (Example 2) and corresponds to lonapegsomatropin.

[0379] Example 2 Effects of 2-week treatment with Compound 1 on metabolic parameters, liver pathology, and transcriptome profile in male DIO-NASH mice The plasma pharmacokinetic profile was determined in a preliminary study following a single injection of Compound 1 (14.4 mg / kg) into C57BL / 6JRj mice, with blood samples collected over 96 hours. Plasma levels of Compound 1 were determined using a sandwich ELISA. All concentrations of Compound 1 are protein (hGH) equivalents.

[0380] C57BL / 6JRj mice (Janvier, France, 5 weeks old) were fed a nonalcoholic steatohepatitis (NASH)-inducing diet (D09100310, SSNIFF, Germany) consisting of 40% fat, 22% fructose, and 2% cholesterol for a total of 32 weeks.

[0381] Liver biopsies were surgically collected from anesthetized animals and fixed in 10% neutral buffered formalin for histology. Based on the evaluation of liver tissue stained with picrosirius red and hematoxylin and eosin (HE), the livers of animals with a steatosis score of 3, an inflammation score of at least 2, and a fibrosis stage of at least 1 according to the Kleiner-based scoring system (Kleiner, 2005 Hepatology. 2005 Jun;41(6):1313-21. doi: 10.1002 / hep.20701. PMID: 15915461) were selected for study. Animals (n=12 per group) were treated for 14 days by subcutaneous injection every 48 hours with either vehicle or compound 1 administered at 14.4mg / kg or 28.8mg / kg. Blood samples were collected 24 and 48 hours after the first dose and again on day 14, 24 hours after the last dose, and the resulting heparinized plasma supernatants were transferred to fresh tubes, immediately frozen on dry ice, and stored at -70°C.

[0382] After 14 days of treatment, livers were collected from anesthetized animals prior to euthanasia. Liver samples were collected for histological evaluation, RNA sequencing (RNAseq), triglyceride, and total cholesterol analysis.

[0383] Paraffin-embedded sections were deparaffinized in xylene and rehydrated in a graded series of ethanol before HE staining (HE, Dako), CD68 (clone ED1, Abcam, catalog Ab31630) and CD11b (AbCam, catalog 133357). IHC was performed using standard procedures. IHC positive staining was quantified by image analysis using VIS software (Visiopharm, Denmark). The number of hepatocytes with lipid droplets and inflammatory foci was determined by a deep learning app developed by Gubra using VIS software.

[0384] Plasma alanine transaminase (ALT) and aspartate transaminase (AST), and liver homogenate triglycerides (TG) and total cholesterol (TC) were measured on a cobas c 501 autoanalyser using commercially available kits (Roche Diagnostics).

[0385] RNA was isolated using a NucleoSpin® kit (MACHEREY-NAGEL) using 10 ng–1 μg of purified RNA in total from each bulk liver sample. cDNA libraries were generated using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs) and then sequenced on a NextSeq 500 using the NextSeq 500 / 550 High Output Kit V2 (Illumina).

[0386] Sequencing data were aligned to the mouse genome (Ensembl database) using Spliced ​​Transcripts Alignment to a Reference (STAR) software. Bioinformatics analysis assessed data quality using standard RNA-sequencing quality control parameters, principal component analysis and hierarchical clustering to assess inter- and intra-group variation, and the R-package DESeq2 to identify differentially expressed genes.

[0387] result Pharmacokinetic evaluation of a preliminary study in normal C57BL / 6JRj mice resulted in a maximum mean plasma concentration of 80 μg / mL at 24 hours. Compound 1 exposure was detected up to 96 hours and accordingly was dosed every 48 hours in subsequent studies in DIO-NASH mice.

[0388] Treatment of diet-induced (DIO)-NASH mice with Compound 1 (14.4 mg / kg and 28.8 mg / kg) resulted in significant reductions in plasma markers of liver injury ALT and AST (73% and 83%, respectively) compared to vehicle. Hepatic triglycerides were reduced by 56-57% with both doses of Compound 1 compared to vehicle. Hepatic total cholesterol was reduced by 30% with the high dose treatment. Reduction in steatosis was supported by histological assessment, where both the lipid area fraction and the number of hepatocytes containing lipid droplets were reduced by 50-61% with both low and high dose Compound 1 treatment compared to vehicle. Table 1 summarizes these results.

[0389] [Table 1]

[0390] The inflammatory state of the liver was examined by histological and gene expression analysis. Immunohistochemical staining for the inflammatory marker cluster of differentiation 11b (CD11b) (expressed by monocytes and macrophages) showed a trend toward a decrease in CD11b (40% decrease, p=0.051) after treatment with Compound 1 (28.8 mg / kg). In support of this, the transcription of ITGAM, which encodes CD11b, was significantly downregulated (47%) by Compound 1 (28.8 mg / kg). These results are summarized in Table 2.

[0391] [Table 2]

[0392] Similarly, expression of the inflammatory markers cluster of differentiation 14 (CD14), monocyte chemoattractant protein-1 (MCP-1), CC motif chemokine receptor 1 (CCR1) and CC motif chemokine receptor 2 (CCR2) (associated with monocyte recruitment), as well as the macrophage marker galectin-3, was downregulated by treatment with Compound 1 (28.8 mg / kg).

[0393] Finally, downregulation of expression of MCP-1, C-X-C motif chemokine ligand 1 (CXCL1), CCR2, C-C motif chemokine receptor 7 (CCR7), and cluster of differentiation 11c (CD11c), all of which correlate with M1 macrophage polarization, was observed (Table 2).

[0394] Expression of the gene encoding alpha smooth muscle actin (α-SMA), an important marker for hepatic stellate cell activation, was significantly decreased after both low and high dose treatment. In addition, there was a significant decrease in gene expression of tissue inhibitor of metalloproteinases 1 (TIMP-1), a fibrosis marker. The results are shown in Table 3.

[0395] [Table 3]

[0396] conclusion Treatment with Compound 1 for 14 days resulted in significant improvement in hepatic steatosis and reduction in plasma levels of the liver enzymes ALT / AST. Furthermore, improvement in liver inflammation, as indicated by reduced recruitment and accumulation of macrophages and monocytes, downregulation of selected genes associated with M1 macrophage polarization, and reduced gene expression of key markers of fibrosis, was found in a mouse model of NASH.

[0397] Abbreviation ALT Alanine transaminase AST Aspartate transaminase CCL2 CC motif chemokine ligand 2 CCR1 CC motif chemokine receptor 1 CCR2 CC motif chemokine receptor 2 CCR7 CC motif chemokine receptor 7 CD11b differentiation antigen group 11b CD14 differentiation antigen group 14 CD68 differentiation antigen group 68 CXCL1 CXC motif chemokine ligand 1 DIO-NASH Diet-induced nonalcoholic steatohepatitis GH Growth Hormone HE Hematoxylin and Eosin NASH Nonalcoholic Steatohepatitis NBF Neutral Buffered Formalin MCP-1 Monocyte chemotactic protein-1 PSR Picro Sirius Red RNAseq RNA sequencing TC Total cholesterol TG Triglycerides TIMP-1 Tissue inhibitor of metalloproteinases 1

Claims

1. Use of long-acting growth hormone (GH) in the manufacture of a medicine for the treatment of diseases induced by inflammation.

2. 2. The use according to claim 1, wherein the inflammation-induced disease is non-alcoholic fatty liver disease (NAFLD).

3. 3. The use according to claim 1 or 2, wherein the inflammation-induced disease is non-alcoholic steatohepatitis (NASH).

4. The use according to any one of claims 1 to 3, wherein the long-acting GH comprises a growth hormone moiety covalently conjugated to one or more chemical moieties.

5. The use according to claim 4, wherein the chemical moiety is a polymer moiety.

6. 6. The use according to claim 4 or 5, wherein the chemical moiety is a PEG-based moiety.

7. The use according to any one of claims 4 to 6, wherein the bond between the growth hormone moiety and the chemical moiety is a stable covalent bond.

8. The use according to any one of claims 4 to 6, wherein the bond between the growth hormone moiety and the chemical moiety is a reversible covalent bond.

9. The use according to any one of claims 1 to 3, wherein the long-acting GH comprises growth hormone non-covalently embedded or encapsulated in a polymer or lipid-containing matrix.

10. The use according to any one of claims 1 to 3, wherein the long-acting growth hormone comprises a growth hormone moiety fused to at least one natural or non-natural amino acid sequence.

11. The use according to any one of claims 1 to 10, wherein administration of long-acting GH causes a rebalance of macrophage phenotype between M1 and M2.

12. The use according to any one of claims 1 to 11, wherein administration of long-acting growth hormone results in changes in one or more markers of liver inflammation selected from the group consisting of cytokines, chemokines, and other transcriptional and histological markers.

13. The use according to any one of claims 1 to 12, wherein the long-acting GH inhibits the recruitment of inflammatory monocytes to the site of inflammation.

14. The use according to any one of claims 1 to 13, wherein the administration of a long-acting growth hormone results in a reduction of adiposity.

15. The use according to any one of claims 1 to 14, wherein the long-acting GH is administered to the patient once a week.

16. The long-acting GH is represented by the formula (C-ii) 【Chemistry 1】 [In the formula, -D is an hGH moiety linked to the remainder of the molecule through the nitrogen of the amine functional group of -D; p1, p2, p3 and p4 are independently integers ranging from 200 to 250. The use according to any one of claims 1 to 6, 8 or 11 to 15,

17. The use according to claim 16, wherein p1, p2, p3 and p4 in formula (C-ii) are independently integers in the range of 210 to 240.

18. The use according to claim 16 or 17, wherein p1, p2, p3 and p4 in formula (C-ii) are independently integers in the range of 220 to 240.

19. The use according to any one of claims 16 to 18, wherein -D is the hGH moiety of SEQ ID NO:

1.

20. 20. The use according to any one of claims 16 to 19, wherein -D is linked to the remainder of the molecule through the nitrogen of the amine functionality provided by the lysine side chain of -D.

21. The use according to any one of claims 1 to 4, 7 or 11 to 15, wherein the long-acting GH is somapasitan.

22. Treatment, (a) administering to a patient having an inflammation-induced disease at least an initial dose of long-acting GH; (b) measuring insulin-like growth factor-1 (IGF-1) levels, and (c) decreasing the dose of long-acting GH by at least 5% if the IGF-1 level is above a standard deviation score of +3 and increasing the dose of long-acting GH by at least 5% if the IGF-1 level is below a standard deviation score of 0. The use according to any one of claims 1 to 21, comprising:

23. Treatment, (a) administering to a patient having an inflammation-induced disease at least an initial dose of long-acting GH; (b) measuring biomarkers indicative of M1 and M2 macrophages; (c) adjusting the dose of long-acting GH based on the change in macrophage phenotype due to M1 reduction or M2 induction as indicated by the biomarkers. The use according to any one of claims 1 to 21, comprising:

24. 24. The use of claim 23, wherein the biomarker indicative of M2 macrophages is selected from the group consisting of IL-2, IL-4, IL-10, IL-13, CCL17, CCL18, CCL22, CCL24, CCL13, CCL16, CXCR1, CXCR2, CD14, CD23, CD36, CD163, mannose receptor (CD206), scavenger receptor A, Chi3l3 / Ym1, Retnla / Fizz-1, and arginase-1.

25. 25. The use according to claim 23 or 24, wherein the dose adjustment in step (c) involves measuring IGF-1 levels and the adjustment of the dose of long-acting GH is such that the IGF-1 level is in the range of 0 to +3 standard deviation scores.

26. The use according to any one of claims 23 to 25, wherein steps (b) and (c) are repeated until macrophage rebalance is achieved.

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