ES2703388T3 - Growth hormones with prolonged efficacy in vivo - Google Patents

Abstract

A conjugate of a growth hormone having the formula (I): A-W-B-GH (I) wherein GH represents a growth hormone compound, A represents an albumin binding residue; W is a chemical group linking A and B, and wherein B represents a hydrophilic spacer selected from: ** Formula ** where * denotes a point of attachment, i.e., an open bond.

Description

DESCRIPTION

Growth hormones with prolonged efficacy in vivo

Field of the invention

The present invention relates to a growth hormone compound bound to an albumin binding residue by means of a hydrophilic spacer, and to methods for the preparation and use of such compounds. These compounds have a prolonged action profile and are useful in therapy.

BACKGROUND OF THE INVENTION

It is well known to modify the properties and characteristics of the peptides by conjugating groups to the peptide that properly change the properties of the peptide. Said conjugation generally requires that some functional group in the peptide react with another functional group in a conjugation group. Typically, amino groups, such as the N-terminal amino group or the g-amino group in lysines, have been used in combination with a suitable acylating reagent as described for GLP-1 in WO2005 / 027978. Alternatively, polyethylene glycol (PEG) or derivatives thereof can be bound to the proteins. For a review, see Exp. Opion. Ther. Patent., 14, 859-894, (2004). It has been shown that the binding of PEG to growth hormone can have a positive effect on the mean plasma half-life of growth hormone, WO 03/044056.

The use of carboxypeptidases to modify the C-terminal end of the peptides has been described above. WO 92/05271 describes the use of carboxypeptidases and nucleophilic compounds to amidate the C-terminal carboxyl group, and WO 98/38285 describes particular Y carboxypeptidase variants suitable for this purpose.

EP 243 929 describes the use of carboxypeptidase to incorporate polypeptides, reporter groups or cytotoxic agents at the C-terminal end of proteins or polypeptides.

WO 2005/035553 describes methods for the selective conjugation of peptides by the enzymatic incorporation of a functional group at the C-terminal end of a peptide.

Previously, transglutaminase has been used to alter the properties of peptides. In the food industry and in particular in the dairy industry many techniques are available for example for binding peptides by the use of transglutaminases. Other documents describe the use of transglutaminase to alter the properties of physiologically active peptides. The documents EP 950665, EP 785276 and Sato, Adv. Drug Delivery Rev. 54, 487 504 (2002) describe the direct reaction between peptides comprising at least one Gln and a PEG with amine functional group or similar ligands in the presence of transglutaminase, and Wada, Biotech. Lett. 23, 1367-1372 (2001) describes the direct conjugation of p-lactoglobulin with fatty acids by means of transglutaminase. The international patent application published as WO 2005/070468 describes the use of transglutaminase to incorporate a handle where conjugation groups can be attached. WO2008 / 03750 discloses GH compounds wherein a 4-aminobenzoyl group is bound to GH by reductive amination.

Growth hormone is a fundamental hormone involved in the regulation not only of somatic growth, but also in the regulation of the metabolism of proteins, carbohydrates and lipids. The main effect of growth hormone is to promote growth. Human growth hormone is a protein of 191 amino acid residues with the sequence:

FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISL LLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLK NYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (sec. With ID #: 1).

The administration of human growth hormone and its closely related variants is used to treat a variety of diseases related to a growth hormone deficiency. Since it is a peptide, growth hormone is administered parenterally, that is, by means of a needle. Growth hormone is also characterized by a relatively short half-life, therefore frequent administrations are required with the corresponding pain and inconvenience for the patient. Therefore, there is still a need to provide growth hormone compounds with improved pharmacological properties, such as for example a prolonged half-life.

The present invention provides novel conjugates of growth hormone with improved pharmacological properties as well as methods for their production.

BRIEF DESCRIPTION OF THE INVENTION

The bioavailability of a pharmaceutical composition administered subcutaneously may be related to the rate of absorption. The ability of a compound to pass through the tight junctions of capillaries subcutaneous can be in part related to its physical and chemical properties as well as the molecular size or hydrodynamic volume of the compound. A protein conjugate such as a pegylated hGH (PEG-hGH) with a 40 kDa PEG has an apparent molecular weight of 150-250 kDa. A molecule of hGH with covalently bound albumin has a molecular weight of 87 kDa, whereas a molecule of hGH with a bound non-covalent albumin will dissociate from albumin at some time and will therefore have a molecular weight of 22 kDa.

It is contemplated that the amount of time in the dissociated state depends, at least in part, on the affinity of the remaining albumin binding. Therefore the rate of absorption of a hGH molecule with a non-covalently bound albumin may be faster than for a PEG-hGH. An increase in the rate of absorption can be obtained when using albumin binding moieties that have lower affinity for albumin.

Additionally, the physical and chemical properties of the linker and / or the spacer that provide binding of the binding moiety to albumin and hGH will influence the functionalities of the compounds.

The present inventors have surprisingly found that growth hormone (GH) compounds can selectively bind to an albumin-binding residue by means of a hydrophilic spacer that separates GH and albumin-binding residue with a chemical moiety. which has a value of mLogP <0 - or a cLogP <0.5 to obtain conjugates of GH with improved pharmacological properties.

In addition, the present invention is based on the observation that the introduction of a binding residue to albumin by means of a hydrophilic spacer in human growth hormone (hGH) can be carried out selectively where a large proportion of the activity can be retained. biological Preferably, an albumin binding residue by means of a hydrophilic spacer is introduced at the positions corresponding to the positions of phenylalanine 1, glutamine 40 and / or glutamine 141 in hGH having the sequence of sec. with no. of ident.1. The use of transglutaminase (TGase), and in particular TGase from Streptoverticillium mobaraenae or Streptomyces lydicus allows a selective introduction of an albumin binding residue by means of a hydrophilic spacer at positions 40 and / or 141, and the remaining 11 Glutamine residues are left unmodified despite the fact that glutamine is a substrate for transglutaminase.

Therefore, in one embodiment of the present invention the growth hormone compound (GH) is bound to an albumin binding residue (A) by means of a hydrophilic spacer (B), wherein the hydrophilic spacer (B) ) is selected from:

where * denotes a point of union, that is, an open link.

Typically, the albumin binding residue is attached to the N-terminus, position 40 or position 141 of the hGH by means of a hydrophilic spacer. In additional embodiments two or three albumin binding residues are attached to the N-terminus, position 40 and / or position 141 of the hGH by means of a hydrophilic spacer.

A further object of the present invention is to provide a method for improving the properties of a GH by conjugating said protein according to the methods of the present invention.

Definitions

In the present context, the term "growth hormone compound" as used herein refers to a growth hormone of mammalian origin, such as a human, bovine or porcine growth hormone, and a recombinant growth hormone. , such as a recombinant human, bovine or porcine growth hormone, and variants of such growth hormones. As used herein "GH" and "growth hormone compound" are interchangeable. When GH is a variant of a growth hormone of mammalian origin, such as hGH and recombinant hGH, it is understood that said variant is the compound obtained by replacing one or more amino acid residues in the growth hormone, for example. sequence, hGH, with another natural or non-natural amino acid; and / or by the addition of one or more natural or unnatural amino acids to the growth hormone, e.g., sequence, hGH; and / or by removing one or more amino acid residues from the growth hormone, e.g., sequence, hGH, wherein either of these steps may optionally be followed by another derivation of one or more amino acid residues. In particular, such substitutions are conservative in the sense that an amino acid residue is replaced by another amino acid residue of the same group, ie by another amino acid residue with similar properties. Amino acids can be conveniently divided into the following groups on the basis of their properties: Basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, cysteine and asparagine), amino acids hydrophobic (such as leucine, isoleucine, proline, methionine and valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine and threonine). Typically, GH has at least 80% identity with hGH, and typically, has at least 20% of the growth hormone activity of hGH as determined in assay I herein.

In the present context, the term "albumin binding residue" as used in the present disclosure means a residue that binds non-covalently to human serum albumin. The albumin binding residue bound to the growth hormone compound (GH) typically has a binding affinity to human serum albumin that is below about 10 pM or even below about 1 pM. There is a known variety of albumin binding residues between the linear and branched lipophilic moieties containing 12-40 carbon atoms, compounds with a cyclopentanofenanthrene skeleton, and / or peptides having 10-45 amino acid residues, and so on. The albumin binding properties can be measured by surface plasmon resonance as described in J. Biol. Chem. 277 (38), 35035-35042, (2002).

The term "hydrophilic spacer" as used in the present disclosure refers to a spacer that separates a growth hormone compound and an albumin binding residue with a chemical moiety comprising at least 5 non-hydrogen atoms, where 30-50% of these are N or O.

In the present context, the term "transamination" and related terms indicate a reaction wherein the nitrogen of the amide in the glutamine side chain is exchanged with the nitrogen of another compound, in particular the nitrogen of another nitrogen-containing nucleophile.

Transglutaminase (E.C.2.3.2.13) is also known as protein-glutamine-Y-glutamyltransferase and catalyzes the general reaction

QC (O) -NH ² (amine acceptor) can represent a peptide or protein containing a glutamine residue and Q'-NH ² (amine donor) represents an amine-containing nucleophile. Alternatively, QC (O) -NH ² and Q'-NH ² may represent an amine acceptor and a lysine-containing peptide or protein, respectively. In the present invention, however, QC (O) -NH ² represents a growth hormone that contains a glutamine residue and Q'-NH ² represents an amine-containing nucleophile as indicated above.

Examples of useful transglutaminases include microbial transglutaminases, such as, for example, Streptomyces mobaraense, Streptomyces cinnamoneum and Streptomyces griseocarneum (all described in US 5,156,956), and Streptomyces lavendulae (described in US 5,252,469) and Streptomyces ladakanum (JP 2003 document 199595). It should be noted that members of the former Streptoverticillium genus are now included in the genus Streptomyces (Kaempfer, J. Gen. Microbiol., 137, 1831-1892 (1991)). Other useful microbial transglutaminases have been isolated from Bacillus subtilis (described in US 5,731,183) and from various Mixomycetes. Other examples of useful microbial transglutaminases are those described in WO 96/06931 (for example Bacillus lydicus transglutaminase ) and WO 96/22366. Useful non-microbial transglutaminases include guinea pig transglutaminase, and transglutaminases from various marine sources such as the Japanese snapper Pagrus major (described in EP-0555649), and the Japanese oyster Crassostrea gigas (described in US 5,736,356).

In the present context, the term "not accessible" indicates that something is absent or de facto absent in the sense that it can not be achieved. When it is noted that the functional groups are not accessible in a protein to be conjugated it is understood that it indicates that said functional group is absent from the protein or, if present, is somehow prevented from taking part in the reactions. By way of example, said functional group can have a deep localization in the structure of the protein so that it is protected from participating in the reaction. It is recognized that whether a functional group is accessible or not depends on the reaction conditions. It can be envisaged that, for example in the presence of denaturing agents or at elevated temperatures the protein can be deployed to expose functional groups that are otherwise not accessible. It should be understood that "not accessible" refers to "not accessible in the reaction condition chosen for the particular interest reaction".

The term "alkane" or "alkyl" denotes a saturated, linear, branched and / or cyclic hydrocarbon. Unless specified with another number of carbon atoms, the term is meant to mean hydrocarbons from 1 to 30 (both inclusive) carbon atoms, such as 1 to 20 (both included), such as from 1 to 10 (both included), for example from 1 to 5 (both included). The terms "alkyl" and "alkylene" refer to the corresponding radical and bi-radical, respectively.

The term "C 1-6 alkyl" refers to a saturated straight or branched chain hydrocarbon having from one to six carbon atoms in an inclusive manner. Examples of such groups include, methyl, 2-propyl, 1-butyl, 2-butyl, 2-methyl-2-propyl, 2-methyl-1-butyl and n-hexyl.

The term "C ^3-10 cycloalkyl" typically refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecanyl and.

The term "alkene" denotes linear, branched and / or cyclic hydrocarbons comprising at least one carbon-carbon double bond. Unless specified with another number of carbon atoms, it is understood that the term denotes hydrocarbons with 2 to 30 (both included) carbon atoms, such as 2 to 20 (both included), such as 2 to 10 (both included), for example 2 to 5 (both included). The terms "alkenyl" and "alkenylene" refer to the corresponding radical and bi-radical, respectively.

The term "alkyne" is understood to mean linear, branched and / or cyclic hydrocarbons comprising at least one carbon-carbon triple bond, and may optionally comprise one or more carbon-carbon double bonds. Unless specified with another number of carbon atoms, it is understood that the term denotes hydrocarbons with 2 to 30 (both included) carbon atoms, such as 2 to 20 (both included), such as 2 to 10 ( both included), for example from 2 to 5 (both included). The terms "alkynyl" and "alkynylene" refer to the corresponding radical and bi-radical, respectively.

The term "homocyclic aromatic compound" is understood to mean aromatic hydrocarbons, such as benzene and naphthalene.

The term "heterocyclic compound" is understood to mean a cyclic compound comprising 5, 6 or 7 ring atoms of which 1, 2, 3 or 4 are heteroatoms selected from N, O and / or S. Examples include aromatic compounds heterocyclics, such as thiophene, furan, pyran, pyrrole, imidazole, pyrazole, isothiazole, isooxazole, pyridine, pyrazine, pyrimidine, pyridazine, as well as their partially or completely hydrogenated equivalents, such as piperidine, pyrazolidine, pyrrolidine, pyroline, imidazolidine, imidazoline, piperazine and morpholine.

The terms "hetero alkane", "hetero alkene" and "heteroalkyne" are understood to mean alkanes, alkenes and alkynes as defined above, in which one or more hetero atoms or group have been inserted into the structure of said moieties. Examples of hetero groups and atoms include -O-, -S-, -S (O) -, -S (O) ² -, -C (O) - -C (S) - and -N (R *) -, wherein R * represents hydrogen or C ¹ -C ⁶ -alkyl. Examples of heteroalkanes include.

The terms "radical" or "biradical" are understood to indicate a compound from which one or two, respectively, hydrogen atoms have been removed. When specifically indicated, a radical can also indicate the residue formed by the formal removal of a larger group of atoms, for example hydroxyl, from a compound.

The term "halogen" is understood to mean the members of the seventh major group of the periodic table, for example F, Cl, Br and I.

In the present context, the term "aryl" is understood to mean a carbocyclic aromatic ring radical or a radical of a fused aromatic ring system wherein at least one of the rings is aromatic. Typical aryl groups include phenyl, biphenylyl, naphthyl.

The terms "heteroaryl" or "hetaryl", as used herein, alone or in combination, refer to an aromatic ring radical with for example 5 to 7 member atoms, or to a radical of an aromatic ring system fused to for example 7 to 18 member atoms, wherein at least one ring is aromatic, which contains one or more heteroatoms such as ring atoms that are selected from heteroatoms of nitrogen, oxygen or sulfur, wherein the N-oxides and sulfur monoxides and sulfur dioxides are permissible heteroaromatic substitutions. Examples include furanyl, thienyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridinyl, pyridazinyl, pyrazinyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothiophenyl, indolyl and indazolyl. .

The term "conjugate" as a noun indicates a modified protein, ie a protein with a moiety attached to it to modify the properties of said protein. As a verb, the term "conjugate" is understood to mean the process of binding a moiety to a protein to modify the properties of said protein.

As used herein, the term "prodrug" denotes biohydrolyzable amides and biohydrolyzable esters and further encompasses a) compounds in which the biohydrolyzable functionality in such a prodrug is included in the compound according to the present invention, and b) compounds that can be oxidized or reduced biologically to a certain functional group for producing pharmacological substances according to the present invention. Examples of these functional groups include 1,4-dihydropyridine, N-alkylcarbonyl-1,4-dihydropyridine, 1,4-cyclohexadiene, fer-butyl.

As used herein, the term "biohydrolyzable ester" is an ester of a pharmacological substance (in this case, a compound according to the invention) that a) does not interfere with the biological activity of the original substance but confers advantageous properties in vivo on that substance such as the duration of action, the onset of action, or b) is biologically inactive but is easily converted in vivo to the biologically active principle by the subject. The advantage is, for example, an increase in solubility or that the biohydrolyzable ester is orally absorbed from the intestine and transformed into a compound according to the present invention in the plasma. Many examples of these are known in the art and include, by way of example, lower alkyl esters (eg, C ¹ -C ⁴ ), lower acyloxyalkyl esters, lower alkoxyacyloxyalkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters, and choline esters.

As used herein, the term "biohydrolyzable amide" is an amide of a pharmacological substance (in this case, a compound according to the present invention) that a) does not interfere with the biological activity of the original substance but confers properties advantageous in vivo on that substance such as the duration of action, the onset of action, or b) is biologically inactive but is easily converted in vivo to the biologically active principle by the subject. The advantage is, for example, increased solubility or that the biohydrolysable amide is orally absorbed from the intestine and transformed into a compound according to the present invention in the plasma. Many examples of these are known in the art and include, by way of example, lower alkyl amides, aminino acid amines, alkoxyacyl amides and alkylaminoalkylcarbonyl amides.

In the present context, the term "pharmaceutically acceptable salt" refers to salts that are not harmful to the patient. Said salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. The acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic acids , ethanesulfonic, tartaric, ascorbic, pamoic, salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic. Other examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts mentioned in J. Pharm. Sci. 66, 2, (1977). Examples of metal salts include lithium, sodium, potassium, magnesium salts. Examples of alkylated ammonium and ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts.

A "therapeutically effective amount" of a compound as used in the present disclosure refers to an amount sufficient to cure, alleviate, or partially arrest the clinical manifestations of a given disease and its complications. An adequate amount to achieve this is defined as "quantity with therapeutic efficacy". The amounts effectively for each purpose will depend on the severity of the disease or injury, as well as the weight and general condition of the subject. It will be understood that the determination of an appropriate dosage can be achieved through the use of routine experimentation, through the construction of a matrix of values and the evaluation of different points in the matrix, which is within the ordinary skills of a physician or veterinarian trained.

The term "treatment" and "treating" as used in the present description refers to the management and care of a patient for the purpose of combating a condition, such as a disease or disorder. The term includes the full spectrum of treatments for a particular condition of which the patient suffers, such as administration of the active compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or calm the symptoms and complications, and / or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention should be understood as the management and care of a patient for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of symptoms or complications. The patient to be treated is preferably a mammal; in particular, a human being, but may also include animals, such as dogs, cats, cows, sheep and pigs.

Description of the invention

The present invention relates to a conjugate of a growth hormone comprising a compound of growth hormone (GH) selectively bound to an albumin binding residue (A) by means of a hydrophilic spacer (B), or a salt , solvate or prodrug of this pharmaceutically acceptable, wherein B is selected from:

and where * denotes a point of union, that is, an open link.

In one embodiment of the present invention the hydrophilic spacer has a value of mLogP <0. The solubility of a hydrophilic spacer can be described by its logP value. LogP, also known as the partition coefficient, is the logarithm of the concentration ratio of a compound in the two phases of a mixture of two immiscible solvents in equilibrium. Typically one of the solvents is water while the second is selected from octan-1-ol, chloroform, cyclohexane and propylene glycol dipelargonate (PGDP). The LogP values measured in these different solvents show differences mainly due to the effects of hydrogen bonding. Octanol can donate and accept hydrogen bonds while cyclohexane is inert. Chloroform can donate hydrogen bonds while PGDP can only accept them.

In another embodiment of the invention, the hydrophilic spacer has a LogP below -0.5 either in octan-1-ol, chloroform, cyclohexane and propylene glycol dipelargonate (PGDP).

In another embodiment, the hydrophilic spacer has a logP below -1 in either octan-1-ol, chloroform, cyclohexane, and propylene glycol dipelargonate (PGDP).

Alternatively, the LogP value can be calculated as mLogP and / or cLogP for the albumin binding portion or the hydrophilic spacer part by the use of published algorithms (J. Am. Chem. Soc., 86 (1964) 5175- 5180 "A New Substituent Constant, g, Derived from Partition Coefficients," CA Lipinski et al., Advanced Drug Delivery Reviews, 23 (1997) 3-25, "Experimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development Settings" and I. Moriguchi, S. Hirono, I. Nakagome, H. Hirano, Chem. and Pharm. Bull., 42 (1994) 976-978 "Comparison of Reliability of log Values for Drugs Calculated by Several Methods". of the present invention the hydrophilic spacer has a cLogP <0.5.

In another embodiment, the growth hormone compound (GH) is bound to an albumin binding residue by means of a hydrophilic spacer (B) as defined in the claims.

In another embodiment, the growth hormone compound (GH) binds to two albumin binding residues by means of one or two hydrophilic spacers (B) as defined in the claims. Therefore, in one example an albumin binding residue is linked by means of a hydrophilic spacer to glutamine at position 40 and the other albumin binding residue is attached by means of a hydrophilic spacer to glutamine at position 141; or alternatively two albumin binding residues are attached by means of a hydrophilic spacer to glutamine at position 40 or 141 or the N-terminus. In yet another embodiment the growth hormone compound (GH) binds to three albumin binding residues by means of one or more hydrophilic spacers.

In one embodiment the hydrophilic spacer comprises at least one OEG moiety, the radical of 8-amino-3,6-dioxaoctanic acid, ie -NH- (CH ² ) ² -O- (CH ² ) ² -O-CH ² -CO)-. In another specific embodiment the hydrophilic spacer comprises at least two OEG moieties. The orientation of the OEG moiety (s) of this type in one embodiment is such that -C (O) - is closer to the growth hormone compound but does not connect with the growth hormone compound and the linker of binding to albumin and -NH- is closer to the binding residue to albumin. In additional embodiments comprising two OEG moieties the two moieties have an identical orientation or a different orientation. In one embodiment two such OEG moieties are located adjacent to each other while in alternative embodiments such OEG moieties are separated by one or more covalently bonded atoms.

In one embodiment the hydrophilic spacer comprises at least one glutamic acid residue. The amino acid glutamic acid comprises two carboxylic acid groups. Its gamma-carboxyl group can be used to form an amide bond with the epsilon-amino group of lysine, or with an amino group of an OEG molecule, if present, or with the amino group of another Glu residue, if present . The alpha-carboxyl group can alternatively be used to form a similar amide bond with the epsilon-amino group of lysine, or with an amino group of one OEG molecule, if present, or with the amino group of another Glu residue, if is present The amino group of Glu can, in turn, form an amide bond with the carboxyl group of the albumin binding residue, or with the carboxyl group of an OEG moiety, if present, or with the gamma carboxyl group or the alpha carboxyl group of another Glu, if present. The linkage of the amino group of a Glu with a gamma carboxyl group of a second Glu can be referred to as a "gamma-Glu" moiety.

In one embodiment the hydrophilic spacer comprises at least one combined OEG-Glu residue (-NH- (CH ² ) ² -O- (CH ² ) ² -O-CH ² -C (O) NH-CH (C (O) OH) - (CH ² ) ² -C (O) -) or at least one combined Glu-OEG moiety (-NH-CH (C (O) OH) - (CH ² ) ² -C (O) NH- ( c H ² ) ² -O- (Ch ² ) ² -O-CH ² -C (O) -) or a combination thereof, wherein such GIu-o Eg and OEG-Glu moieties can be separated by one or more atoms linked covalently or directly linked together by an amide bond of Glu that forms a gammal-Glu.

It is described that the hydrophilic spacer can have the formula

-X ¹ -X ² -X ³ -X ⁴ -

where

X1 is -W1 - [(CHR1) l1-W2] m1 - {[(CH2) n1E1] m2 - [(CHR2) l2-W3] m3} n2-,

X2 is - [(CHR3) l3-W4] m4 - {[(CH2) n3E2] m5 - [(CHR4) 14 -W5] m6} n4-,

X3 is - [(CHR5) l5-W6] m7-,

X ⁴ is -F-D1- (CH2) i6-D2-,

I1, I2, I3, I4, I5 and I6 are independently selected from 0-16,

m1, m3, m4, m6 and m7 are independently selected from 0-10,

m2 and m5 are independently selected from 0-25,

n1, n2, n3 and n4 are independently selected from 0-16,

F is aryl, hetaryl, pyrrolidine-2,5-dione or a valence bond, wherein the aryl and

Hetaryl groups are optionally substituted with halogen, -CN, -OH, -C (O) OH,

-C (O) NH ^2, -S (O) ² OH or C ^1-6 alkyl,

R1, R2, R3, R4 and R5 are independently selected from hydrogen, -C (O) OH,

-C (O) NH ^2, -S (O) OH, -S (O) ² OH, -NH-C (= NH) -NH ^2, C ^1-6 -alkyl, aryl or hetaryl; wherein the alkyl, aryl and hetaryl groups are optionally substituted with halogen, -C (O) OH,

-C (O) NH ² , -S (O) OH, -S (O) ² OH, -CN or -OH,

D1, D2, E1 and E2 are independently selected from -O-, -NR6-, -N (COR7) - or a valence bond; wherein R6 and R7 independently represent hydrogen or C ^1-6 alkyl,

W ¹ to W ⁶ are independently selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) ² NHC (O) -, -OC (O) NH-, -NHC (O) O-, -C (O) CH ² -, -CH ² C (O) -, - C (O) CH = CH-, -CH = CHC (O) -, - (CH ² ) s ¹ -, -C (O) -, -C (O) O-, -OC (O) -, or a valence link; where s1 is 0 or 1.

It is disclosed that D1 can be selected from -O-, -NR6-, -N (COR7) - or a valence bond,

D2 is -NR6-, E1 and E2 are independently selected from -O-, -NR6-, -N (COR 7) - or a valence bond and R6 and R7 independently represent hydrogen or C ^1-6 alkyl,

In addition, a conjugate of a growth hormone of the formula (I) is described: A-W-B-GH (I)

where

GH represents a growth hormone compound,

B represents a hydrophilic spacer,

W is a chemical group that links A and B, and

A represents a residue of binding to albumin; Y

pharmaceutically acceptable salts, solvates and prodrugs thereof.

As described above, the growth hormone compound (GH) can be bound to two albumin binding residues by means of a hydrophilic spacer, consequently a conjugate of a growth hormone of the formula (II) is further described:

A-W-B-GH-B'-W-A '(II)

where

GH represents a growth hormone compound,

W is a chemical group that links A and B,

W is a chemical group that binds A 'and B',

A and A 'independently represent an albumin binding residue,

B and B 'independently are hydrophilic spacers, and

pharmaceutically acceptable salts, solvates and prodrugs thereof.

In the conjugate of formula (II) W is selected from the same groups as W, A 'is selected from the same groups as A and B' is selected from the same groups as B, and it should be understood that W and W, A and A ', and B and B' are independently selected from any of the respective groups as defined below. Therefore, any of the modalities of W, A, and B below are also modes of W, A ', and B'. In addition, any of the embodiments described herein independently refers both to the conjugates of formula (I) and (II), as well as the broad aspect and modalities thereof when appropriate.

In a GH embodiment it is a variant of hGH, wherein a variant is understood to be the compound obtained by replacing one or more amino acid residues in the hGH sequence with another natural or non-natural amino acid; and / or by the addition of one or more natural or unnatural amino acids to the hGH sequence; and / or by removing one or more amino acid residues from the hGH sequence, wherein any of these steps may optionally be followed by an additional derivation of one or more amino acid residues. In particular, such substitutions are conservative in the sense that an amino acid residue is replaced by another amino acid residue of the same group, ie by another amino acid residue with similar properties. Amino acids can be conveniently divided into the following groups on the basis of their properties: Basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, cysteine and asparagine), amino acids hydrophobic (such as leucine, isoleucine, proline, methionine and valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine and threonine).

In another embodiment, GH represents a growth hormone compound comprising an amino acid sequence having at least 90% identity with the amino acid sequence of human growth hormone (hGH) (sec. With ident. No .: 1) ). In additional embodiments, the GH has at least 80%, such as at least 85%, such as at least 95% identity with hGH. In additional embodiments, said identities with hGH are associated with at least 20%, such as at least 40%, such as at least 60%, such as at least 80% of the growth hormone activity of hGH as determined in the test I in the present. Any of the sequence identity modalities can be combined with any of the activity modalities, such as a GH that has at least 80% identity with hGH and is associated with at least 60% of the growth hormone activity of hGH ; a GH that has at least 90% identity with hGH and is associated with at least 40% of the growth hormone activity of hGH; a GH that has at least 95% identity with hGH and is associated with at least 80% of the growth hormone activity of hGH, and so on.

Still in another modality, GH is hGH (sec with ident #: 1).

It is described that the hydrophilic spacer B can have the formula

-X ¹ -X ² -X ³ -X ⁴ -

where

X1 is -W1 - [(CHR1) I1-W2] m1 - {[(CH2) n1E1] m2 - [(CHR2) I2-W3] m3} n2-,

X2 is - [(CHR3) I3-W4] m4 - {[(CH2) n3E2] m5 - [(CHR4) I4-W5] m6} n4-,

X3 is - [(CHR5) I5-W6] m7-,

X ⁴ is -F-D1- (CH2) I6-D2-,

I1, I2, I3, I4, I5 and I6 are independently selected from 0-16,

m1, m3, m4, m6 and m7 are independently selected from 0-10,

m2 and m5 are independently selected from 0-25,

n1, n2, n3 and n4 are independently selected from 0-16,

F is aryl, hetaryl, pyrrolidine-2,5-dione or a valence bond, wherein the aryl and

Hetaryl groups are optionally substituted with halogen, -CN, -OH, -C (O) OH,

-C (O) NH ² , -S (O) ² OH or C ^1-6 -alkyl,

R1, R2, R3, R4 and R5 are independently selected from hydrogen, -C (O) OH,

-C (O) NH ² , -S (O) OH, -S (O) ² OH, -NH-C (= NH) -NH ² , C ^1-6 -alkyl, aryl or hetaryl; wherein the alkyl, aryl and hetaryl groups are optionally substituted with halogen, -C (O) OH,

-C (O) NH ² , -S (O) OH, -S (O) ² OH, -CN or -OH,

D1, D2, E1 and E2 are independently selected from -O-, -NR6-, -N (COR7) - or a valence bond; where R6

and R7 independently represent hydrogen or C ^1-6 -alkyl,

W1 to W6 are independently selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -,

-S (O) ² NHC (O) -, -OC (O) NH-, -NHC (O) O-, -C (O) CH ² -, -CH ² C (O) -, -C (O ) CH = CH-, -CH = CHC (O) -, - (CH ² ) s ² -, -C (O) -, -C (O) O-, -OC (O) -, or a bond of Valencia; where s2 is 0 or 1.

In another embodiment W ¹ is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) ² NHC (O) - or a valence bond. Typically, W ¹ is selected from -C (O) NH-, -NHC (O) -, -C (O) n Hs (O) ² -,

In another embodiment W ² is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) ² NHC (O) - or a valence bond. Typically, W ² is selected from -C (O) NH-, -NHC (O) -, -C (O) n Hs (O) ² -,

In another embodiment W3 is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) ² NHC (O) - or a valence bond. Typically, W ³ is selected from -C (O) NH-, -NHC (O) -, -C (O) n Hs (O) ² -,

In another embodiment W4 is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) ² NHC (O) - or a valence bond. Typically, W ⁴ is selected from -C (O) NH-, -NHC (O) -, -C (O) n Hs (O) ² -,

In another embodiment W5 is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) ² NHC (O) - or a valence bond. Typically, W ⁵ is selected from -C (O) NH-, -NHC (O) -, -C (O) n Hs (O) ² -,

In another embodiment W6 is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) ² NHC (O) - or a valence bond. Typically, W6 is selected from -C (O) NH-, -NHC (O) -, -C (O) n Hs (O) ² -,

In another embodiment, R 1 is selected from hydrogen, -C (O) OH, -C (O) NH ² , -S (O) ² OH or C ^1-6 -alkyl; where the group

alkyl is optionally substituted with -C (O) OH, -C (O) NH ² , -S (O) ² OH. Typically, R 1 is selected from -C (O) OH, -C (O) NH ² , or C ^1-6 -alkyl; wherein the alkyl group is optionally substituted with -C (O) OH, -C (O) NH ² , or -S (O) ² OH.

In another embodiment R2 is selected from hydrogen, -C (O) OH, -C (O) NH ² , -S (O) ² OH or C ^1-6 -alkyl; where the group

alkyl is optionally substituted with -C (O) OH, -C (O) NH ² , -S (O) ² OH. Typically, R 2 is selected from -C (O) OH, -C (O) NH ² , or C ^1-6 -alkyl; wherein the alkyl group is optionally substituted with -C (O) OH, -C (O) NH ² , or -S (O) ² OH.

In another embodiment R3 is selected from hydrogen, -C (O) OH, -C (O) NH ² , -S (O) ² OH or C ^1-6 -alkyl; where the group

alkyl is optionally substituted with -C (O) OH, -C (O) NH ² , -S (O) ² OH. Typically, R 3 is selected from -C (O) OH, -C (O) NH ² , or C ^1-6 -alkyl; wherein the alkyl group is optionally substituted with -C (O) OH, -C (O) NH ² , or -S (O) ² OH.

In another embodiment R 4 is selected from hydrogen, -C (O) OH, -C (O) NH ² , -S (O) ² OH or C ^1-6 -alkyl; where the group

alkyl is optionally substituted with -C (O) OH, -C (O) NH ² , -S (O) ² OH. Typically, R 4 is selected from -C (O) OH, -C (O) NH ² , or C ^1-6 -alkyl; wherein the alkyl group is optionally substituted with -C (O) OH, -C (O) NH ² , or -S (O) ² OH.

In another embodiment R 5 is selected from hydrogen, -C (O) OH, -C (O) NH ² , -S (O) ² OH or C ^1-6 -alkyl; where the group

alkyl is optionally substituted with -C (O) OH, -C (O) NH ² , -S (O) ² OH. Typically, R 5 is selected from -C (O) OH, -C (O) NH ² , or C ^1-6 -alkyl; wherein the alkyl group is optionally substituted with -C (O) OH, -C (O) NH ² , or -S (O) ² OH.

In another embodiment E1 and E2 are independently selected from -O-, -NR6-, -N- (COR7) - or a valence bond. R6

and R7 independently represent hydrogen or C ^1-6 -alkyl.

In another embodiment E1 is selected from -O- or -NR6- or a valence bond. Typically, E1 is selected from -O-.

In another embodiment E2 is selected from -O- or -NR6- or a valence bond. Typically, E2 is selected from -O-.

In another mode E1 and E2 are both -O-.

In another modality E1 and E2 are both -NR6-.

In another embodiment F is phenyl, pyrrolidine-2,5-dione or a valence bond.

In another embodiment D1 is selected from -O-, -NR6-, -N (COR7) - or a valence bond.

In another embodiment D1 is selected from -O- or -NR6- or a valence bond. Typically, D1 is selected from -NR6. In another embodiment D2 is selected from -O-, -NR6-, -N (COR7) - or a valence bond.

In another embodiment D2 is selected from -O- or -NR6- or a valence bond. Typically, D2 is selected from -NR6.

R6 and R7 independently represent hydrogen or Ci-6-alkyl,

In another embodiment I1 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment I2 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment I3 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment I4 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment I5 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment I6 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment m1 is 0-6, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment m2 is 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In another mode m3 is 0-5, such as 0, 1, 2, 3, 4, 5 or 6.

In another mode m4 is 0-5, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment m5 is 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In another embodiment m6 is 0-5, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment m7 is 0-5, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment n1 is 0-10, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment n2 is 0-10, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment n3 is 0-10, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment n4 is 0-10, such as 0, 1, 2, 3, 4, 5 or 6.

In another embodiment X ¹ is -W1 - [(CHR1) n-W2] m1 - {[(CH2) n1O] m2 - [(CHR 2) l2-W3] m3} N2- and X ² is - [(CHR 3) l3 -W4] m4 - {[(CH2) n3O] m5 - [(CHR4) i4-W5] m6} n4-, where - {[(CH2) n1O] m2 - [(CHR2) i2-W3] m3} n2 - and - {[(CH2) n3O] m5 - [(CHR4) i4-W5] m6} n4- are selected from,

where * is understood to denote a point of union, that is, an open link.

In another embodiment the molar weight of said hydrophilic spacer is in the range of 80 Daltons (D) to 1500 D or in the range of 500 D to 1100 D.

Still in another modality W has the formula

-W7-Y-,

where

Y is - (CH2) I7-C3-10-cycloalkyl-W8- or a valence bond,

I7 is 0-6,

W7 is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -,

-C (O) NHS (O) 2-, -S (O) 2NHC (O) -, -OC (O) NH-, -NHC (O) O-, -C (O) CH2-, -CH2C ( O) -, -C (O) CH = CH-, -CH = CHC (O) -, - (CH2) s3-, -C (O) -, -C (O) O-, -o C (O ) -, or a valence link; where s3 is 0 or 1,

W8 is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, - C (O) NHS (O) ² -, - S (O) ² NHC (O) -, -OC (O) NH-, -NHC (O) O-, -C (O) CH ² -, -CH ² C (O) -, -C (O) CH = CH-, -CH = CHC (O) -, - (CH ² ) s ⁴ -, -C (O) -, -C (O) O-, -OC (O) -, or a valence bond ; where s4 is 0 or 1.

In a W Y modality it is - (CH2) I7-cyclohexyl-W8-.

In another modality, Y is a valence bond.

In a W7 modality it is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) ² NHC (O) - or a valence bond. Typically, W ⁷ is selected from -C (O) NH-, -NHC (O) -, -C (O) NHS (O) ² . In another embodiment W8 is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) ² NHC (O) - or a valence bond. Typically, W8 is selected from -C (O) NH-, -NHC (O) -, -C (O) NHS (O) ² . In another modality, l7 is 0 or 1.

In another embodiment the hydrophilic spacer B of the present invention is selected from

where * is understood to denote a point of union, that is, an open link.

The albumin binding residue (substituent A in formula (I) or (II) above) linked to the growth hormone compound of the present invention is a lipophilic residue, which binds non-covalently to albumin. Typically, the albumin binding residue is negatively charged at physiological pH, and has a binding affinity to human serum albumin that is below about 10 pM or even below about 1 pM.

In another embodiment of the growth hormone compound of the present invention the albumin binding residue is selected from a straight chain alkyl group, a branched alkyl group, a group having a wcarboxylic acid group or an acid isosteroid. w-carboxylic Typically, the albumin binding residue has from 6 to 40 carbon atoms. In another embodiment, the albumin binding residue has from 8 to 26 carbon atoms. In another embodiment, the albumin binding residue has from 8 to 20 carbon atoms.

In another embodiment A has from 14 to 26 carbon atoms and comprises a w-carboxylic acid group. In another embodiment A has from 14 to 26 carbon atoms and comprises an isostere of w-carboxylic acid, such as a tetrazole.

In another modality A is selected from

where * denotes the union to B to W.

The hydrophilic spacer (B) is preferably introduced at a position of the growth hormone compound (GH) in a selective manner to be able to control whether one, two, or three albumin binding residues (A) should be incorporated into the composed of growth hormone. The hydrophilic spacer (B) can be attached to a side chain of an amino acid of the GH compound. Said side chain of an amino acid may be a side chain of an amino acid that was chemically modified, of the GH compound. Another, of said side chain of an amino acid may be a side chain of an amino acid that was enzymatically modified, of the GH compound. Preferably, a transglutaminase is used to introduce a hydrophilic spacer into the glutamine residue at the position corresponding to position 40 or 141 in sec. with no. of ident. 1. Another way of selectively introducing a hydrophilic spacer is into the N-terminal residue of the growth hormone compound, such as hGH (sec.with ident # 1).

In the growth hormone conjugate of the formula (I) the fragment A-W-B can be linear or branched. In one embodiment, A-W-B is not a linear peptide.

In another embodiment the albumin binding residue by means of a hydrophilic spacer is attached to the glutamine residue in the position corresponding to position 40 in sec. with no. of ident. one.

In another embodiment the albumin binding residue by means of a hydrophilic spacer is attached to the glutamine residue in the position corresponding to position 141 in sec. with no. of ident. one.

In another embodiment, the albumin binding residue by means of a hydrophilic spacer is attached to the N-terminal residue of the growth hormone compound, such as hGH (sec.with ident # 1).

In another embodiment the albumin binding residue by means of a hydrophilic spacer is attached to the glutamine residue in the position corresponding to position 40 and to the glutamine residue in the position corresponding to position 141 in sec. with no. of ident. one.

In another embodiment the albumin binding residue by means of a hydrophilic spacer is attached to the glutamine residue at the position corresponding to position 40 and to the N-terminal residue of the growth hormone compound, such as hGH (sec. identification number 1).

In another embodiment the albumin binding residue by means of a hydrophilic spacer is attached to the glutamine residue at the position corresponding to position 141 and to the N-terminal residue of the growth hormone compound, such as hGH (sec. identification number 1).

The growth hormone conjugates of the present invention are selected from

In another aspect the present invention relates to a conjugate of a growth hormone comprising a growth hormone compound (GH) linked to an albumin binding residue by means of a hydrophilic spacer, or a salt, solvate or prodrug of this pharmaceutically acceptable for use in therapy. In addition, in the conjugate of a growth hormone of the present invention Gh, the albumin binding residue, and the hydrophilic spacer are selected from any of the above embodiments, in particular the conjugate of a growth hormone has the formula ( I) or (II).

In another aspect the present invention relates to a pharmaceutical composition comprising a conjugate of a growth hormone comprising a compound of growth hormone (GH) bound to an albumin binding residue by means of a hydrophilic spacer, or a a pharmaceutically acceptable salt, solvate or prodrug thereof, optionally in combination with an acceptable pharmaceutical excipient.

The term "identity" as known in the art, refers to a relationship between the sequences of two or more proteins, as determined by comparison of the sequences. In the art, "identity" also refers to the degree of sequence relationship between the proteins, as determined by the number of matches between the strands of two or more amino acid residues. The "identity" measures the percent of identical matches between the smallest of two or more sequences with the alignments of gaps (if any) directed by a particular mathematical model or a computer program (ie, "algorithms"). ). The identity of related proteins can be easily calculated by known methods. Such methods include those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math., 48, 1073, (1988).

The preferred methods for determining identity are designed to give the greatest match between the sequences analyzed. Methods for determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, which includes GAP (Devereux et al., Nucl.Acid.Res., 12, 387, (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLAs Tp, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215, 403-410, (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIH Bethesda, MD 20894, Altschul et al., Supra ). The well-known algorithm of Smith Waterman can also be used to determine identity.

For example, by using the GAP computer algorithm (Genetics Computer Group, University of Wisconsin, Madison, Wis.), Two proteins, for which the percent sequence identity is to be determined, are aligned for an optimal match of their respective amino acids (the "matching range", as determined by the algorithm). A gap opening penalty (calculated as 3 times the average diagonal, the "average diagonal" is the average of the diagonal of the comparison matrix used, the "diagonal" is the score or number assigned to each match perfect of amino acids by the comparison matrix in particular) and a gap extension penalty (which is usually {the fraction (1/10)} times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 together with the algorithm. The algorithm also uses a standard comparison matrix (see Dayhoff et al., Atlas of Protein Sequence and Structure, vol.5, supp.3 (1978) for the PAM 250 comparison matrix, Henikoff et al., Proc. Natl. Acad. Sci USA, 89, 10915-10919, (1992) for the BLOSUM comparison matrix 62).

Preferred parameters for a protein sequence comparison include the following:

Algorithm: Needleman et al., J. Mol. Biol, 48, 443-453, (1970); Comparison matrix: BLOSUM 62 by Henikoff et al., Proc. Natl. Acad. Sci. USA, 89, 10915-10919, (1992); Penalty for gap: 12, penalty for length of hole: 4, Threshold of similarity: 0.

The GAP program is useful with the above parameters. The aforementioned parameters are the default parameters for protein comparisons (together with the absence of penalty for gaps at the ends) with the use of the GAP algorithm.

The compounds of the present invention have improved pharmacological properties compared to the corresponding unconjugated growth hormone, which is also referred to as the parent compound. The examples of such pharmacological properties include functional half-life in vivo, immunogenicity, renal filtration, protease protection and albumin binding.

The term "functional in vivo half-life " is used in its normal meaning, ie, the time when 50% of the biological activity of the GH or GH conjugate is still present in the target body / organ, or the time when the activity of the GH or GH conjugate is 50% of its initial value As an alternative to determine the functional half-life in vivo, the "half-life in vivo in plasma" can be determined, that is, the time when 50% of the GH or GH conjugate circulates in the plasma or bloodstream before being eliminated. The determination of the half-life in plasma is often simpler than determining the functional half-life and the magnitude of the half-life in plasma is usually a good indication of the magnitude of the functional half-life in vivo. Alternative terms to the half-life in plasma include serum half-life, average lifespan in circulation, circulating half-life, serum elimination, plasma elimination and elimination half-life.

The term "increase" as used in conjunction with the functional half-life in vivo or the half-life in plasma is used to indicate that the relevant half-life of the GH conjugate significantly increased statistically with respect to that of the Original GH, as determined under comparable conditions For example the relevant half-life can be increased by at least about 25%, such as by at least about 50%, for example, by at least about 100%, 150%, 200 %, 250%, or 500% In one embodiment, the compounds of the present invention show an increase in the half-life of at least about 5 hours, preferably at least about 24 hours, more preferably at least about 72 hours , and most preferably at least about 7 days, with respect to the half-life time of the original GH.

The measurement of the average life time in plasma in vivo can be carried out in several ways as described in the literature. An increase in plasma half-life in vivo can be quantified as a decrease in clearance (CL) or as an increase in mean residence time (MRT). The conjugated GH of the present invention for which CL decreases to less than 70%, such as less than 50%, such as less than 20%, such as less than 10% of the CL of the original GH as determined in A suitable assay is said to have an increased in vivo plasma half-life. The conjugated GH of the present invention for which the MRT increases to more than 130%, such as more than 150%, such as more than 200%, such as more than 500% of the MRT of the original GH in a suitable assay is it says that it has an increased half-life in plasma in vivo . The elimination and the average residence time can be assessed in standard pharmacokinetic studies by the use of suitable test animals. Within the capabilities of a person skilled in the art is to choose a test animal suitable for a given protein. The tests on humans, of course, represent the final test. Suitable test animals include male Sprague-Dawley rats, mice and all normal macaque monkeys. Typically mice and rats are injected in a single subcutaneous bolus, whereas monkeys can be injected in a single subcutaneous bolus or in a single iv dose. The amount injected depends on the test animal. Subsequently, blood samples are drawn in a period of one to five days as appropriate for the evaluation of CL and MRT. The blood samples are conveniently analyzed by ELISA techniques.

The term "immunogenicity" of a compound refers to the ability of the compound, when administered to a human being, to elicit a detrimental immune response, whether humoral, cellular, or both.In any human subpopulation, there may be individuals exhibiting sensitivity to particular proteins administered Immunogenicity can be measured by quantification of the presence of antibodies against growth hormone and / or growth hormone-sensitive T cells in a sensitive individual, using conventional methods known in the art. In one embodiment, the conjugated GH of the present invention shows a decrease in immunogenicity in a sensitive individual of at least about 10%, preferably at least about 25%, more preferably at least about 40% and most preferably at least approximately 50%, with respect to the immunogenicity of the individual of the GH origin to the.

The terms "protease protection" or "protease protease" as used herein indicate that the conjugated GH of the present invention is more resistant to peptidase or plasma proteases than the original GH. It is known that proteases and peptidases enzymes present in plasma are involved in the degradation of circulating proteins, such as circulating peptide hormones, such as growth hormone.

Growth hormone may be susceptible to degradation, for example, by thrombin, plasmin, subtilisin and serine proteinase similar to chymotrypsin. Assays for the determination of the degradation of these proteases are described in J. Biotech., 65, 183, (1998). In one embodiment, the hydrolysis rate of the GH conjugate is less than 70%, such as less than 40%, such as less than 10% of that of the original GH.

The most abundant protein component in the circulating blood of mammalian species is serum albumin, which is normally present at a concentration of about 3 to 4.5 grams per 100 milliliters of whole blood. Serum albumin is a blood protein of approximately 70,000 daltons that has numerous important functions in the circulatory system. It functions as a transporter of a variety of organic molecules that are found in the blood, as the main transporter of various metabolites such as fatty acids and bilirubin through the blood, and, because of its abundance, as an osmotic regulator of circulating blood. Serum albumin has a half-life of more than a week, and one approach to increasing the plasma half-life of proteins has been to conjugate the protein to a group that binds to serum albumin. The albumin binding property can be determined as described in J. Med. Chem., 43, 1986, (2000).

The conjugates of the growth hormone of formula (I) or (II) exert a growth hormone activity and as such can be used in the treatment of diseases or conditions that will benefit from an increase in the amount of circulating growth hormone. It is noted that any reference herein to methods of treatment refers to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a treatment method.

In particular, the invention provides for use in a method for the treatment of growth hormone deficiency (GHD); Turner syndrome; Prader-Willi syndrome (PWS); Noonan syndrome; Down's Syndrome; chronic kidney disease, juvenile rheumatoid arthritis; cystic fibrosis, HIV infection in children receiving HAART treatment (children with HIV / HALS); children born small for gestational age (SGA); small stature in children born with very low birth weight (VLBW) except SGA; skeletal dysplasia; hypochondroplasia; achondroplasia; idiopathic small stature (ISS); GHD in adults; fractures in or of long bones, such as tibia, fibula, femur, humerus, radius, ulna, clavicle, metacarpus, metatarsus, and digits; fractures in or of spongy bones, such as the skull, base of the hand, and base of the foot; patients after tendon or ligament surgery in for example the hand, knee, or shoulder; patients who have or suffer from osteogenesis due to distraction; patients after a hip or disc replacement, meniscus repair, spinal fusion or prosthetic fixation, such as in the knee, hip, shoulder, elbow, wrist or jaw; patients in whom osteosynthesis materials have been fixed, such as nails, screws and plates; patients without union or with a bad union of the fractures; patients after an osteotomy, for example of the tibia or the 1st toe; patients after implanting grafts; degeneration of articular cartilage in the knee caused by trauma or arthritis; osteoporosis in patients with Turner syndrome; osteoporosis in men; adult patients on chronic dialysis (APCD); cardiovascular disease associated with poor nutrition status in APCD; reversal of cachexia in APCD; cancer in APCD; chronic obstructive pulmonary disease in APCD; HIV in APCD; elderly with APCD; chronic liver disease in APCD, fatigue syndrome in APCD; Crohn's disease; affected liver function; men with HIV infections; short bowel syndrome; central obesity; HIV-associated lipodystrophy syndrome (HALS); male infertility; patients after elective major surgery, alcohol / drug detoxification or neurological trauma; aging; Weak old age osteoarthritis; cartilage with traumatic damage; erectile dysfunction; fibromyalgia; memory disorders; depression; traumatic brain injury; subarachnoid hemorrhage; very low birth weight; metabolic syndrome; glucocorticoid myopathy; or small stature due to glucocorticoid treatment in children, the method comprising administering to a patient in need thereof a therapeutically effective amount of a growth hormone conjugate according to formula (I) or (II).

In another aspect, the disclosure provides a method for accelerating the healing of muscle tissue, nerve tissue or wounds; the acceleration or improvement of blood flow to the injured tissue; or the decrease in infection rate in damaged tissue, the method comprises administering, to a patient in need thereof, a therapeutically effective amount of a conjugate of a growth hormone of formula (I) or (II).

In another embodiment, the invention relates to the use of a conjugate of a growth hormone of formula (I) or (II) in the production of a medicament for the treatment of diseases that benefit from an increase in the plasma level of the growth hormone, as the diseases mentioned above.

A typical parenteral dose is in the range of 10-9 mg / kg to about 100 mg / kg of body weight per administration. Typical administration doses are from about 0.0000001 to about 10 mg / kg body weight per administration. The exact dose will depend for example on the indication, the medication, the frequency and mode of administration, the sex, the age and the general condition of the subject to be treated, the nature and severity of the disease or condition that is being treated. will treat, the desired effect of the treatment and other factors evident to the person skilled in the art.

Typical dosing frequencies are twice a day, once a day, twice daily, twice weekly, once a week or even at longer dosing intervals. Due to the extended half-lives of the fusion proteins of the present invention, a dosing regimen with long dosing intervals, such as twice a week, once a week or even with longer dosing intervals is a particular embodiment of the invention.

Many diseases are treated with the use of more than one medication in the treatment, whether they are administered concomitantly or sequentially. Therefore, the use of a conjugate of a growth hormone of formula (I) or (II) in therapeutic methods for the treatment of one of the aforementioned diseases in combination with one or more other therapeutically active compounds normally used is described. in the treatment of these diseases. By analogy, the use of a conjugate of a growth hormone of formula (I) or (II) in combination with other therapeutically active compounds used is further described. normally in the treatment of one of the diseases mentioned above in the production of a medicament for said disease.

General methods of preparation

Enzymatic Conjugation:

In the preparation of a growth hormone conjugate of the present invention, typically at least one of the covalent bonds established in the preparation of an AWB-GH conjugate of formula (I) is prepared by the use of an enzyme as illustrated in the examples below. This enzyme, for example, can be selected from the group consisting of transglutaminases, serine proteases and cysteine proteases. Typically, said enzyme is a transglutaminase. Said transglutaminase for example can be selected from the group consisting of microbial transglutaminases, tissue transglutaminases and factor XIII and variants thereof. In another embodiment, said enzyme is a cysteine protease. A cysteine protease of this type, for example, can be selected from the group consisting of papain, sortasa A and sortasa B. In another embodiment, said enzyme is a serine protease. This serine protease, for example, can be selected from the group consisting of carboxypeptidase Y (CPY) (PCT Application No. WO2005 / 035553 contains a general description of protein modification by the use of CPY), trypsin and chymotrypsin.

The conjugate of a growth hormone of the present invention can be prepared by many different methods, examples are shown below.

The present invention further provides methods for preparing conjugates of A-W-B-GH of formula (I).

Transglutaminase

As noted above, at least one of the covalent bonds established in the preparation of an AWB-GH conjugate of the present invention can be prepared by the use of a transglutaminase. Transglutaminases may include microbial transglutaminases such as that isolated from the Streptomyces species; S. mobaraense, S. cinnamoneum, S. griseocarneum (US5156956), S. lavendulae (US5252469) and Streptomyces ladakanum (JP2003 / 199569). Other useful microbial transglutaminases have been isolated from Bacillus subtilis (described in US5731183) and from various Mixomycetes. Other examples of useful microbial transglutaminases are those described in WO96 / 06931 (for example Bacillus lydicus transglutaminase) and WO96 / 22366. Useful non-microbial transglutaminases include guinea pig transglutaminase, and transglutaminases from various marine sources such as the Japanese snapper Pagrus major (described in EP0555649), and the Japanese oyster Crassostrea gigas (described in US5736356). Functional analogs and their derivatives may also be useful.

Typically, the TGase used in the methods of the invention is a microbial transglutaminase. In one embodiment, the TGase is from S. mobaraense or a variant thereof, for example as described in WO2007 / 020290 and WO2008 / 020075. In another embodiment, the TGase is from S. ladakanum or a variant thereof, for example as described in WO2008 / 020075.

The conjugation of hGH to AWB according to the present invention can be achieved by TGase-mediated modification which leads to a selective alteration at specific positions of lysine (Lys) or glutamine (Gln) in the sequence of the GH compound depending on the substrate used. The use of amines as substrates will lead to the modification of glutamines while the use of primary amides will lead to the modification of lysines. hGH (sec. with ident 1) has 9 lysine residues at positions 38, 41, 70, 115, 140, 145, 158, 168 and 172 and 13 glutamine residues at positions 22, 29, 40 , 46, 49, 68, 69, 84, 91, 122, 137, 141 and 181. Not all are readily available for modification or suitable for modifications as this will lead to decreased protein binding power of binding to growth hormone, therefore leads to reduced biological activity. The structure of protein crystals by X-ray between hGH and its binding protein (pdb: 3HHR) reveals that at least 4 lysines (38, 41, 168 and 172) take part in binding to the binding protein and potentially only one of glutamines (Gln 46). This makes the glutamines more attractive as a target for the selective introduction of an albumin binding linker. These structural considerations are further supported by the findings summarized by N. Chéne and others in Reprod. Nutr. Develop. 29, 1-25 (1989) where it is concluded that the chemical modifications affecting the lysines have been found to have a negative effect on the biological activity in vivo and on the capacity of binding to the hepatic receptors of GH.

Chemistry I

In one aspect the present invention relates to a preparation of a growth hormone conjugate of formula (VI) wherein a GH compound is treated with an aldehyde or ketone derived from a property modifying group to produce an amine, imine or a hemiaminal.

In another aspect the present invention relates to a preparation of a conjugate of a growth hormone of formula (VI) comprising the treatment of an aldehyde or ketone derived from the compound of GH with an aniline or heteroarylamine derived from a group modifying the properties to produce an amine, imine or a hemiaminal.

In one embodiment, an aldehyde derived from the compound of GH (IV) is treated with an aniline or heteroarylamine derived from a group modifying the properties (V).

The term "aldehyde (or ketone) derived from a compound of GH" or "an aldehyde or ketone derived from the compound of GH" is meant to indicate a GH compound to which a functional group of aldehyde or ketone has been covalently bound, or a GH compound on which an aldehyde or ketone functional group has been generated The preparation of aldehydes derived from a GH compound, such as the compound (IV) illustrated below is well known to those skilled in the art, and any of these known methods can be used to prepare the aldehyde derived from a compound of GH (IV) necessary for the embodiment of the invention described herein.

In one embodiment, the conjugate A-W-B-hGH (VI) is prepared as illustrated below:

The enzymatic reaction with GH mediated by TGase (I) results in the modification of the Gln at position 141 and / or 40 to produce GH (II). The conjugation of oxidized GH with periodate (II) with A-W-B-NH2 (V) is produced by means of reductive alkylation. Reductive alkylation is exemplified herein and is well known in the art and results in g H (VI) compounds modified at the Gln (141) and / or 40 position.

Chemistry II

In one embodiment, the AWB-hGH conjugate is prepared as illustrated below:

The derivatization process as shown above provides a binding linker to albumin bound to a hGH compound in the Phe (1) position.

It is generally considered that a close relationship with the natural peptide is an advantage with therapeutic interventions comprising the administration of variants or analogs of this natural peptide as it minimizes the risk, for example, of any unwanted generation of antibodies.

Many nucleophilic compounds are known that can be incorporated into the peptides according to the methods of the present invention, and the α-amino acids are one of those types of nucleophilic compounds. However, for the purpose of the present invention, it is preferred to select the nucleophilic compound so that the transacylated compound that is formed is not a substrate for the applied enzyme. In other words, it is preferred to apply a nucleophilic compound that effectively blocks any other reaction of the enzyme. An example of such compounds are the amides of α-amino acids since the carboxy-amidated peptides are not substrates of the carboxypeptidases.

It is recognized that the fact that a compound is a substrate for a given enzyme or not, in principle, depends on the conditions, for example the time frame, in which the reaction takes place. Given sufficient time, many compounds are, in fact, substrates for an enzyme although under normal conditions they are not considered as such. When it is stated above that the transaclylated compound itself should not be a substrate of the enzyme it is understood that this indicates that the transaclylated compound itself is not a substrate for the enzyme to the extent that the following reactions in the method of the present invention. If the transacylated compound is, in fact, a substrate for the enzyme, the enzyme can be removed or inactivated, for example by enzymatic inhibitors, after the transacylation reaction.

Pharmaceutical compositions

Another purpose is to provide a pharmaceutical composition comprising a conjugate of a growth hormone of the present invention, such as a conjugate of a growth hormone of formula (I) or (II), which is present at a concentration of 10-15. mg / ml to 200 mg / ml, such as for example 10-10 mg / ml to 5 mg / ml and wherein said composition has a pH of 2.0 to 10.0. The composition may further comprise pharmaceutical excipients, such as a buffer system, preservative (s), tonicity agent (s), chelating agent (s), stabilizers and surfactants. In one embodiment of the invention, the pharmaceutical composition is an aqueous composition, ie a composition comprising water. Said composition is typically a solution or a suspension. In a further embodiment of the invention, the pharmaceutical composition is an aqueous solution. The term "aqueous composition" is defined as a composition comprising at least 50% w / w of water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50% w / w of water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w / w of water.

In another embodiment, the pharmaceutical composition is a lyophilized composition, to which the physician or patient adds solvents and / or diluents before use.

In another embodiment, the pharmaceutical composition is a dry composition (for example, lyophilized or spray-dried) ready for use without any prior dissolution.

In another aspect the invention relates to a pharmaceutical composition comprising an aqueous solution of a conjugate of a growth hormone, such as a conjugate of a growth hormone of formula (I) or (II), and a buffer, wherein said GH conjugate is present in a concentration of 0.1-100 mg / ml or higher, and wherein said composition has a pH of from about 2.0 to about 10.0.

In another embodiment of the invention the pH of the composition is selected from the list consisting of 2.0, 2.1, 2.2, 2.3,

In a further embodiment of the invention the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, hydrogen disodium phosphate, sodium phosphate and tris (hydroxymethyl) -aminomethane, bicine, tricine, melic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment of the invention.

In a further embodiment of the invention, the composition further comprises a pharmaceutically acceptable preservative. In a further embodiment of the invention, the preservative is selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl phydroxybenzoate. , 2-phenylethanol, benzyl alcohol, chlorobutanol and thiomersal, bronopol, benzoic acid, imidaurea, chlorhexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesin (3p-chlorphenoxypropane-1,2-diol) or its mixtures In a further embodiment of the invention the preservative is present in a concentration of 0.1 mg / ml to 20 mg / ml. In a further embodiment of the invention the preservative is present in a concentration of 0.1 mg / ml to 5 mg / ml. In a further embodiment of the invention the preservative is present in a concentration of 5 mg / ml to 10 mg / ml. In a further embodiment of the invention the preservative is present in a concentration of 10 mg / ml to 20 mg / ml. Each of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical compositions is well known to the experts. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.

In a further embodiment of the invention, the composition further comprises an isotonic agent. In another embodiment of the invention the isotonic agent is selected from the group consisting of a salt (for example sodium chloride), a sugar or alcohol sugar, an amino acid (for example L-glycine, L-histidine, arginine, lysine, isoleucine , aspartic acid, tryptophan, threonine),

an alditol (for example glycerol (glycerin), 1,2-propanediol (propylene glycol), 1,3-propanediol, 1,3-butanediol) polyethylene glycol (for example PEG 400), or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glycans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin can be used. , cyclodextrin, soluble starch, hydroxyethyl starch and Na carboxymethylcellulose. In one embodiment, the sugar additive is sucrose. The alcoholic sugar is defined as a C4-C8 hydrocarbon having at least one -OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol and arabitol. In one embodiment, the alcoholic sugar additive is mannitol. The sugars or alcoholic sugars mentioned above can be used individually or in combination. There is no fixed limit for the amount used, provided that the sugar or the alcoholic sugar is soluble in the liquid preparation and does not adversely affect the stabilizing effects achieved with the use of the methods of the invention. In one embodiment, the concentration of the sugar or the alcoholic sugar is between about 1 mg / ml and about 150 mg / ml. In a further embodiment of the invention, the isotonic agent is present in a concentration of 1 mg / ml to 50 mg / ml. In a further embodiment of the invention, the isotonic agent is present in a concentration of 1 mg / ml to 7 mg / ml. In a further embodiment of the invention, the isotonic agent is present in a concentration of 8 mg / ml to 24 mg / ml. In a further embodiment of the invention, the isotonic agent is present in a concentration of 25 mg / ml to 50 mg / ml. Each of these specific isotonic agents constitutes an alternative embodiment of the invention. The use of an isotonic agent in pharmaceutical compositions is well known to the experts. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.

In a further embodiment of the invention, the composition further comprises a chelating agent. In a further embodiment of the invention, the chelating agent is selected from salts of ethylenediaminetetraacetic acid (EDTA), citric acid and aspartic acid, and mixtures thereof. In a further embodiment of the invention, the chelating agent is present in a concentration of 0.1 mg / ml to 5 mg / ml. In a further embodiment of the invention, the chelating agent is present in a concentration of 0.1 mg / ml to 2 mg / ml. In a further embodiment of the invention, the chelating agent is present in a concentration of 2 mg / ml to 5 mg / ml. Each of these specific chelating agents constitutes an alternative embodiment of the invention. The use of a chelating agent in pharmaceutical compositions is well known to the experts. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.

In a further embodiment of the invention, the composition further comprises a stabilizer. The use of a stabilizer in pharmaceutical compositions is well known to the experts. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.

More particularly, the compositions of the invention are liquid stabilized pharmaceutical compositions whose components with therapeutic activity include a protein that possibly shows aggregate formation during storage in liquid pharmaceutical compositions. By the term "aggregate formation" is meant a physical interaction between protein molecules that results in the formation of oligomers, which may remain soluble, or large visible aggregates that precipitate out of the solution. By the term "during storage" is meant a liquid pharmaceutical composition or composition which, once prepared, is not immediately administered to a subject. In contrast, after preparation, it is packaged for storage, either in liquid form, in the frozen state, or in dry form for subsequent reconstitution in a liquid form or other form suitable for administration to a subject. By the term "dry form" is meant the liquid pharmaceutical composition or the composition that is dried either by freeze drying (i.e. lyophilization; see, for example, Williams and Polli, J. Parenteral Sci. Technol. 38, 48-59, (1984)), spray drying (see Masters (1991) in Spray-Drying Handbook (5th ed., Longman Scientific and Technical, Essez, UK), pp. 491 676; Broadhead et al. (1992) Drug Devel, Ind. Pharm., 18, 1169-1206, (1992), and Mumenthaler et al., Pharm. Res.

11, 12-20 (1994)), or air-drying (Carpenter and Crowe, Cryobiology 25, 459-470, (1988); and Roser, Biopharm., 4, 47, 53, (1991)). The formation of aggregates by a protein during the storage of a liquid pharmaceutical composition can adversely affect the biological activity of that protein, which results in the loss of therapeutic efficacy of the pharmaceutical composition. In addition, the formation of aggregates can cause other problems such as blocking of tubes, membranes or pumps when the pharmaceutical composition containing the protein is administered by the use of an infusion system.

The pharmaceutical compositions of the invention may further comprise an amount of an amino acid base sufficient to decrease aggregate formation by the protein during storage of the composition. By "amino acid base" is meant an amino acid or a combination of amino acids, wherein any given amino acid is present in its free base form or in its salt form. When a combination of amino acids is used, all amino acids can be present in their free base forms, all can occur in their salt forms, or some can be present in their free base forms, while others are present in their salt forms. In one embodiment, the amino acids for use in the preparation of the compositions of the invention are those carrying a charged side chain, such as arginine, lysine, aspartic acid and glutamic acid. Any stereoisomer (i.e., L or D isomer, or mixtures thereof) of a particular amino acid (methionine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof) or combinations of these stereoisomers or glycine or an organic base such as imidazole may be present in the pharmaceutical compositions of the invention provided that the particular amino acid or organic base is present in either its free base form or its salt form. In one embodiment, the stereoisomer L of an amino acid is used. In one embodiment, the stereoisomer L is used. The compositions of the invention can also be formulated with analogs of these amino acids. By "amino acid analogue" is meant an amino acid derivative of natural origin that produces the desired effect of decreasing aggregate formation by the protein during storage of the liquid pharmaceutical compositions of the invention. Suitable arginine analogs include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine, suitable methionine analogs include ethionine and butionine and suitable cysteine analogs include S-methyl-L cysteine. As with the other amino acids, amino acid analogs are incorporated into the compositions either in their free base form or in their salt form. In a further embodiment of the invention, amino acids or amino acid analogs are used in a concentration, which is sufficient to prevent or delay aggregation of the protein.

In a further embodiment of the invention, methionine (or other amino acids or sulfuric acid analogs) may be added to inhibit the oxidation of the methionine residues to methionine sulfoxide when the protein acting as a therapeutic agent is a protein comprising at least one residue of methionine susceptible to such oxidation. By "inhibit" is meant the minimum accumulation of oxidized methionine species over time. Inhibition of methionine oxidation results in increased retention of the protein in its proper molecular form. Any stereoisomer of methionine (L or D isomer) or any combination thereof can be used. The amount that must be added must be an amount sufficient to inhibit the oxidation of the methionine residues so that the amount of methionine sulphoxide is acceptable to the regulatory agencies. Typically, this means that the composition contains no more than about 10% to about 30% methionine sulfoxide. Generally, this can be achieved by the addition of methionine so that the ratio between the methionine added and the methionine residues varies from about 1: 1 to about 1000: 1, such as 10: 1 to about 100: 1.

In a further embodiment of the invention, the composition further comprises a stabilizer selected from the group of high molecular weight polymers or low molecular weight compounds. In a further embodiment of the invention the stabilizer is selected from polyethylene glycol ( e.g. , PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy / hydroxycellulose or its derivatives ( e.g. , HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulfur-containing substances such as monothioglycerol, thioglycolic acid and 2-methylthioethanol, and different salts ( e.g. , sodium chloride). Each of these specific stabilizers constitutes an alternative embodiment of the invention.

The pharmaceutical compositions may further comprise additional stabilizing agents, which further improve the stability of a therapeutically active protein therein. Stabilizing agents of particular interest for the present invention include methionine and EDTA, which protect the protein against oxidation of methionine, and a nonionic surfactant, which protects the protein against aggregation associated with freezing, thawing or mechanical shearing.

In a further embodiment of the invention, the composition further comprises a surfactant. In another embodiment of the invention the surfactant is selected from a detergent, ethoxylated castor oil, polyglycolized glycerides, acetylated monoglycerides,

sorbitan esters and fatty acid, polyoxypropylene-polyoxyethylene block polymers (for example, poloxamers such as Pluronic® F68, poloxamer 188 and 407, Triton X-100), polyoxyethylene sorbitan esters and fatty acid, derivatives of polyoxyethylene and polyethylene as alkylated and alkoxylated derivatives (polysorbates, for example Tween-20, Tween-40, Tween-80 and Brij-35), monoglycerides or their ethoxylated derivatives, diglycerides or their polyoxyethylene derivatives, alcohols, glycerol, lectins and phospholipids (eg, phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, diphosphatidyl glycerol and sphingomyelin), phospholipid derivatives (eg, dipalmitoyl phosphatidic acid) and lysophospholipids (eg palmitoyl lysophosphatidyl-L-serine and 1-acyl) -sn-glycero-3-phosphate esters of ethanolamine, choline, serine or threonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkyl ether) - derivatives of lysophosphatidyl and phosphatidylcholine, for example lauroyl and myristoyl derivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and modifications of the polar head group, ie hills, ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositol, and DODAC, DOTMA, DCP, BISHOP positively charged, lysophosphatidylserine and lysophosphatidyltreonine, and glycerophospholipids (eg, cephalins), glyceroglycolipids (eg, galactopyranoside), sphingoglycolipids (eg, ceramides, gangliosides), dodecylphosphocholine, chicken egg lysolecithin, fusidic acid derivatives - (eg sodium taurodihydrofusidate, etc.), fatty acids and long chain C ⁶ -C salts ¹² (e.g., oleic acid and caprylic acid), acylcarnitines and derivatives, lysine, arginine or histidine, Na-acylated, or acylated lysine or arginine derivatives in the side chain, Na-acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, Na-acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, DSS (sodium docusate, CAS Registry no. [577-11-7]), calcium docusate, record c As núm. [128-49-4]), potassium docusate, CAS Registry no. [7491 09-0]), SDS (sodium dodecyl sulfate or sodium lauryl sulfate), sodium caprylate, cholic acid or derivatives thereof, bile acids and their salts and conjugates of glycine or taurine, ursodeoxycholic acid, sodium cholate, sodium deoxycholate, sodium taurocholate, sodium glycocholate, N-hexadecyl-N, N-dimethyl-3-ammonium-1-propanesulfonate, monovalent surfactants (alkyl-aryl sulfonates) anionic, zwitterionic surfactants (for example N-alkyl- N, N-dimethylammonium-1-propanesulfonates, 3-colamido-1-propyldimethylammonium-1-propanesulfonate, cationic surfactants (quaternary ammonium bases) (for example cetyltrimethylammonium bromide, cetylpyridinium chloride), nonionic surfactants (e.g. , dodecyl pD-glucopyranoside), poloxamines (for example, from Tetronic), which are tetrafunctional block copolymers derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine, or the surfactant can be selected from the derivative group two of imidazoline, or their mixtures. Each of these specific surfactants constitutes an alternative embodiment of the invention.

The use of a surfactant in pharmaceutical compositions is well known to the experts. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.

It is possible that other ingredients may be present in the pharmaceutical composition of the present invention. These additional ingredients may include wetting agents, emulsifiers, antioxidants, dough agents, tonicity modifiers, chelating agents, metal ions, oil vehicles, proteins ( e.g. , human serum albumin, gelatin or proteins) and a zwitterion (e.g. an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine). These additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical composition of the present invention.

Pharmaceutical compositions containing a conjugate of a growth hormone according to the present invention can be administered to a patient in need of such treatment at various sites, for example, at topical sites, eg, skin and mucosal sites, at sites that elude absorption, for example, administration in an artery, in a vein, in the heart and in sites that involve absorption, for example, administration to the skin, under the skin, in a muscle or in the abdomen.

The administration of pharmaceutical compositions according to the invention can be carried out through several routes of administration, for example, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through of the bronchioles and alveoli or their combination, epidermal, dermal, transdermal, vaginal, rectal, ocular, for example through the conjunctiva, urethral, and parenteral to patients who need such treatment.

The compositions of the present invention can be administered in various dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsions, foams, ointments, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules, for example , hard gelatine capsules and soft gelatin capsules, suppositories, rectal capsules, drops, gels, sprays, powders, aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, solution for injection, transforming solutions in situ, for example, in situ gelation, in situ placement, in situ precipitation, in situ crystallization, infusion solution, and implants.

The compositions of the invention may additionally be combined, or linked, for example, by covalent, hydrophobic and electrostatic interactions, with a pharmaceutical carrier, drug delivery system and advanced drug delivery system to further improve the stability of the conjugate of the drug. growth hormone, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any of their combinations. Examples of carriers, drug delivery systems and advanced drug delivery systems include polymers, for example, cellulose and derivatives, polysaccharides, e.g., dextran and derivatives, starch and derivatives, poly (vinyl alcohol), acrylate polymers and methacrylate, polylactic and polyglycolic acid and its block copolymers, polyethylene glycols, carrier proteins, for example, albumin, gels, for example, thermogelling systems, for example block copolymer systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticles, liquid crystals and its dispersions, phase L2 and its dispersions, well known to those skilled in the art of phase behavior in lipid and water systems, polymeric micelles, multiple emulsions, self-emulsifiers, self-emulsifiers, cyclodextrins and their derivatives, and dendrimers.

The compositions of the present invention are useful in the composition of solids, semisolids, powder and solutions for the pulmonary administration of the growth hormone conjugate, through the use, for example, of a fixed dose inhaler, a dry powder inhaler. and a nebulizer, all of which devices are well known to those skilled in the art.

The compositions of the present invention are specifically useful in the composition of controlled, sustained, prolonged, delayed and slow release drug delivery systems. More specifically, the compositions are useful in the composition of controlled release and parenteral sustained release systems (both systems lead to a reduction in many times the amount of administrations), well known to those skilled in the art. Even more preferably, they are controlled release and sustained release systems administered subcutaneously. Without limiting the scope of the invention, examples of useful controlled release systems and compositions are hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres, nanoparticles.

Methods for producing controlled release systems useful for the compositions of the present invention include, crystallization, condensation, co-crystallization, precipitation, coprecipitation, emulsification, dispersion, high pressure homogenization, encapsulation, spray drying, microencapsulation, coacervation, phase separation. , evaporation of the solvent to produce microspheres, extrusion and supercritical fluid processes. General reference is made to the manual of Pharmaceutical Controlled Release (Wise, DL, ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Composition and Delivery (MacNally, EJ, ed. Marcel Dekker, New York, 2000).

Parenteral administration can be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a syringe similar to a pen. Alternatively, parenteral administration can be performed by means of an infusion pump. Another option is a composition that can be a solution or suspension for the administration of the conjugate of a growth hormone in the form of a nasal or pulmonary atomizer. As yet another option, the pharmaceutical compositions containing the growth hormone conjugate of the invention can also be adapted to transdermal administration, for example, by injection without a needle or from a patch, optionally an iontophoretic patch, or transmucosal administration, by example, buccal.

The term "stabilized composition" refers to a composition with increased physical stability, increased chemical stability or increased physical and chemical stability.

The term "physical stability" of the protein composition as used in the present disclosure refers to the tendency of the protein to form biologically and / or insoluble inactive aggregates of the protein as a result of exposure of the protein to thermomechanical stress and / or interaction with interfaces and surfaces that are destabilizing, such as surfaces and hydrophobic interfaces. The physical stability of the aqueous protein compositions is evaluated by visual inspection and / or turbidity measurements after exposing the composition poured into suitable containers ( eg , cartridges or jars) to mechanical / physical stress ( e.g. , agitation). ) at different temperatures during various periods of time. The visual inspection of the compositions is done in a sharp light focused with a dark background. The turbidity of the composition is characterized by a visual rating by assigning a turbidity degree score for example on a scale of 0 to 3 (a composition that does not show turbidity corresponds to a visual score of 0, and a composition that visually shows a turbidity in daylight corresponds to a visual score of 3). A composition is classified as physically unstable with respect to protein aggregation, when it shows a visual turbidity in daylight. Alternatively, the turbidity of the composition can be assessed by simple measurements of turbidity well known to the experts. The physical stability of aqueous protein compositions can be further evaluated by the use of a spectroscopic agent or probe of the conformational state of the protein. The probe is preferably a small molecule that preferentially binds to an unnatural conformer of the protein. An example of a small molecular protein spectroscopic probe is thioflavin T. Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and perhaps also other protein configurations, Thioflavin T results in a new excitation maximum at about 450 nm and an enhanced emission at approximately 482 nm when bound to a fibrillar protein form. Unbound thioflavin T is essentially non-fluorescent at these wavelengths.

Other small molecules can be used as probes for changes in the structure of the protein from natural to unnatural states. For example, probes of "hydrophobic patches" that preferentially bind to patches hydrophobes exposed from a protein. Hydrophobic patches are generally buried within the tertiary structure of a protein in its natural state, but are exposed as a protein begins to unfold or denature. Examples of these small molecular spectroscopic probes are hydrophobic, aromatic dyes, such as anthracene, acridine, phenanthroline. Other spectroscopic probes are metal-amino acid complexes, such as complexes with the cobalt metal of hydrophobic amino acids, such as phenylalanine, leucine, isoleucine, methionine and valine.

The term "chemical stability" of the protein composition as used in the present disclosure refers to covalent chemical changes in the structure of the protein that lead to the formation of chemical degradation products with a possible lower biological potency and / or a Potential increase in immunogenic properties compared to the structure of the natural protein. Various chemical degradation products can be formed depending on the type and nature of the natural protein and the environment to which the protein is exposed. The removal of chemical degradation most likely can not be completely avoided and often increased amounts of chemical degradation products are observed during storage and use of the protein composition as is well known to the skilled artisan. Most proteins are prone to deamidation, a process in which the amide group on the side chain is hydrolyzed into glutaminyl or asparaginyl residues to form a free carboxylic acid. Other degradation pathways involve the formation of high molecular weight transformation products where two or more protein molecules are covalently linked together by transamidation and / or disulfide interactions leading to the formation of degradation products of dimers, oligomers and bound polymers covalently (Stability of Protein Pharmaceuticals, Ahern.TJ & MC, Plenun Press, New York, 1992). Oxidation (of, for example, methionine residues) can be mentioned as another variant of chemical degradation. The chemical stability of the protein composition can be assessed by measuring the amount of chemical degradation products at various time points after exposure to different environmental conditions (often the formation of degradation products can be accelerated, for example, by the increase in temperature). The amount of each individual degradation product is often determined by separation of the degradation products depending on the size of the molecule and / or the charge by the use of various chromatography techniques (e.g., SEC-HPLC and / or RP -HPLC).

Therefore, as noted above, a "stabilized composition" refers to a composition with increased physical stability, increased chemical stability or increased physical and chemical stability. In general, a composition must be stable during use and storage (in accordance with recommended use and storage conditions) until the expiration date is reached.

In one embodiment of the invention, the pharmaceutical composition comprising the growth hormone conjugate of formula (I) or (II) is stable for more than 6 weeks of use and for more than 3 years of storage.

In another embodiment of the invention, the pharmaceutical composition comprising the growth hormone conjugate of formula (I) or (II) is stable for more than 4 weeks of use and for more than 3 years of storage.

In a further embodiment of the invention, the pharmaceutical composition comprising the growth hormone conjugate of formula (I) or (II) is stable for more than 4 weeks of use and for more than two years of storage.

In yet another embodiment of the invention, the pharmaceutical composition comprising the growth hormone conjugate of formula (I) or (II) is stable for more than 2 weeks of use and for more than two years of storage.

The terms "a" and "an" and "the" and similar referents as used in the context of the description of the invention should be interpreted to cover both the singular and the plural, unless otherwise indicated in the present description or context contradicts it clearly.

The mention of ranges of values in the present description is only intended to serve as a quick writing method to refer individually to each separate value within the range, unless otherwise indicated in the present description, and each separate value is incorporated in the description as if individually mentioned in the present description. Unless stated otherwise, all of the exact values provided in the present description are representative of the corresponding approximate values (e.g., all of the illustrative exact values provided with respect to a particular factor or measurement can be considered to also provide a measure corresponding approximate, modified by "approximately", where appropriate).

All the methods described in the present description can be performed in any suitable order unless otherwise indicated in the present description or the context clearly contradicts it in any other way.

The use of any and all examples, or of an illustrative language (eg, "such as") provided in the present disclosure, is merely intended to better illuminate the invention. No language in the specification should be construed as indicating that any element is essential to the practice of the invention unless it is explicitly stated.

The citation and incorporation of patent documents in the present description is done only for convenience and does not reflect any point of view about the validity, patentability and / or enforceability of such patent documents.

The description further includes the modalities mentioned hereinafter which should not be construed as a description of the claimed invention.

A conjugate of a growth hormone comprising a growth hormone compound (GH) bound to an albumin binding residue by means of a hydrophilic spacer, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

2. The conjugate of mode 1 where the hydrophilic spacer has a mLogP <0 or a cLogP <0.5.

3. The conjugate of mode 1 or 2 wherein the growth hormone compound (GH) is bound to an albumin binding residue by means of a hydrophilic spacer.

4. The conjugate of mode 1 or 2 wherein the growth hormone compound (GH) binds to two or three albumin binding residues by means of a hydrophilic spacer.

5. The conjugate of any of the modalities 1-4 wherein the hydrophilic spacer has the formula

-X ¹ -X ² -X ³ -X ⁴ -where

X1 is -W1 - [(CHR1) I1-W2] m1 - {[(CH2) n1E1] m2 - [(CHR2) I2-W3] m3} n2-,

X2 is - [(CHR3) I3-W4] m4 - {[(CH2) n3E2] m5 - [(CHR4) I4-W5] m6} n4-,

X3 is - [(CHR5) I5-W6] m7-,

X ⁴ is F-D1- (CH2) I6-D2-,

I1, I2, I3, I4, I5 and I6 are independently selected from 0-16,

m1, m3, m4, m6 and m7 are independently selected from 0-10,

m2 and m5 are independently selected from 0-25,

n1, n2, n3 and n4 are independently selected from 0-16,

F is aryl, hetaryl, pyrrolidine-2,5-dione or a valence bond, wherein the aryl and hetaryl groups are optionally substituted with halogen, -CN, -OH, -C (O) OH,

-C (O) NH ² , -S (O) ² OH or C ^1-6 -alkyl,

R1, R2, R3, R4 and R5 are independently selected from hydrogen, -C (O) OH,

-C (O) NH ² , -S (O) OH, -S (O) ² OH, -NH-C (= NH) -NH ² , C ^1-6 -alkyl, aryl or hetaryl; wherein the alkyl, aryl and hetaryl groups are optionally substituted with halogen, -C (O) OH,

-C (O) NH ² , -S (O) OH, -S (O) ² OH, -CN, or -OH,

D1, D2, E1 and E2 are independently selected from -O-, -NR6-, -N (COR7) - or a valence bond; wherein R6 and R7 independently represent hydrogen or C ^1-6 -alkyl,

W ¹ to W ⁶ are independently selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, - CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) ² NHC (O) -, -OC (O) NH-, -NHC (O) O-, -C (O) CH ² -, -CH ² C (O) -, - C (O) CH = CH-, -CH = CHC (O) -, - (CH ² ) s ¹ -, -C (O) -, -C (O) O-, -OC (O) -, or a valence link; where s1 is 0 or 1,

or a pharmaceutically acceptable salt, amide, or ester thereof.

6. A conjugate of a growth hormone of the formula (I):

A-W-B-GH (I)

where

GH represents a growth hormone compound,

B represents a hydrophilic spacer,

W is a chemical group that links A and B, and

A represents a residue of binding to albumin; Y

pharmaceutically acceptable salts, solvates and prodrugs thereof.

7. The conjugate of mode 6 wherein GH represents a growth hormone compound comprising an amino acid sequence having at least 90% identity to the amino acid sequence of human growth hormone (hGH) (sec. Ident number: 1).

8. The conjugate of mode 6 or 7, where GH is hGH (sec with ident.num.:1).

9. The conjugate of any of the modes 6-8 where B has the formula

-X ¹ -X ² -X ³ -X ⁴ -where

X1 is -W1 - [(CHR1) I1-W2] m1 - {[(CH2) n1E1] m2 - [(CHR2) I2-W3] m3} n2-,

X2 is - [(CHR3) I3-W4] m4 - {[(CH2) n3E2] m5 - [(CHR4) I4-W5] m6} n4-,

X3 is - [(CHR5) I5-W6] m7-,

X ⁴ is F-D1- (CH2) I6-D2-,

I1, I2, I3, I4, I5 and I6 are independently selected from 0-16,

m1, m3, m4, m6 and m7 are independently selected from 0-10,

m2 and m5 are independently selected from 0-25,

n1, n2, n3 and n4 are independently selected from 0-16,

F is aryl, hetaryl, pyrrolidine-2,5-dione or a valence bond, wherein the aryl and hetaryl groups are optionally substituted with halogen, -CN, -OH, -C (O) OH,

-C (O) NH ² , -S (O) ² OH or C ^1-6 -alkyl,

R1, R2, R3, R4 and R5 are independently selected from hydrogen, -C (O) OH,

-C (O) NH ² , -S (O) OH, -S (O) ² OH, -NH-C (= NH) -NH ² , C ^1-6 -alkyl, aryl or hetaryl; wherein the alkyl, aryl and hetaryl groups are optionally substituted with halogen, -C (O) OH,

-C (O) NH ² , -S (O) OH, -S (O) ² OH, -CN or -OH,

D1, D2, E1 and E2 are independently selected from -O-, -NR6-, -N (COR7) - or a valence bond; wherein R6 and R7 independently represent hydrogen or C ^1-6 -alkyl,

W ¹ to W ⁶ are independently selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) ² NHC (O) -, -OC (O) NH-, -NHC (O) O-, -C (O) CH ² -, -CH ² C (O) -, - C (O) CH = CH-, -CH = CHC (O) -, - (CH ² ) s ² -, -C (O) -, -C (O) O-, -OC (O) -, or a valence link; where s2 is 0 or 1.

10. The conjugate of any of the modes 6-9, where W has the formula

-W ⁷ -Y-,

where

Y is - (CH2) i7-C3-i0-cycloalkyl-W8- or a valence bond,

I7 is 0-6,

W7 is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -,

-C (O) NHS (O) 2-, -S (O) 2NHC (O) -, -OC (O) NH-, -NHC (O) O-, -C (O) CH2-, -CH2C ( O) -, -C (O) CH = CH-, -CH = CHC (O) -, - (c H2) s3-, -C (O) -, -C (O) O-, -o C ( O) -, or a valence link; where s3 is 0 or 1,

W8 is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -,

-C (O) NHS (O) 2-,

-S (O) 2NHC (O) -, -OC (O) NH-, -NHC (O) O-, -C (O) CH2-, -CH2C (O) -, -C (O) CH = CH -,

-CH = CHC (O) -, - (CH ² ) s ⁴ -, -C (O) -, -C (O) O-, -OC (O) -, or a valence bond; where s4 is 0 or 1.

11. The conjugate of any of the modes 6-10, wherein I1, I2, I3, I4, I5 and I6 independently are 0-6.

12. The conjugate of any of the modes 6-11, where m1, m3, m4, m6 and m7 independently are 0-6.

13. The conjugate of any of the modes 6-12, where m2 and m5 independently are 0-10.

14. The conjugate of any of embodiments 6-13, wherein n1, n2, n3 and n4 independently are 0-10, such as 0-6.

15. The conjugate of any of the embodiments 6-14, wherein D1 and D2 are independently selected from -O- or -NR6- or a valence bond.

16. The conjugate of any of the modes 6-15, where D1 is NR6-.

17. The conjugate of any of the modes 6-16, where D2 is NR6-.

18. The conjugate of any of the modes 6-17, where D1 and D2 are both -O-.

19. The conjugate of any of the modes 6-18, wherein D1 and D2 are both -NR6-.

20. The conjugate of any of the embodiments 6-19, wherein E1 and E2 are independently selected from -O- or -NR6- or a valence bond.

21. The conjugate of any of the modes 6-20, where E1 is NR6-.

22. The conjugate of any of the modes 6-21, where E2 is NR6-.

23. The conjugate of any of the modes 6-22, where E1 and E2 are both -O-.

24. The conjugate of any of the modes 6-23, where E1 and E2 are both -NR6-.

25. The conjugate of any of the embodiments 6-24, wherein W ¹ to W ⁸ are independently selected from the group consisting of -C (O) NH-, -NHC (O) -, -C (O) NHCH ² - , -CH ² NHC (O) -,

-C (O) NHS (O) ² -, -S (O) ² NHC (O) - or a valence bond.

26. The conjugate of any of embodiments 6-25, wherein R1, R2, R3, R4 and R5 are independently selected from hydrogen, -C (O) OH, -C (O) NH ^2, -S (O) ² OH or C ^1-6 -alkyl; wherein the alkyl group is optionally substituted with -C (O) OH, -C (O) NH 2, -S (O) 2 OH.

27. The conjugate of any of the modes 6-26, where - {[(CH2) n1E1] m2 - [(CHR2) I2-W3] m3} n2- and

- {[(CH ² ) n3E2] m5 - [(CHR4) I4-W5] m6} n4-, where E1 and E2 are O, are selected from

where * is understood to denote a point of union, that is, an open link.

28. The conjugate of any of the modes 6-27, where A is selected from

where * denotes the union to B to W.

29. The conjugate of any of embodiments 1-28, wherein the albumin binding residue by means of a hydrophilic spacer is attached to the glutamine residue at the position corresponding to position 40 in sec. with no. of ident. one.

30. The conjugate of any of embodiments 1-29, wherein the albumin binding residue by means of a hydrophilic spacer is attached to the glutamine residue at the position corresponding to position 141 in sec. with no. of ident. one.

31. The conjugate of any of embodiments 1-30, wherein the albumin binding residue by means of a hydrophilic spacer is attached to the N-terminal residue of the growth hormone compound, such as hGH (sec. of ident 1).

32. A conjugate of a growth hormone of the formula (II):

A-W-B-GH-B'-W-A '(N)

where

GH represents a growth hormone compound,

W is a chemical group that links A and B,

W 'is a chemical group that binds A' and B ',

A and A 'independently represent an albumin binding residue,

B and B 'independently are hydrophilic spacers, and

pharmaceutically acceptable salts, solvates and prodrugs thereof.

33. The conjugate of mode 32, wherein GH represents a growth hormone compound comprising an amino acid sequence having at least 90% identity to the amino acid sequence of human growth hormone (hGH) (sec. with ID number: 1).

34. The conjugate of mode 33, where GH is hGH (sec with ident.num.:1).

35. The conjugate of any of the modes 32-34, wherein W 'is selected from W, A' is selected from A and B 'is selected from B.

36. The conjugate of any of the embodiments 32-35, wherein W and W ', A and A', and B and B 'are independently selected from their respective definitions of any of the modes 6-31.

37. The conjugate of any of the above embodiments, wherein the molar weight of said hydrophilic spacer is in the range of 80 D to 1500 D or in the range of 500 D to 1100 D.

38. The conjugate of any of the above embodiments, wherein said albumin binding residue is a lipophilic residue.

39. The conjugate of any of the above embodiments, wherein said albumin binding residue binds non-covalently to albumin.

40. The conjugate of any of the above embodiments, wherein said albumin binding residue is negatively charged at physiological pH.

41. The conjugate of any of the above embodiments, wherein said albumin binding residue has a binding affinity to human serum albumin that is below about 10 pM or below about 1 pM.

42. The conjugate of any of the above embodiments, wherein said albumin binding residue is selected from a straight chain alkyl group, a branched alkyl group, a group having a w-carboxylic acid group or an isoster w-carboxylic acid.

43. The conjugate of any of the above embodiments, wherein said albumin binding residue has from 6 to 40 carbon atoms, from 8 to 26 carbon atoms or from 8 to 20 carbon atoms.

44. The conjugate of any of the above embodiments, wherein said albumin binding residue is a peptide, such as a peptide comprising less than 40 amino acid residues.

45. The conjugate of any of the preceding embodiments, wherein said compound is selected from

46. The conjugate of any of the 1-45 modalities for use in therapy.

47. The conjugate of any of embodiments 1-45 for use in a method for treating Turner syndrome; Prader-Willi syndrome (PWS); Noonan syndrome; Down's Syndrome; chronic kidney disease, juvenile rheumatoid arthritis; cystic fibrosis, HIV infection in children receiving HAART treatment (children with HIV / HALS); children born small for gestational age (SGA); small stature in children born with very low birth weight (VLBW) except SGA; skeletal dysplasia; hypochondroplasia; achondroplasia; idiopathic small stature (ISS); GHD in adults; fractures in or of long bones, such as tibia, fibula, femur, humerus, radius, ulna, clavicle, metacarpus, metatarsus, and digits; fractures in or of spongy bones, such as the skull, base of the hand, and base of the foot; patients after tendon or ligament surgery in, for example, the hand, knee, or shoulder; patients who have or suffer from osteogenesis due to distraction; patients after a hip or disc replacement, meniscus repair, spinal fusion or prosthetic fixation, such as in the knee, hip, shoulder, elbow, wrist or jaw; patients in whom osteosynthesis materials have been fixed, such as nails, screws and plates; patients without union or with a bad union of the fractures; patients after an osteotomy, for example of the tibia or the 1st toe; patients after implanting grafts; degeneration of articular cartilage in the knee caused by trauma or arthritis; osteoporosis in patients with Turner syndrome; osteoporosis in men; adult patients on chronic dialysis (APCD); cardiovascular disease associated with poor nutrition status in APCD; reversal of cachexia in APCD; cancer in APCD; chronic obstructive pulmonary disease in APCD; HIV in APCD; elderly with APCD; chronic liver disease in APCD, fatigue syndrome in APCD; Crohn's disease; affected liver function; men with HIV infections; short bowel syndrome; central obesity; HIV-associated lipodystrophy syndrome (HALS); male infertility; patients after elective major surgery, alcohol / drug detoxification or neurological trauma; aging; Weak old age osteoarthritis; cartilage with traumatic damage; erectile dysfunction; fibromyalgia; memory disorders; depression; traumatic brain injury; subarachnoid hemorrhage; very low birth weight; metabolic syndrome; glucocorticoid myopathy; small stature due to treatment with glucocorticoids in children, accelerated healing of muscle tissue, nerve tissue or wounds; the acceleration or improvement of blood flow to the injured tissue; or the decrease in the infection rate in damaged tissue.

48. A pharmaceutical composition comprising a conjugate of any of embodiments 1-45, optionally in combination with an acceptable pharmaceutical excipient.

49. A method of preparing a conjugate of any of embodiments 1-45, the method comprises introducing an albumin binding linker to a growth hormone produced recombinantly or analogously to a growth hormone by means of the TGase method.

50. The use of a conjugate of any of the 1-45 modalities in the production of a medicament for the treatment of a growth hormone deficiency (GHD).

51. The use of a conjugate of any of modalities 1-45 in the production of a medicament for the treatment of Turner syndrome; Prader-Willi syndrome (PWS); Noonan syndrome; Down's Syndrome; chronic kidney disease, juvenile rheumatoid arthritis; cystic fibrosis, HIV infection in children receiving HAART treatment (children with HIV / HALS); children born small for gestational age (SGA); small stature in children born with very low birth weight (VLBW) except SGA; skeletal dysplasia; hypochondroplasia; achondroplasia; idiopathic small stature (ISS); GHD in adults; fractures in or of long bones, such as tibia, fibula, femur, humerus, radius, ulna, clavicle, metacarpus, metatarsus, and digits; fractures in or of spongy bones, such as the skull, base of the hand, and base of the foot; patients after tendon or ligament surgery in, for example, hand, knee, or shoulder; patients who have or suffer from osteogenesis due to distraction; patients after a hip or disc replacement, meniscus repair, spinal fusions or prosthetic fixation, such as in the knee, hip, shoulder, elbow, wrist or jaw; patients in whom osteosynthesis material has been fixed, such as nails, screws and plates; patients without union or with a bad union of fractures; patients after an osteotomy, for example, of the tibia or the 1st toe; patients after implanting grafts; degeneration of articular cartilage in the knee caused by trauma or arthritis; osteoporosis in patients with Turner syndrome; osteoporosis in men; adult patients on chronic dialysis (APCD); disease cardiovascular disease associated with malnutrition status in APCD; reversal of cachexia in APCD; cancer in APCD; chronic obstructive pulmonary disease in APCD; HIV in APCD; elderly with APCD; chronic liver disease in APCD, fatigue syndrome in APCD; Crohn's disease; affected liver function; men with HIV infections; short bowel syndrome; central obesity; HIV-associated lipodystrophy syndrome (HALS); male infertility; patients after elective major surgery, alcohol / drug detoxification or neurological trauma; aging; Weak old age osteoarthritis; cartilage with traumatic damage; erectile dysfunction; fibromyalgia; memory disorders; depression; traumatic brain injury; subarachnoid hemorrhage; very low birth weight; metabolic syndrome; glucocorticoid myopathy; small stature due to treatment with glucocorticoids in children, accelerated healing of muscle tissue, nerve tissue or wounds; the acceleration or improvement of blood flow to a damaged tissue; or the decrease in the infection rate in damaged tissue.

The description herein of any aspect or mode of the invention by the use of terms such as "comprising", "having", "including" or "containing" with reference to an element or elements is intended to provide support for a similar aspect or modality of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise indicated or that the context clearly contradicts ( e.g. , a composition described herein as comprising a particular element is to be understood as also describing a composition consisting of that element, unless otherwise indicated or context clearly contradicts it).

The present invention is further illustrated by the following examples.

EXAMPLES

Abbreviations:

amu = units of atomic mass

hr (s) = hour (s)

Hz = hertz

l = liter (s)

M = molar

mbar = millibar

mg = milligram (s)

min = minute (s)

ml = milliliter (s)

mM = millimolar

mm = millimeter (s)

mmol = millimole (en)

nmol = nanomol (en)

mol = mol (en)

N = normal

nm = nanometer (s)

sec = second (s)

ppm = parts per million

ESI = electrospray ionization

i.v. = intravenous line

m / z = mass ratio in relation to the load

MS = mass spectrometry

HPLC = high pressure liquid chromatography

RP = reverse phase

HPLC-MS = high pressure liquid chromatography - NMR mass spectrometry = nuclear magnetic resonance spectroscopy

p.o. = orally

rt or RT = room temperature

s.c. = subcutaneous route

tr = retention time

Boc = tere-butyloxycarbonyl

OtBu = tere butyl ester

tBu = tere butyl

Boc-4-ABZ-OH = 4- tere- butoxycarbonylamino-benzoic acid

DCM = dichloromethane, CH ² Cl ² , methylene chloride

DIC = diisopropylcarbodiimide

DIPEA = N, N-diisopropylethylamine

DMF = N, N-dimethylformamide

DMSO = dimethylsulfoxide

DTT = dithiothreitol

EDAC = 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride

Et ² O = diethyl ether

EtOAc = ethyl acetate

Fmoc = 9H-fluoren-9-ylmethoxycarbonyl

Fmoc-Glu-O-f-Bu = N-Fmoc-glutamic-1-t-butyl ester

Fmoc-Lys (Mtt) -OH = (S) -6 - [(diphenyl-p-tolyl-methyl) -amino] -2- (9H-fluoren-9-ylmethoxycarboxylamino) -hexanoic acid

Fmoc-OEG-OH = (2 [2- (Fmoc-amino) ethoxy] ethoxy) acetic acid

H ² O = water

HOBt = 1-hydroxybenzotriazole

MeCN = acetonitrile

MeOH = methanol

NaCl = sodium chloride

NaOH = sodium hydroxide

NMP = N-methylpyrrolidin-2-one

OEG = (2 [2- (amino) ethoxy] ethoxy) acetic acid

TFA = trifluoroacetic acid

THF = tetrahydrofuran

TIS = triisopropylsilane

CDCl3 = dechloric chloroform

CD ³ OD = tetradeuterio methanol

DMSO-cfó = hexadeuterio dimethylsulfoxide

The examples also use the following general methods:

General method for preparing hGH compounds.

The gene encoding the growth hormone compound was inserted recombinantly into a plasmid vector. A suitable strain of E. coli was subsequently transformed by the use of the plasmid vector. hGH or GH variants can be expressed with an N-terminal methionine or as a MEAE fusion of which the MEAE sequence is subsequently cleaved.

The cell stock was prepared in 25% glycerol and stored at -80 ° C. The glycerol stock strain was inoculated onto plates with LB and subsequently incubated at 37 ° C overnight. The content of each plate was washed with LB medium and diluted in 500 ml of LB medium to determine expression. The cultures were incubated at 37 ° C with shaking at 220 rpm until an OD ⁶⁰⁰ of 0.6 was reached. The subsequent induction was performed by using 0.2 mM IPTG at 25 ° C for 6 hrs. The cells were finally harvested by centrifugation.

Cells were subsequently suspended in 10 mM Tris-HCl, pH = 9.0 containing 0.05% Tween 20, 2.5 mM EDTA, 10 mM cysteamine and 4 M urea, and lysed with the use of a disruptor of cells at 30 kPSI. The supernatant was collected by centrifugation and subsequently subjected to chromatographic purification.

Purification was performed by the use of ion exchange chromatography and hydrophobic interaction, followed by removal of the tag peptide by the use of human dipeptidyl peptidase I (hDPPI) expressed from CHO cell. The final purification was achieved by isoprecipitation and ion exchange chromatography. Purification can also be achieved by the use of ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, size exclusion chromatography and membrane-based separation techniques known to one skilled in the art.

Chemical characterization of the protein of the purified growth hormone compounds.

The intact purified protein was analyzed by the use of MALDI-MS. The mass observed corresponded to the theoretical mass deduced from the amino acid sequence.

The expected disulfide linking bonds can be demonstrated by mapping peptides with the use of trypsin and AspN digestion followed by MALDI-MS analysis of the digestion before and after the reduction of the disulfide bonds with DTT.

Capillary electrophoresis

Capillary electrophoresis was carried out using an Agilent Technologies 3DCE system (Agilent Technologies). Data acquisition and signal processing were performed through the use of Agilent Technologies 3DCE ChemStation. The capillary was a "capillary with extended light path" of 64.5 cm (56.0 cm efficient length) 50 pm i.d. of Agilent. UV detection was performed at 200 nm (16 nm Bw, Reference 380 nm and 50 nm Bw). The run electrolyte was 50 mM phosphate buffer pH 7 (method A). The capillary was conditioned with 0.1 M NaOH for 3 min, then with Milli-Q water for 2 min and with the electrolyte for 3 min. After each run, the capillary was rinsed with milli-Q water for 2 min, then with phosphoric acid for 2 min, and with milli-Q water for 2 min. The hydrodynamic injection was performed at 50 mbar for 4.0 sec. The voltage was 25 kV. The temperature of the capillary was 30 oC and the run time was 10.5 min.

Maldi-Tof mass spectrometry

Molecular weights were determined by using the Autoflex Maldi-Tof instrument (Bruker). The samples were prepared by the use of alpha-cyano-4-hydroxy cinnamic acid as a matrix.

RP-HPLC

The RP-HPLC analysis was performed on an Agilent 1100 system using a C-18 Vydac 218TP544.6 mm x 250 mm 5 pm silica column (The Separations Group, Hesperia). Detection was by UV at 214 nm, 254 nm, 280 nm and 301 nm. The column was equilibrated with 0.1% trifluoroacetic acid / H ² O and the sample was eluted by a suitable gradient from 0 to 90% acetonitrile against 0.1% trifluoroacetic acid / H ² O.

LC-MS

The LC-MS analysis was performed on a PE-Sciex API 100 or 150 mass spectrometer equipped with two Perkin Elmer Series 200 micropumps, a Perkin Elmer Series 200 automatic sampler, a 785A UV detector from Applied Biosystems and a dispersion detector of light Sedex 75 Evaporative. A C-18 Waters Xterra silica column 3.0 mm x 50 mm 5p was eluted at 1.5 ml / min at room temperature. It was equilibrated with 5% MeCN / 0.1% TFA / H ² O and eluted for 1.0 min with 5% MeCN / 0.1% TFA / H ² O and then with a linear gradient to MeCN 90% / 0.1% TFA / H ² O for 7 min. The detection was by UV detection at 214 nm and Evaporative light scattering. A fraction of the eluate from the column was introduced into the ionspray interface of a PE-Sciex API 100 mass spectrometer. The mass range 300-2000 amu was scanned every 2 seconds during the run.

Quantification of the protein

Protein concentrations were estimated by measuring the absorbance at 280 nm with the use of a NanoDrop ND-1000 UV spectrophotometer.

Enzymatic mapping of peptides for the determination of derivation site (s)

Peptide mapping was performed by using a reduced and alkylated Asp-N digestion of the protein. First the protein was treated with DTT and iodoacetamide according to standard procedures. The alkylated product was purified by the use of HPLC. Subsequently, the purified alkylated product was digested overnight with endoprotease Asp-N (Boehringer) at an enzyme: substrate ratio of 1: 100. Digestion was separated by HPLC using a C-18 column and a standard TFA / MeCN buffer system. The resulting map of the peptide was compared with that of the non-derived hGH and fractions with different retention times were collected and further analyzed by the use of Maldi-tof mass spectrometry.

SDS PAGE

Poly-acrylamide and SDS gel electrophoresis was performed using NuPAGE 4% -12% Bis-Tris gels (Invitrogen NP0321BOX). The gels were stained with silver (Invitrogen LC6100) or stained with Coomassie (Invitrogen LC6065) and where relevant they were also stained for PEG with barium iodide as described by MM Kurfurst in Anal. Biochem. 200 (2), 244-248, (1992).

Protein chromatography

Protein chromatography was performed on an Ákta Explorer chromatographic system and columns of GE Health Care. The anion exchange was performed by using a Q-Sepharose HP 26/10 column. The starting buffer was 20 mM triethanolamine buffer pH 8.5 and the elution buffer was 0.2 M NaCl starting buffer. The compounds were typically eluted with a gradient of 0-75% elution buffer with 15 volumes of column. The salting and the buffer exchange was carried out using a HiPrep 26/10 column.

The TGase used in the examples is the microbial transglutaminase of Streptoverticillium mobaraense according to US5156956.

Calculation of LogP

The LogP values can be calculated as mLogP and / or cLogP for the albumin binding portion and / or the hydrophilic spacer part by the use of published algorithms (J. Am. Chem. Soc., 86 (1964) 5175- 5180 "A New Substituent Constant, t, Derived from Partition Coefficients", CA Lipinski et al., Advanced Drug Delivery Reviews, 23 (1997) 3-25, "Experimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development Settings" and I. Moriguchi, S. Hirono, I. Nakagome, H. Hirano, Chem. and Pharm. Bull., 42 (1994) 976-978 "Comparison of Reliability of log Values for Drugs Calculated by Several Methods". clogP -logP description of Pomona College (partition coefficient in octanol / water) is calculated with Sybyl 7.0 of Tripos (http://www.tripos.com) version 4.2 of the algorithm of clogP and version 22 of its fragment of database associated as provided by BioByte Corp (http://www.biobyte.com/).

Assay (I) Assay in BAF-3GHR to determine growth hormone activity

BAF-3 cells (a murine line of pro-B lymphoid cells derived from the bone marrow) were originally dependent on IL-3 for growth and survival. Il-3 activates JAK-2 and STAT which are the same mediators as active GH after stimulation. After transfection of the human growth hormone receptor, the cell line became a cell line dependent on growth hormone. This clone can be used to evaluate the effect of different growth hormone samples on the survival of BAF-3GHR.

The BAF-3GHR cells are cultured in deprivation medium (culture medium without growth hormone) for 24 hours at 370C, 5% CO ² .

The cells are washed and resuspended in deprivation medium and plated. 10 pl of the growth hormone compound or human growth hormone in different concentrations or control are added to the cells, and the plates are incubated for 68 hours at 37 ° C, 5% CO ² .

AlamarBlue® is added to each well and the cells are then incubated for another 4 hours. AlamarBlue® is a redox indicator, and is reduced by innate reactions to cellular metabolism and, therefore, provides an indirect measure of the number of viable cells.

Finally, the metabolic activity of the cells is measured in a plate fluorescence reader. The absorbance in the samples is expressed in% of cells not stimulated with growth hormone or control compound and from the concentration-response curves the activity can be calculated (amount of a compound that stimulates the cells with 50%).

Assay to measure the rate of degradation by proteases of GH and conjugates of hGH compounds

The compound of interest is digested by a relevant protease (trypsin, chymotrypsin, pepsin, elastase, factor VIIa, factor Xa, proteinase K, carboxy peptidase, DPPIV, neutral endopeptidase, granzyme B, proline-endopeptidase, staphylococcal peptidase I, thermolysin, thrombin , Arg-C proteinase, Asp-N endopeptidase, caspase 1-10, clostripain, enterokinase, glutamyl endopeptidase, granzyme B, LysC, LysN, proline-endopeptidase and staphylococcal peptidase I or tissue extracts) in a suitable buffer (for example PBS or Ammonium bicarbonate) at 37 ° C for up to 24 hrs. The proteolytic degradation is evaluated by an HPLC assay.

Proteolytic digestion:

100 pl of a solution of a test compound at 1 mg / ml in ammonium bicarbonate buffer is degraded by the enzyme for up to 24 hrs at 37 ° C. Sub-samples are taken at various time points and the proteolytic reaction is stopped by acidifying the sample at a 10-fold dilution in 1% TFA. These diluted samples are analyzed by reverse phase HPLC to estimate the degree of proteolytic digestion.

HPLC method:

10 pl of the above solution is injected onto a Vydac C42x150 mm reverse phase eluted column with a linear gradient of 0.1% TFA in water to 100% acetonitrile containing 0.1% TFA for a period of 30 min. at a flow rate of 0.2 ml / min. Peak detection is performed at 214 nm of UV absorption. The percentage (%) of the intact compound at the time point t = T is calculated from the peak area at the time point t = T (At) and the peak area at = 0 (A ⁰ ) as (At / Aü) x100%. The percentage (%) of the intact compound is plotted against time using the GraphPad Prism software see. 5.01. The half-life time (Ty) is calculated as a phase decrease also by the GraphPad Prism software. Examples of enzymes that can be used are elastase (porcine pancreas from Sigma) and chymotrypsin (Roche sequencing grade). An example of a buffer is 50 mM ammonium bicarbonate, pH = 8.5.

Pharmacokinetics

The pharmacokinetics of the compounds of the examples are investigated in Sprague Dawley rats, males, after a single intravenous (i.v.) and / or subcutaneous (s.c.) administration.

The test compounds are diluted to a final concentration of 1 mg / ml in a dilution buffer consisting of: glycine 20 mg / ml, mannitol 2 mg / ml, NaHCO32.5 mg / ml, with adjustment of pH to 8, two.

The test compounds are studied in male Sprague Dawley rats weighing 250 g. The test compounds are administered as a single injection either i.v. in the vein of the tail or s.c. in the neck with a 25 G needle at a dose of 60 nmol / kg of body weight.

For each test compound the blood sampling is done according to the following program presented in table 1.

Table 1. Blood sampling program for each test compound.

At each sampling time, 0.25 ml of blood is removed from the tail vein by the use of a 25 G needle. The blood samples are taken in an EDTA coated test tube and stored on ice until centrifugation at 1200 x G for 10 min at 4 ° C. The plasma is transferred to a Micronic tube and stored at -20 ° C until analysis.

The concentrations of the test compound are determined by a sandwich ELISA with the use of a polyclonal guinea pig anti-hGH antibody as a receptor, and biotinylated hGH binding protein (soluble part of the human GH receptor) as a detector. The detection limit of the assay was 0.2 nM.

A non-compartmental pharmacokinetic analysis is performed on the half-time concentration profiles of each test compound by using WinNonlin Professional (Pharsight Inc., Mountain View, CA, United States). Estimates of pharmacokinetic parameters of terminal half-life (t) and mean residence time (MRT) are calculated.

In vivo dose-response study in Sprague Dawley rats with surgical removal of the pituitary gland

The dose-response relationship in vivo is studied in Sprague Dawley rats, males, with surgical removal of the pituitary gland. The rat with surgical removal of the pituitary gland is an animal model of well-known and recognized growth hormone deficiency, where there is no production of growth hormone after surgical removal of the pituitary gland. This also leads to low circulating levels of insulin-like growth factor-1 (IGF-1) another important clinical characteristic of growth hormone deficiency in humans.

Hypophysectomy is performed in 4-week-old male rats weighing 90-100 g. The animals enter the study 3 to 4 weeks after surgery with a weight of 100-110 g. Animals with body weight gain of more than 10% during the 3-4 weeks after surgery do not enter the study.

Dose-response studies are usually performed with the use of five dose levels of the test compound from 1-150 nmol / rat.

Elimination

Elimination rates can be measured in pigs, usually at least five crossbred LYD female pigs. The pigs are weighed, fasted and a special "pig jacket" is placed to carry the gamma counter and transmitter and they are placed in individual pens before the start of the study. All pigs are fasted for 18 hours before the study.

The animals are dosed (60 nmol) subcutaneously on the left and right side of the neck respectively with a 28G Novopen3® needle and a NovoFine® with a fine black stop for the needle. The depth of the injection is 5 mm. The test solutions (which include the compounds) are diluted in a buffer consisting of: glycine 20 mg / ml, mannitol 2 mg / ml, NaHCO 32.4 mg / ml, with adjustment of pH to 8.2.

Iodination with 125I is carried out by Chemistry & Isotope Lab. Novo Nordisk A / S. The final radioactive formulation has a specific radioactive activity of 3 pCi / ml and is provided

in 3 ml of Penfills. The solutions were stored at 2-8 ° C until use.

The elimination of radioactive deposits is measured by portable equipment for approximately 24-48 hours.

Preparation of albumin binding groups

Example 1

OEG tetrazole linker (I):

OEG linker tetrazole (I) was synthesized according to scheme 1.

Scheme 1

2 g of Rink amide resin (2 g, 0.6 mMol / g) were weighed into a flask. The resin was swollen with NMP (3 x 30 ml) for 2 hrs.

Removal of the Fmoc group: The resin was stirred with 25% piperidine in NMP (30 ml) for 10 min. The resin was drained and treated with 25% piperidine in NMP (30 ml) for 1 hour followed by draining and washing with NMP (6 x 30 ml).

Fmoc-Lys (Mtt) -OH and HOBT were weighed into a flask, dissolved in bromine blue phenol in NMP (30 ml, 0.5 mM). This solution was added to the previously drained resin followed by the addition of DIC. The reaction was stirred at room temperature for 21 hrs. The resin was drained and washed with NMP (6 x 30 ml) followed by washing with DCM (3 x 30 ml).

The resin was treated with hexafluorisopropanol (20 ml) for 10 min. It was stirred for 10 min. The resin was drained and washed with DCM (3 x 30 ml). The resin was treated with hexafluorisopropanol (20 ml) for 10 min again and stirred for 10 min. The resin was drained and washed with DCM (3 x 30 ml) followed by draining and washing with NMP (3 x 30 ml).

Boc-4-ABZ-OH and HOBT were weighed into a flask, dissolved in bromine blue phenol in NMP (30 ml, 0.5 mM). This solution was added to the previously drained resin followed by the addition of DIC. The reaction was stirred at room temperature. The resin was drained and washed with NMP (6 x 30 ml).

Removal of the Fmoc group: The resin was stirred with 25% piperidine in NMP (10 ml) for 10 min. The resin was drained and treated with 25% piperidine in NMP (10 ml) for 1 hour followed by draining and washing with NMP (6 x 15 ml).

Fmoc-OEG-OH and HOBT were weighed into a flask, dissolved in bromine blue phenol in NMP (15 ml, 0.5 mM). This solution was added to the drained resin followed by the addition of DIC. The reaction was stirred at room temperature for 23 hrs. The resin was drained and washed with NMP (6 x 15 ml).

Removal of the Fmoc group: The resin was stirred with 25% piperidine in NMP (10 ml) for 10 min. The resin was drained and treated with 25% piperidine in NMP (10 ml) for 1 hour followed by draining and washing with NMP (6 x 15 ml).

Fmoc-Glu-O-f-Bu and HOBT were weighed into a flask, dissolved in bromine blue phenol in NMP (15 ml, 0.5 mM). This solution was added to the drained resin followed by the addition of DIC. The reaction was stirred at room temperature for 18 hrs. The resin was drained and washed with NMP (6 x 15 ml).

Removal of the Fmoc group: The resin was stirred with 25% piperidine in NMP (10 ml) for 10 min. The resin was drained and treated with 25% piperidine in NMP (10 ml) for 1 hour followed by draining and washing with NMP (6 x 15 ml).

Fmoc-Glu-O-f-Bu and HOBT were weighed into a flask, dissolved in 15 ml of 0.5 mM blue bromine phenol in NMP. This solution was added to the drained resin followed by the addition of DIC. The reaction was stirred at room temperature for 18 hrs. The resin was drained and washed with NMP (6 x 15 ml).

Removal of the Fmoc group: The resin was stirred with 25% piperidine in NMP (10 ml) for 10 min. The resin was drained and treated with 25% piperidine in NMP (10 ml) for 1 hour followed by draining and washing with NMP (6 x 15 ml).

Fmoc-OEG-OH and HOBT were weighed into a flask, dissolved in bromine blue phenol in NMP (15 ml, 0.5 mM). This solution was added to the drained resin followed by the addition of DIC. The reaction was stirred at room temperature.

The resin was drained and washed with NMP (6 x 15 ml).

Removal of the Fmoc group: The resin was stirred with 25% piperidine in NMP (10 ml) for 10 min. The resin was drained and treated with 25% piperidine in NMP (10 ml) for 1 hour followed by draining and washing with NMP (6 x 15 ml).

In a flask were weighed 4- (16-1H-tetrazol-5-yl-hexadecanoylsulfamoyl) -butyric acid and HOBT, were dissolved in bromine blue phenol in NMP (15 ml, 0.5 mM). This solution was added to the drained resin followed by the addition of DIC. The reaction was stirred at room temperature for 21 hrs. The resin was drained and washed with NMP (6 x 15 ml NMP) followed by draining and washing with DCM (6 x 15 ml).

The resin was cleaved with a mixture of 95% TFA in water (10 ml) DCM (0.25 ml) and TIPS (0.25 ml). The resin was stirred for 2 hrs at room temperature and filtered into cold diethyl ether (75 ml). The resulting precipitate was isolated by centrifugation followed by washing with diethyl ether (3 x) and dried under vacuum for 48 hrs to yield 300 mg of the crude compound.

TOF-MS: Rt = 4.7 min, mass 1268.71

The crude compound was purified on HPLC-prep (GILSON). T2145-10; 30-> 80% MeCN. The mixed fractions were evaporated to dryness in a rotary evaporator and the residue was dissolved in H ² O / MeCN 1: 1 and lyophilized overnight to yield 170 mg decomposed.

Example 2

In a manner similar to that described in Example 1 above, the following compound can be prepared by the use of FMOC-Lys (Mtt) -OH and Wang resin.

Calculated mass 1124.33

Example 3

In a manner similar to that described in Example 1 above and as shown below, the following compound was prepared with the use of Boc-Gly-PAM resin as the starting material.

TOF-MS: mass 1128.38

Example 4

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

TOF-MS: mass 1333.64

Example 5

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

TOF-MS: mass 1320.67

Example 6

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

TOF-MS: mass 2114.64

Example 7

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

TOF-MS: mass 1534.82

Example 8

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

TOF-MS: mass 823.05

Example 9

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Wang Resin.

TOF-MS: mass 980.22

Example 10

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

TOF-MS: mass 851.10

Example 11

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

TOF-MS: mass 1258.51

Example 12

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Wang Resin.

TOF-MS: mass 1269.49

Example 13

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

TOF-MS: mass 841.04

Example 14

In a manner similar to that described in Example 1 above, the following compound can be prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

Calculated mass 863.07

Example 15

In a manner similar to that described in Example 1 above, the following compound can be prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

Calculated mass 855.07

Example 16

In a manner similar to that described in Example 1 above, the following compound can be prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

Calculated mass 883.12

Example 17

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

TOF-MS: mass 1123.35

Example 18

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

TOF-MS: Rt = 4.7 min, mass 1267.45

Example 19

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FM0C-Lys (Mtt) -0H and Rink amide resin.

TOF-MS: mass 1310.67

Example 20

In a manner similar to that described in Example 1 above, the following compound can be prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

Calculated mass 1308.58

Example 21

In a manner similar to that described in Example 1 above, the following compound can be prepared by the use of FMOC-Glu (ODmab) -OH and 2-chlorotryl chloride resin.

Calculated mass: 1235.56

Example 22

In a manner similar to that described in Example 1 above, the following compound can be prepared by the use of FMOC-Glu (ODmab) -OH and 2-chlorotryl chloride resin.

Calculated mass: 1173.40

Example 23

In a manner similar to that described in Example 1 above, the following compound was prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

TOF-MS: mass 703.93

Example 24

In a manner similar to that described in Example 1 above, the following compound can be prepared by the use of FMOC-Lys (Mtt) -OH and Rink amide resin.

Calculated mass 689.90

Example 25

In a manner similar to that described in Example 1 above, the following compound can be prepared by the use of FMOC-Glu (ODmab) -OH and 2-chlorotryl chloride resin.

Calculated mass 1182.34

Preparation of albumin binding GH compounds

Example 26

1. Coupling of the albumin binding group of general formula A-W-B1-NH2 to the transmethylated and oxidized GH compound (I)

The use of a transglutaminase to bind an aldehyde handle to the GH in glutamine residues has been previously described in WO2005 / 070468. The method can be used according to the present invention for the binding of a linker based on an albumin binding group of formula AW-B1-NH2. The TGase used is the microbial transglutaminase of Streptoverticillium mobaraense according to US5156956.

The reaction can be carried out in the following manner: GH (I) is transaminated initially with 1,3-diamino-2-propanol (II) as described in WO2005 / 070468:

In the next stage, to the transaminated GH (III) periodate is added. Oxidation is typically done at a low temperature, such as 4-10 ° C for 30 min. optionally in the dark. The periodate can oxidize methionine residues in GH to their corresponding residues of methionine sulfoxide. To minimize this risk of oxidation, small molecule organic thioethers may be added during the periodate oxidation. A suitable organic thioether is 3-methylthiopropan-1-ol but the skilled person will be able to suggest others.

Oxidation of a transaminated GH compound (III):

The buffer change can be performed to obtain an acid solution required for efficient reduction with sodium cyano borohydride. Typically, an excess of A-W-B1-NH2 amine is used, and sodium cyanoborohydride can be added in smaller portions over time.

Reductive amination of (IV) with a linker of the albumin binding group (V):

The last reaction can be done as follows:

To a solution of oxidized transaminated GH (IV) is added a solution of the linker of the albumin binding group (V) in a mixture of AcOH (1.5 ml) and 50 mM MES (0.5 ml) at pH 6 , 00. The resulting reaction mixture is stirred gently at RT for 30 min. at which time a solution of NaCNBH3 (15 pl, (22 mg of NaCNBH3 dissolved in 500 pl of Milli-Q AcOH water (15 pl)) is added. The sample is covered with tin foil and stirred overnight at RT.

The conjugate can be isolated by anion exchange chromatography in the following manner:

The acetic acid is removed by changing the buffer with pure water (3X) with the use of Amicon Ultra15 devices (UltraK 10K tubes) by centrifugation at 4000 rpm / min. for 3 x 8 min. Then the buffer is changed to 20 mM TEA, pH: 8.50 by the use of Amicon filter devices and diluted to a final volume of 50 ml with 20 mM TEA, before loading onto a HiLoad Q column. Sepharose, 26/10. The column is initially washed with 20 mM TEA, pH 8.50 (buffer A) and then eluted with 20 mM TEA, 500 mM NaCl, pH 8.50 (buffer B) by using a gradient of 0-100% (B) in 20 HP, with a flow rate of 2 ml / min. The buffer of the mixed fractions was changed 5 times to 10 mM ammoniumbicarbonate buffer in pure water with the use of Amicon Ultra15 devices (UltraK 10K tubes) by centrifugation at 4000 rpm / min. for 3 x 8 min

Example 27

In a manner similar to that described in example 26 above, the following compound can be prepared by the use of an albumin binding group from Example 2.

Calculated mass 23473.81

Example 28

In a manner similar to that described in Example 26 above, the following compound was prepared by the use of an albumin binding group from Example 4.

TOF-MS: mass 23,428

Example 29

In a manner similar to that described in example 26 above, the following compound was prepared by the use of an albumin binding group from Example 5.

TOF-MS: mass 23472.40

Example 30

In a manner similar to that described in Example 26 above, the following compound was prepared by the use of an albumin binding group from Example 6.

TOF-MS: mass 24265.71

Example 31

In a manner similar to that described in Example 26 above, the following compound was prepared by the use of an albumin binding group from Example 7.

TOF-MS: mass 23686.83

Example 32

In a manner similar to that described in example 26 above, the following compound was prepared by the use of an albumin binding group from Example 8.

TOF-MS: mass 22974.75

Example 33

In a manner similar to that described in example 26 above, the following compound was prepared by the use of an albumin binding group from Example 9.

TOF-MS: mass 23 131.31

Example 34

In a manner similar to that described in Example 26 above, the following compound was prepared by the use of an albumin binding group from Example 10.

TOF-MS: mass 23002

Example 35

In a manner similar to that described in example 26 above, the following compound was prepared by the use of an albumin binding group from Example 11.

TOF-MS: mass 23419.59

Example 36

In a manner similar to that described in example 26 above, the following compound was prepared by the use of an albumin binding group from Example 12.

TOF-MS: mass 23420.58

Example 37

In a manner similar to that described in example 26 above, the following compound was prepared by the use of an albumin binding group from Example 13.

TOF-MS: mass 22992.13

Example 38

In a manner similar to that described in Example 26 above, the following compound can be prepared by using an albumin binding group from Example 14.

Calculated mass 23015.15

Example 39

In a manner similar to that described in example 26 above, the following compound can be prepared by using an albumin binding group from Example 15.

Calculated mass 23006.15

Example 40

In a manner similar to that described in Example 26 above, the following compound can be prepared by using an albumin binding group from Example 16.

Calculated mass 23034.18

Example 41

In a manner similar to that described in Example 26 above, the following compound was prepared by using an albumin binding group from Example 17.

TOF-MS: mass 23273,97

Example 42

In a manner similar to that described in Example 26 above, the following compound was prepared by using an albumin binding group from Example 18.

TOF-MS: mass 23333

Example 43

In a manner similar to that described in example 26 above, the following compound can be prepared by the use of an albumin binding group from Example 19.

Calculated mass 23461.75

Example 44

In a manner similar to that described in example 26 above, the following compound can be prepared by the use of an albumin binding group from Example 20.

Calculated mass 23459.67

Example 45

In a manner similar to that described in Example 26 above, the following compound can be prepared by using an albumin binding group from Example 21.

Calculated mass 23386.65

Example 46

In a manner similar to that described in example 26 above, the following compound can be prepared by the use of an albumin binding group from Example 22.

Calculated mass 23324.48

Example 47

In a manner similar to that described in Example 26 above, the following compound was prepared by the use of an albumin binding group from Example 23.

TOF-MS: mass 22841

Example 48

In a manner similar to that described in example 26 above, the following compound can be prepared by the use of an albumin binding group from Example 24.

Calculated mass 22826.97

Example 49

In a manner similar to that described in Example 26 above, the following compound can be prepared by using an albumin binding group from Example 25.

Example 50

Coupling of a binding linker to albumin of general formula A-W-B1-NH2 to the transmethylated and oxidized GH compound (I)

The use of a transglutaminase to bind an aldehyde handle to the GH in glutamine residues has been previously described in WO2005 / 070468. The method can be used according to the present invention for the binding of a linker based on an albumin binding group of formula AW-B1-NH2. The TGase used is the microbial transglutaminase of Streptoverticillium mobaraense according to US5156956.

The reaction can be carried out in the following manner: GH (I) is transaminated initially with 1,3-diamino-2-propanol (II) as described in WO2005 / 070468:

The transaminated hGH Gln40 (III) was obtained from Example 26 as a by-product of the purification by CIE chromatography.

In the next stage, to the transaminated GH (III) periodate is added. Oxidation is typically done at a low temperature, such as 4-10 ° C for 30 min. optionally in the dark. The periodate can oxidize methionine residues in GH to their corresponding residues of methionine sulfoxide. To minimize this risk of oxidation, small molecule organic thioethers may be added during the periodate oxidation. A suitable organic thioether is 3-methylthiopropan-1-ol but the skilled person will be able to suggest others.

Oxidation of a transaminated GH compound (III):

The buffer change can be performed to obtain an acid solution required for efficient reduction with sodium cyano borohydride. Typically, an excess of A-W-B1-NH2 amine is used, and sodium cyanoborohydride can be added in smaller portions over time.

Reductive amination of (IV) with a linker of the albumin binding group (V):

The last reaction can be done as follows:

To a solution of oxidized transaminated GH (IV) is added a solution of bile acid linker (V) in a mixture of AcOH (1.5 ml) and 50 mM MES (0.5 ml) at pH 6.00. The resulting reaction mixture is stirred gently at RT for 30 min. at which time a solution of NaCNBH3 (15 pl, (22 mg of NaCNBH3 dissolved in 500 pl of Milli-Q AcOH water (15 pl)) is added. The sample is covered with tin foil and stirred overnight at RT. The conjugate can be isolated by anion exchange chromatography in the following manner:

The acetic acid is removed by changing the buffer with pure water (3X) with the use of Amicon Ultra15 devices (UltraK 10K tubes) by centrifugation at 4000 rpm / min. for 3 x 8 min. Then the buffer is changed to 20 mM TEA, pH: 8.50 by the use of Amicon filter devices and diluted to a final volume of 50 ml with 20 mM TEA, before loading onto a HiLoad Q column. Sepharose, 26/10. The column is initially washed with 20 mM TEA, pH 8.50 (buffer A) and then eluted with 20 mM TEA, 500 mM NaCl, pH 8.50 (buffer B) by using a gradient of 0-100% (B) in 20 HP, with a flow rate of 2 ml / min. The buffer of the mixed fractions was changed 5 times to 10 mM ammoniumbicarbonate buffer in pure water with the use of Amicon Ultra15 devices (UltraK 10K tubes) by centrifugation at 4000 rpm / min. for 3 x 8 min

In a manner similar to that described in example 50 above, the following compound was prepared by the use of an albumin binding group from Example 6.

TOF-MS: Rt = 4.7 min, mass 23473.81

Example 51

1. Coupling of a GH (I) compound N-terminally with an albumin binding group (II)

(A) Reductive alkylation of (I) with an albumin-binding aldehyde (II)

The albumin binding group (II) was obtained as described in example 3.

2- (C20diácido-Trx-yGlu-Glu-OEG-OEG-Gly-Glycine amide) -ethyl-Na1-hGH

hGH (23 mg) was dissolved in Hepes buffer (2.3 ml 0.25 mM pH 7.0).

C ²⁰ diacid-Trx-yGlu-Glu-OEG-OEG-Gly-Gly-dimethylacetal (2 mg, see example 3 above) was treated with TFA (50 pl) for 6 min. and evaporated to dryness in vacuo. The residue was extracted again with EtOH (200 pl) and evaporated to dryness in vacuo. The residue was dissolved in DMF (100 pl) and added to the hGH solution. A precipitate formed and was redissolved by addition of DMF (1 ml). After 1 hour a solution of NaCNBH3 (20 mg, in 0.5 ml of MeCN (230 pl)) was added portionwise and left to stand for 20 hrs. The reaction was quenched by the addition of AcOH (2 mL) and diluted with water to a total volume of 20 mL and purified on prep HPLC. in a C18 column with a gradient of MeCN / 0.1% TFA of 40-80% against 0.1% TFA in water. The last elution peak was collected, diluted from 70% MeCN to 10% with water and lyophilized to yield 4.51 mg of the compound.

TOF-MS: mass 23237.6.

Example 52

The GH compounds as described herein were analyzed in one or more of the assays described herein above.

The EC50 values based on a BAF assay as well as the half-life (Ty) and the mean residence time (MRT) were determined and are included in Table 2 below.

The elimination rate for the selected compounds was measured in five crossbred LYD female pigs as described above. The results are presented as AUC (0-45 hrs) in Table 2.

The property that increases the solubility expressed as cLogP of space (B) as determined by the method described above is also included in table 2.

The binding affinity to albumin as determined by Biacor and or HPLC is also included in Table 2.

Also included are data related to a pegylated hGH compound. PEG-hGH is identical to the compound described in Example 1 of WO2006024953A2.

Claims (1)

A conjugate of a growth hormone having the formula (I): A-W-B-GH (I) where GH represents a growth hormone compound, A represents a residue of binding to albumin; W is a chemical group that links A and B, and wherein B represents a hydrophilic spacer selected from:

where * denotes a point of union, that is, an open link. The conjugate according to claim 1, wherein W has the formula -W7-Y-, where Y is - (CH2) I7-C3-10-cycloalkyl-W8- or a valence bond, I7 is 0-6, W7 is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, - S (O) ² NHC (O) -, -OC (O) NH-, -NHC (O) O-, -C (O) CH ² -, -CH ² C (O) -, -C (O) CH = CH-, -CH = CHC (O) -, - (CH ² ) s ³ -, -C (O) -, -C (O) O-, -OC (O) -, or a valence bond ; where s3 is 0 or 1, and W8 is selected from -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) 2NHC (O) -, -OC (O) NH-, -NHC (O) O-, -C (O) CH2-, -CH2C (O) -, -C (O) CH = CH -, -CH = CHC (O) -, - (CH2) s4-, -C (O) -, -C (O) O-, -OC (O) -, or a valence bond; where s4 is 0 or 1. The conjugate according to claim 2, wherein W ⁷ and W ⁸ are independently selected from the group consisting of -C (O) NH-, -NHC (O) -, -C (O) NHCH ² -, -CH ² NHC (O) -, -C (O) NHS (O) ² -, -S (O) ² NHC (O) - and a valence bond. The conjugate according to any of claims 1-3, wherein A is selected from

where * denotes the union to B to W. 5. The conjugate according to any of claims 1-4, wherein GH is hGH (sec. of ident.:1). 6. The conjugate according to any of claims 1-4, wherein GH is at least 90% identical to hGH (sec.with ident.ID.:1). 7. The conjugate according to any of claims 1-4, wherein GH is at least 95% identical to hGH (sec.with ident.ID.:1) and has at least 80% of the hormone activity of growth of hGH as determined by trial I. The conjugate according to any of claims 1-7, wherein the albumin binding residue by means of a hydrophilic spacer, B, is attached to the glutamine residue in the position corresponding to position 40 in the sec. . with no. of ident. 1, the glutamine residue in the position corresponding to position 141 in sec. with no. of ident. 1 or the N-terminal residue of the growth hormone compound. The conjugate according to any of claims 1-7, wherein the albumin binding residue by means of a hydrophilic spacer, B, is attached to the glutamine residue in the position corresponding to position 40 in the sec. . with no. of ident. one. The conjugate according to any of claims 1-7, wherein the albumin binding residue by means of a hydrophilic spacer, B, is attached to the glutamine residue at the position corresponding to position 141 in the sec. . with no. of ident. one. 11. The conjugate according to claim 1, wherein said compound is selected from:

12. A pharmaceutical composition comprising a conjugate according to any of claims 1-10, optionally in combination with an acceptable pharmaceutical excipient. 13. A conjugate according to any of claims 1-10 for use in a Turner syndrome treatment method; Prader-Willi syndrome (PWS); Noonan syndrome; Down's Syndrome; chronic kidney disease, juvenile rheumatoid arthritis; cystic fibrosis, HIV infection in children receiving HAART treatment (children with HIV / HALS); children born small for gestational age (SGA); small stature in children born with very low birth weight (VLBW) except SGA; skeletal dysplasia; hypochondroplasia; achondroplasia; idiopathic small stature (ISS); GHD in adults; fractures in or of long bones, such as tibia, fibula, femur, humerus, radius, ulna, clavicle, metacarpus, metatarsus, and digits; fractures in or of spongy bones, such as the skull, base of the hand, and base of the foot; patients after tendon or ligament surgery in, for example, hand, knee, or shoulder; patients who have or suffer from osteogenesis due to distraction; patients after a hip or disc replacement, meniscus repair, spinal fusions or prosthetic fixation, such as in the knee, hip, shoulder, elbow, wrist or jaw; patients in whom osteosynthesis material has been fixed, such as nails, screws and plates; patients without union or with a bad union of fractures; patients after an osteotomy, for example, of the tibia or the 1st toe; patients after implanting grafts; degeneration of articular cartilage in the knee caused by trauma or arthritis; osteoporosis in patients with Turner syndrome; osteoporosis in men; adult patients on chronic dialysis (APCD); cardiovascular disease associated with poor nutrition status in APCD; reversal of cachexia in APCD; cancer in APCD; chronic obstructive pulmonary disease in APCD; HIV in APCD; elderly with APCD; chronic liver disease in APCD, fatigue syndrome in APCD; Crohn's disease; affected liver function; men with HIV infections; short bowel syndrome; central obesity; HIV-associated lipodystrophy syndrome (HALS); male infertility; patients after elective major surgery, alcohol / drug detoxification or neurological trauma; aging; Weak old age osteoarthritis; cartilage with traumatic damage; erectile dysfunction; fibromyalgia; memory disorders; depression; traumatic brain injury; subarachnoid hemorrhage; very low birth weight; metabolic syndrome; glucocorticoid myopathy; small stature due to treatment with glucocorticoids in children, accelerated healing of muscle tissue, nerve tissue or wounds; the acceleration or improvement of blood flow to a damaged tissue; or the decrease in the infection rate in damaged tissue. 14. The use of a conjugate according to any of claims 1-10 in the production of a medicament for the treatment of Turner syndrome; Prader-Willi syndrome (PWS); Noonan syndrome; Down's Syndrome; chronic kidney disease, juvenile rheumatoid arthritis; cystic fibrosis, HIV infection in children receiving HAART treatment (children with HIV / HALS); children born small for gestational age (SGA); small stature in children born with very low birth weight (VLBW) except SGA; skeletal dysplasia; hypochondroplasia; achondroplasia; idiopathic small stature (ISS); GHD in adults; fractures in or of long bones, such as tibia, fibula, femur, humerus, radius, ulna, clavicle, metacarpus, metatarsus, and digits; fractures in or of spongy bones, such as the skull, base of the hand, and base of the foot; patients after tendon or ligament surgery in, for example, hand, knee, or shoulder; patients who have or suffer from osteogenesis due to distraction; patients after a hip or disc replacement, meniscus repair, spinal fusions or prosthetic fixation, such as in the knee, hip, shoulder, elbow, wrist or jaw; patients in whom osteosynthesis material has been fixed, such as nails, screws and plates; patients without union or with a bad union of fractures; patients after an osteotomy, for example, of the tibia or the 1st toe; patients after implanting grafts; degeneration of articular cartilage in the knee caused by trauma or arthritis; osteoporosis in patients with Turner syndrome; osteoporosis in men; adult patients on chronic dialysis (APCD); Associated cardiovascular disease with poor nutrition status in APCD; reversal of cachexia in APCD; cancer in APCD; chronic obstructive pulmonary disease in APCD; HIV in APCD; elderly with APCD; chronic liver disease in APCD, fatigue syndrome in APCD; Crohn's disease; affected liver function; men with HIV infections; short bowel syndrome; central obesity; HIV-associated lipodystrophy syndrome (HALS); male infertility; patients after elective major surgery, alcohol / drug detoxification or neurological trauma; aging; Weak old age osteoarthritis; cartilage with traumatic damage; erectile dysfunction; fibromyalgia; memory disorders; depression; traumatic brain injury; subarachnoid hemorrhage; very low birth weight; metabolic syndrome; glucocorticoid myopathy; small stature due to treatment with glucocorticoids in children, accelerated healing of muscle tissue, nerve tissue or wounds; the acceleration or improvement of blood flow to a damaged tissue; or the decrease in the infection rate in damaged tissue.