RU2802215C2 - Pharmaceutical composition and a method of treatment of growth hormone-related diseases in humans - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to recombinant human growth hormone (rhGH) prodrug conjugates, wherein the molecular weight of the prodrug without hGH-NH is at least 30 kDa and a maximum is 120 kDa. Also a method of treatment of diseases associated with growth hormone in humans using the above-described conjugates is described.

EFFECT: obtaining a pharmaceutical composition and a method of treatment of growth hormone-related diseases in humans.

9 cl, 11 dwg, 27 ex, 2 tbl

Description

Technical field

This invention relates to the field of medicine, more specifically to a pharmaceutical composition and method for the treatment of diseases associated with growth hormone in humans.

Prior Art

Growth hormone (hereinafter: GH) is a hormone that stimulates the growth and reproduction of cells in humans and other animals. It is a 191-amino acid, single-chain polypeptide hormone that is synthesized, stored, and secreted by somatotrophic cells in the lateral wings of the anterior pituitary gland. The hormone is also known as somatotropin when related to recombinant DNA growth hormone and is referred to as "rhGH". Growth hormone has many functions in the body, the most significant of which is to increase height during childhood, and there are several diseases that can be treated with therapeutic use of GH. The effect of growth hormone deficiency varies greatly depending on the age at which it occurs. In children, lack of growth and short stature are the main manifestations of GH deficiency. It can also cause sexual underdevelopment. In adults, the effects of deficiency are less pronounced and may include deficiencies in strength, energy, and bone mass, as well as increased cardiovascular risk. There are many causes of GH deficiency, including mutations in certain genes, congenital malformation involving the hypothalamus and/or anterior pituitary gland, and damage to the pituitary gland through trauma, surgery, or disease. Deficiency is treated with external GH. All GH currently in use are biosynthetic versions of human GH obtained by recombinant DNA. GH is used as a replacement therapy in children and adults who suffer from GH deficiency, either in childhood (after completion of the growth phase) or in adulthood (usually as a result of an acquired pituitary tumor). In such patients, improvements may include a reduction in adipose tissue mass, an increase in lean mass, an improvement in bone density, an improvement in lipid profile, a reduction in cardiovascular risk factors, and an improvement in psychosocial health. Genentech Inc (US) was the first to clone rhGH and described it in EP-B 22242. As of 2006, synthetic growth hormones available in the United States and Europe (and their manufacturers) include Nutropin (Genentech), Humatrope (Eli Lilly), Genotropin (Pfizer), Norditropin (Novo Nordisk), Saizen (Merck Serono), and Omnitropin (Sandoz).

Although molecular biology techniques greatly increase the availability of many proteins and/or polypeptides (hereinafter referred to as proteins), the therapeutic utility of these proteins is often hindered by other factors such as short plasma half-life due to renal and receptor-mediated clearance, aggregation, proteolytic degradation, poor bioavailability and physical properties that preclude the effectiveness of the formulations.

The mechanism for improving protein availability is to fuse the protein with compounds used to make derivatives, which include, but are not limited to, polyethylene glycol and polypropylene glycol. Some of these recognized benefits include: reduced immunogenicity and antigenicity, increased duration of action, and altered pharmacokinetic properties. [Veronese, F.M. "Enzymes for Human Therapy: Surface Structure Modifications," Chimica Oggi, 7:53-56 (1989)] (Ref. 5).

PEGylation of rhGH can improve the performance of the molecule for medical use by increasing the in vivo half-life (allowing for lower dose or dosing frequency), improving its stability, and reducing its antigenicity, or a combination of the two.

In general, this type of molecular modification is well known in the art and there are many patents available from the literature that describe this concept. For example, PEGylated erythropoietin (EPO) from Hofmann La Roche is described in EP-B 1196443, which claims a specific linker containing a PEG covalently linked to EPO, PEGylated interferon alpha, described in EP-B 975369 from Nektar/La Roche, and another PEGylated interferon alpha in EP-B 1562634 from Hofmann La Roche.

It is believed that in vivo clearance of rhGH occurs by two mechanisms presented below. The first is renal clearance, in which rhGH is cleared from the circulation by renal glomerular filtration. The renal clearance of rhGH is well documented and PEGylation of synthetic rhGH is therefore an obvious choice to solve this problem. Renal clearance is 25-53% of total rhGH clearance (Girard, J. Mehls, O.J. Clin. Invest. 1994 March; 93(3): 1163-1171, ref. 3).

The second mechanism is hepatic clearance (in the liver). GH uptake by the liver is induced by receptor-mediated endocytosis followed by lysosomal degradation.

The third mechanism is receptor-mediated clearance in other tissues such as cartilage chondrocytes. By reducing the binding affinity of GH to the GR receptor by PEGylation, receptor-mediated clearance is reduced. However, there are certain problems with the administration of rhGH. One of the main disadvantages of subcutaneous administration of rhGH is the occurrence of lipoatrophy in treated patients.

Lipoatrophy is the medical term for localized fat loss. Compositions for subcutaneous administration of rhGH cause problems of lipoatrophy, which is presumably caused by a high local concentration of the growth hormone complex at the injection site.

At A. et al. in a publication in J. Pediatr. Endocrinol. Metab. 1999 Jan-Feb; 12(1):95-7 has a description of such a case history (ref. 1). This is a report from a patient with an isolated GH deficiency due to a 6.7 kb deletion. gene who received a high dose of rhGH and developed localized lipoatrophy at the injection sites without detecting any antibodies 6 years after treatment. The etiology of lipoatrophy is thought to be due to a direct lipolytic effect of high doses of rhGH.

It is believed that lipoatrophy associated with the administration of rhGH is caused by the activity of rhGH itself, higher concentrations and prolonged exposure. Such high concentrations occur at the injection site.

The chance that high growth hormone activity accumulates near the injection site becomes even higher if rhGH is PEGylated due to the increased residence time. When PEGylated rhGH formulations are used, the tissue is exposed to prolonged and enhanced exposure to growth hormone due to the fact that the PEGylated conjugate has the activity required for pharmacological action, and the diffusion of the conjugate is limited due to the size of the conjugate. The result is increased lipolysis at the injection site.

WO-A 2005/079838 describes PEGylated hGH, where the hGH moiety is attached to the polyethylene glycol polymer via an amino functional group, which can be considered a strong bond due to the stability of the amino group. An example of such a PEGylated hGH compound that causes lipoatrophy is PHA-794428. Compound PHA-794428 is PEGylated rhGH and is also described in WO-A 2005/079838 from Pharmacia (purchased by Pfizer) and also described in Horm. Res. 2006; 65 (suppl. 4): 1-213, CF1-98 GH/IGF Treatment in "First in-human study of PEGylated recombinant human growth hormone", Philip Harris et al. (reference 4).

According to clinical trial information published at www.clinicaltrials.gov, the trial ended on December 10, 2007. Pfizer's decision to terminate the program was due to injection site lipoatrophy that was noted in the clinical Phase 2 trial following a single injection of PHA 794428.

WO-A 2006/102659 (Nektar) also describes and proposes rhGH-PEG conjugates (linear and branched type) via an amine bond. The problem solved in WO-A 2006/102659 is described in paragraph [0005] on page 2. According to the applicant, the problem to be solved is a reduced dosing frequency. Since rhGH therapy typically requires daily injections, patients, and pediatric patients in particular, dislike the inconvenience and discomfort associated with such a regimen. The solution described in Nektar's WO-A is to provide new PEG-rhGH conjugates.

In Table 6 of [0257] WO-A, it can be seen that PEG-rhGH conjugates have quite low in vitro activity compared to natural growth hormone without PEG. Despite low in vitro activity, PEGylated rhGH conjugates are active in vivo. More details are given in [0261]: "Although preliminary in vitro results suggest that increasing the amount of PEG attached to hGH reduces its ability to stimulate the hGH receptor, based on preliminary in vivo results, the decrease in bioavailability appears to be more than balanced by increased plasma half-life and/or availability, which leads to the conclusion that the conjugates presented here have superior pharmacodynamic action in vivo compared to unmodified rhGH at an identical dosing regimen."

WO-A 2006/102659 (Nektar) does not describe self-cleaving linkers - i. it is simply noted that the PEG-rhGH conjugates are active in vivo, although their in vitro activity is significantly reduced. The problem of lipoatrophy is not considered. A solution to the problems of obtaining the desired properties of reduced lipoatrophy and reduced frequency of injections in a PEGylated hGH conjugate is the use of a prodrug. A prodrug is any compound that undergoes biotransformation before exerting a pharmacological effect. Prodrugs can thus be considered as drugs containing specialized non-toxic protecting groups used in the transition state to modify or remove undesirable properties in the parent molecule. In this case, the polymeric carrier can temporarily reduce the activity of growth hormone and, therefore, reduce the likelihood of tissue lipolysis. Transient conjugation to a polymeric carrier will, at the same time, increase the half-life of the conjugate and therefore provide prolonged delivery of hGH.

Many macromolecular prodrugs have been described in the literature in which the macromolecular carrier is linked via a labile ester group to the drug (e.g., Y. Luo, MR Ziebell, GD Prestwich, "A Hyaluronic Acid - Taxol Antitumor Bioconjugate Targeted to Cancer Cells", Biomacromolecules 2000, 1 , 208-218, J Cheng et al., Synthesis of Linear, beta-Cyclodextrin Based Polymers and Their Camptothecin Conjugates, Bioconjugate Chem. 2003, 14, 1007-1017, R. Bhatt et al., Synthesis and in Vivo Antitumor Activity of Poly(L-glutamic acid) Conjugates of 20(S)-Campththecin, J. Med. Chem. 2003, 46, 190-193; R. B. Greenwald, A. Pendri, C. D. Conover, H. Zhao, Y. H. Choe, A. Martinez B. Testa, J. M: Mayer in Hydrolysis in Drug and Prodrug Metabolism, Wiley-VCH, 2003, Chapter 8). In these cases, the conjugated functional group of the bioactive compound is a hydroxyl group or a carboxylic acid.

Particularly for biomacromolecules, but also for low molecular weight polymeric prodrugs, it may be desirable to link the macromolecular carrier to the amino groups (ie N-termini or amino groups of protein lysine) of the bioactive substance. This is especially applicable if masking the bioactivity of a drug requires conjugation of a specific amino group of the bioactive substance, for example, an amino group located in the active site, or a region or epitope involved in receptor binding. Also during prodrug preparation, amino groups can be more chemoselectively targeted and serve to better control carrier-drug conjugation due to their greater nucleophilicity compared to hydroxyl or phenolic groups. This is especially true for proteins, which can contain a huge variety of different reactive functional groups. In this case, non-selective conjugation reactions produce undesirable product mixtures that require significant characterization or purification and can reduce the yield of the reaction and the therapeutic efficacy of the product.

Prodrug activation can occur either by enzymatic or non-enzymatic cleavage of a labile bridge between the carrier and the drug molecule, or by a combination of both methods, for example, an enzymatic step followed by a non-enzymatic rearrangement.

WO-A 2005/099768 describes PEGylated linker molecules with self-cleaving linkers for a large group of biomolecules, including somatotropins (point 6). In WO-A 2005/099768, the problem to be solved is interindividual fluctuations and unpredictable action of prodrug activation when enzymatic mechanisms are involved (page 12, lines 17-30). This application describes an aromatic linker as a solution, which may be based on PEG. Such a PEG linker binds the drug in such a way that the activity of the drug is significantly reduced. It is activated only when the drug is released, which is initiated by hydrolysis. Hydrolysis can be controlled chemically. No special emphasis is placed on GH and related problems such as lipoatrophy associated with this solution.

Briefly, none of the above references describe the decision to develop aro-long acting rhGH based on a prodrug conjugate that can be administered less frequently without increasing the incidence of lipoatrophy.

Thus, it is an object of the present invention to provide such a prodrug, or a pharmaceutical composition containing said prodrug, to reduce the frequency of administration of rhGH using PEG-conjugated rhGH without significant occurrence of lipoatrophy.

The essence of the invention

Therefore, the present invention provides a pharmaceutical composition comprising suitable pharmaceutical excipients and also comprising a clinically effective human in vivo PEGylated recombinant human growth hormone prodrug conjugate rhGH, wherein the PEG is linked to rhGH via a self-hydrolyzable (self-cleaving) transient linker; wherein said prodrug conjugate is characterized in that:

(1): the conjugate has a GH activity that is less than 5% of natural growth hormone without PEG; And

(2): The rate of self-hydrolysis of the linker is such that the in vivo half-life is from 10 hours to 600 hours.

Property (1) provides a low incidence of lipoatrophy when taking the prodrug, despite the fact that it has a significantly longer time of action in vivo. Without wishing to be bound by theory, the present inventors believe that if a prodrug has greater GH activity, that product will still induce lipoatrophy much more frequently than currently marketed rhGH products.

Property (2) provides for the release of rhGH (without PEG) gradually over time, so that the rhGH pharmaceutical product is administered less frequently than human growth hormone, for example, only once a week or once a month instead of daily administration, while while maintaining full efficacy compared to rhGH.

Preferably, the in vivo half-life is up to 5 times shorter, for example 2, 3, 4 or 5 times shorter than the corresponding in vitro half-life of the PEGylated hGH prodrug conjugate. More preferably, the in vivo half-life is up to 3 times shorter than the corresponding in vitro half-life of the PEGylated hGH prodrug conjugate. Most preferably, the in vivo half-life is up to 2-fold shorter than the corresponding in vitro t half-life of the PEGylated hGH prodrug conjugate.

The present invention relates to PEGylated rhGH prodrugs, in particular rhGH PEGylated carrier prodrugs, including cascade carrier prodrugs. Prodrugs can be defined as therapeutic agents that are inactive per se but are predictably converted to active metabolites (see B. Testa, J.M: Mayer in Hydrolysis in Drug and Prodrug Metabolism, Wiley-VCH, 2003, p. 4). In carrier-bound prodrug systems, many medical agents are inactive or show reduced biological activity when the polymer is covalently conjugated to the drug molecule. In such cases, a temporary drug-carrier bond is used such that the medical agent is released from the polymeric carrier in vivo, restoring its biological activity. The reduced biological activity of the prodrug compared to the isolated drug is an advantage if slow or controlled release of the drug is desired. In this case, a relatively large amount of the prodrug can be administered without associated side effects and risk of overdose. The release of the drug occurs over time, thereby reducing the need for repeated and frequent administration of the drug.

In polymeric carrier bound prodrugs, the biologically active moieties are often linked to the polymeric carrier moiety by a labile bridge formed between the carrier moiety and the functional group of the drug molecule. Cleavage of the prodrug carrier creates a molecular compound (drug) with increased bioactivity and at least one by-product, the carrier. This by-product may be biologically inert (eg PEG). After cleavage, the biologically active compound exposes at least one previously conjugated and thus masked or protected functional group, and the presence of this group generally contributes to the biological activity. GR activity can be measured using methods known in the art. In this aspect, particular attention is paid to example 1. Based on the fact that some temporary linkers used in accordance with this invention may have an in vitro half-life of less than 3000 hours, appropriate measurements of GH activity are carried out indirectly, through the determination of the activity of GH of the corresponding PEG conjugate containing a permanent linker instead of a temporary linker. This can be done as described in WO 2006102659 at page 74, paragraph 0240, the biological activity of rhGH and the conjugates described here should be assessed in vitro using the NB2-II rat lymphoma cell proliferation assay. Briefly, rat lymphoma-derived NB2-II cells are incubated with rhGH, which results in the binding of the rhGH molecule to its receptor on the cell surface. Binding to the receptor induces a signal transduction cascade that gives rise to cell proliferation. The results of the analysis are based on the determined protein content and 100% biological activity of unmodified rhGH.

Preferably, the protocol described in Example 24 is used to measure GH activity.

The in vitro half-life can be measured using methods known in the art. Here, particular attention is paid to example 2. Therefore, the second aspect of the present invention relates to a clinically effective amount of a pharmaceutical composition containing the PEGylated rhGH prodrug of the first aspect, for use in a method of treating diseases associated with GH in humans.

This second aspect may alternatively be formulated as a method of treating a GH-associated disease in a human, comprising administering to the human a clinically effective amount of a pharmaceutical composition comprising the PEGylated rhGH prodrug of the first aspect.

In a situation where the residual activity of the prodrug (as, for example, when using transiently PEG-conjugated rhGH) is significantly reduced compared to natural human GH, lipolytic effects may not occur even with long-term use.

The compounds described herein, referred to as PEGylated rhGH prodrugs, are conjugates that may have significantly reduced residual activity compared to human GH. To exert therapeutic benefit, rhGH must be released from the prodrug conjugate, which requires the described prodrugs to undergo an activation step (e.g., 1,6-release mechanisms) referred to here as self-cleavage, cleaving the PEG moiety from the drug. The 1,6 release mechanism is described in WO-A 2005/099768. Without wishing to be bound by theory, the present inventors believe that the transiently PEGylated rhGH conjugates described herein significantly reduce lipoatrophy due to the low activity of the PEGylated rhGH conjugates before the PEG is gradually cleaved off by a self-cleaving linker. This property ensures that the prodrug will not cause lipoatrophy more frequently than human GH or other permanently PEGylated rhGH compounds described above.

The problem underlying the present invention is also solved by a pharmaceutical composition containing suitable pharmaceutical excipients and also containing a human growth hormone (hGH) prodrug conjugate of formula (AA)

Where

hGH-NH is a residue of hGH;

L ^a is a functional group that is self-hydrolyzable (self-cleaving) by a group G ^a that causes self-cleavage; And

S ⁰ is a polymer chain having a molecular weight of at least 5 kDa and containing at least a first BS ¹ branching structure, and at least a first BS ¹ branching structure, contains at least a second polymer chain S ¹ having a molecular weight of at least at least 4 kDa, where at least one of S ⁰ , BS ¹ , S ¹ also contains a group ^{G a} causing self-cleavage, and where the branching structure BS ¹ also contains at least a third polymer chain S ² having a molecular weight of at least 4 kDa, or at least one of S ⁰ , S ¹ contains at least a second BS ² branching structure containing at least a third S ² polymer chain having a molecular weight of at least 4 kDa, and where the molecular weight of the prodrug conjugate without hGH-NH is at least 25 kDa and not more than 1000 kDa, preferably at least 25 kDa and not more than 500 kDa, even more preferably at least 30 kDa and not more than 250 kDa, even more preferably at least 30 kDa and not more than 120 kDa, even more preferably at least 40 kDa and not more than 100 kDa, even more preferably at least 40 kDa and not more than 90 kDa. Surprisingly, it has been found that the residual activity of a prodrug according to the invention can be effectively reduced by the use of a polymeric carrier having at least 3 chains of a certain minimum length (as determined by their molecular weight) and thus, in combination with a transient linker as described herein, solves the problem of obtaining an hGH prodrug that can be administered less frequently without increasing the risk of lipoatrophy. The prodrug must be water soluble. For at least bis-conjugated prodrugs, the problem can be solved by a pharmaceutical composition containing suitable pharmaceutical excipients and also containing a human growth hormone (hGH) prodrug conjugate of formula (AB)

Where

n is 2, 3 or 4; preferably 2;

hGH(-MH) _n is a residue of hGH;

each L is independently a permanent functional group L ^p or a functional group L ^a that is self-hydrolyzable (self-cleaving) through the group G ^a causing self-cleavage; and each S ⁰ is independently a polymer chain having a molecular weight of at least 5 kDa, where S ⁰ is optionally branched, since it contains at least a first BS ¹ branching structure, where at least the first BS ¹ branching structure contains at least a second polymer chain S ¹ having a molecular weight of at least 4 kDa, where at least one of S ⁰ , BS ¹ , S ¹ also contains a self-cleavage group G ^a , and where the molecular weight of the prodrug conjugate without hGH-NH is at least 25 kDa and not more than 1000 kDa, preferably at least 25 kDa and not more than 500 kDa, even more preferably at least 30 kDa and not more than 250 kDa, even more preferably at least 30 kDa and not more than 120 kDa, even more preferably at least 40 kDa and not more than 100 kDa, even more preferably at least 40 kDa and not more than 90 kDa. Another aspect of this invention relates to a prodrug conjugate as defined above.

Preferred embodiments of the present invention are described below by way of example only.

DEFINITIONS

Before discussing the detailed embodiments of the present invention, descriptions of certain terms associated with the main aspects of the present invention are provided.

In general, all specific technical terms used here are to be understood as understood by those skilled in the art in the given technical context.

rhGH, or hGH, or GH, or hGH residue refers to human growth hormone. NH-hGH is the residue of hGH, where -NH- of -NH-hGH is the amino group of hGH. The term "activity" is understood as the ability of a growth hormone or its conjugate to elicit a biological response when administered to a mammal, for example, in an in vivo model, or to elicit a measurable response in an in vitro model, as described in the examples.

In a prodrug system, the activity measured has two components, one from the released free drug and one from the not yet cleaved prodrug conjugate. To separate the activity of the prodrug conjugate, the term "residual activity" is used here, which means the portion of the measured prodrug activity that can be attributed to the prodrug conjugate.

The term "self-cleavage" is understood as the rate-limiting hydrolytic cleavage of the bond between the transient linker and the rhGH drug molecule in aqueous buffered solution under physiological conditions of pH 7.4 and 37°C. Self-cleavage does not require the presence of an enzyme. This self-cleavage or self-hydrolysis is controlled by a self-cleaving group that is part of the prodrug molecule. Such a self-cleaving group may be present as such or in a masked form such that it needs to be unmasked before the self-hydrolysis mechanism can start.

The rate of self-hydrolysis of the linker refers to the rate of cleavage of the hGH-PEGylated prodrug in vivo. Enzymatic or other action almost always results in prodrug linker hydrolysis being faster in vivo than in vitro, and hGH PEG prodrug has been found to cleave autohydrolytically if the in vivo half-life of the prodrug is up to 5 times shorter than the corresponding half-life of the conjugate. PEGylated hGH prodrug in vitro.

The term "transient link" or "transient linker" describes the lability of the bond between PEG and rhGH in a PEGylated rhGH prodrug. In such temporal relationships, rhGH is released from the corresponding prodrug with an in vivo linker half-life of up to 1200 hours.

The term "conjugate" is understood as one or more PEG molecules covalently linked to a drug, which in this case is human growth hormone.

The term "temporary conjugate" refers to PEGylated hGH prodrugs containing at least one temporary linkage.

The term "permanent conjugate" refers to PEGylated hGH conjugates or prodrugs wherein the PEG polymer is linked to hGH via linkages, with an in vitro half-life of at least 3000 hours. The in vitro half-life or in vitro half-life of the linker is equal to the release of 50% hGH from the hGH PEGylated product in buffer at pH 7.4 and 37°C.

The terms "in vivo half-life" or "in vivo linker half-life" is understood to mean the time interval in which 50% of the original proportion of growth hormone is released from the PEGylated hGH prodrug after administration to the human body, calculated taking into account the half-life of the corresponding compound conjugate, as described in example 2.

The term "conjugate half-life" is understood as the time interval during which 50% of the PEGylated constant hGH conjugate as defined above is cleared from the circulation.

The term "lipoatrophy" is understood as the medical term for local loss of adipose tissue. In this context, "lipoatrophy" refers to lipoatrophy at the injection site, which means the passage of tissue lipolysis near the injection site.

The term "prodrug" is understood to mean any compound that undergoes transformation prior to its full pharmacological effect. The classification of prodrug systems is given in the IUPAC definitions (http://www.chem.qmul.ac.uk/iupac/medchem, accessed March 8, 2004):

Prodrug

A prodrug is any compound that undergoes biotransformation prior to demonstrating its pharmacological action. Prodrugs can be considered as drugs containing specialized non-toxic protecting groups, applied temporarily to change or remove undesirable properties in the original molecule.

Double prodrug (or pro-prodrug)

A dual prodrug is a biologically inactive molecule that is converted in vivo in two steps (enzymatically and/or chemically) into active species.

Carrier bound prodrug (carrier prodrug)

A carrier-bound prodrug is a prodrug that contains a temporary bond of the active substance to a temporary carrier group that gives improved physicochemical or pharmacokinetic properties, and that can be easily removed in vivo, usually by hydrolytic cleavage.

Cascade prodrug

A cascaded prodrug is a prodrug for which cleavage of the carrier group becomes effective only after the activating group has been unmasked.

Biotransformation

Biotransformation is the chemical transformation of substances by living organisms or enzyme preparations.

Accordingly, a cascading self-hydrolysis group becomes effective only after certain self-hydrolysis-inducing structural elements are unmasked. One or more cascaded unmasking steps may be carried out as required to open self-hydrolysis-causing building blocks. At least one of the unmasking steps may be based on a biotransformation step. The term "pharmaceutical composition comprising a clinically effective in vivo human amount of a PEGylated recombinant human growth hormone (rhGH) prodrug" means an amount that is high enough to produce a desired clinical effect in a human after administration of the pharmaceutical composition to a human, e.g., the desired clinical effect regarding the treatment of GH-associated disease. In this context, one skilled in the art will be able to adjust the amount of PEGylated recombinant human growth hormone (rhGH) prodrug administered to obtain the desired clinical effect. The term "physiological state" is understood to mean any in vitro or in vivo state identical or similar to the pH and temperature of the human body. More specifically, the physiological state refers to a state with a pH of about pH 7.4 (pH 6.8 - pH 7.8) and a temperature of about 37°C (from 35°C to 40°C). The term "linker" is often used in publications in the field of bioconjugation, and it broadly describes the chemical structures used to link two molecular compounds. Such a relationship may be permanent or temporary. A transient linker is a linker in which the conjugation of the drug to the PEG molecule is reversible. This implies that cleavage of the linker releases the drug in its natural and active form. Structural differentiation of the structural unit of the temporary linker from the polymer carrier can be difficult in the case of carrier-bound prodrugs, especially if the polymer is permanently attached to the clinker and therefore no degradation products of the linker are released as a result of cleavage of the prodrug. The structural definition of a linker becomes even more complex if the linker functions both as a self-cleaving group and as a branching structural unit. Therefore, in the context of this invention, the term "linker" can be used as a synonym for the combination of a functional group L ^a and a group that causes self-cleavage G ^a . In cases where supported prodrugs are described in which the carrier is a branched PEG, it is preferable to use structural descriptions based on combinations of L ^a and G ^a . In such a case, the cleavage group G ^a is considered to be part of the carrier polymer. Changing the chemical nature of G ^a makes it possible to design the self-cleavage properties of the corresponding carrier-bound prodrug to a large extent. The term "permanent linker" refers to a PEG conjugate with an hGH-donated primary amino group via the formation of an aliphatic amide or aliphatic carbamate. If conventional PEGylation reagents are used, the resulting conjugates are usually very resistant to hydrolysis and the rate of amide or carbamate bond cleavage will be too slow for therapeutic use of the prodrug system. However, such a permanent linker conjugate is useful for investigating the therapeutic applicability of prodrug conjugates, as they allow the assessment of residual activity.

If such stable bonds are used in a prodrug, cleavage of the functional group is not possible in a therapeutically useful amount of time without enzymatic catalysis.

The term "water-soluble prodrug" means a prodrug that is soluble in water under physiological conditions. Typically, a water-soluble prodrug will transmit at least 75%, more preferably at least 95% of the light transmitted by the same solution after filtration. By weight, the water-soluble polymer is preferably at least about 35% (by weight) soluble in water, even more preferably at least about 50% (by weight), even more preferably at least about 70% (by weight), even more preferably at least about 85% (by weight), even more preferably at least about 95% (by weight) or completely soluble in water.

The term "PEG" or "pegylation residue" is used herein for suitable water-soluble polymers characterized by repeating units. Suitable polymers may be selected from the group consisting of polyalkyloxy polymers, hyaluronic acid and its derivatives, polyvinyl alcohols, polyoxazolines, polyanhydrides, poly(orthoesters), polycarbonates, polyurethanes, polyacrylic acids, polyacrylamides, polyacrylates, polymethacrylates, polyorganic phosphazenes, polysiloxanes, polyvinylpyrrolidone , polycyanoacrylates and polyesters.

The PEG chains may be composed of a linking group, a polymeric group and an end group.

For branched monoconjugates of PEGylated hGH prodrugs, the critical distance defines the shortest distance between the site of attachment of the PEG chain S ⁰ to ^La and the first branching structure BS ¹ measured as connected atoms.

The term "PEG load" is understood as a description of the molecular weight of a polymer chain consisting of many repeating structural units attached to hGH. Total PEG load refers to the total molecular weight of all polymer carrier chains attached to hGH on a molecular basis.

Detailed description of the invention

Pharmaceutical composition containing suitable pharmaceutical excipients

As known to the person skilled in the art, the pharmaceutical composition contains pharmaceutically acceptable excipients and/or carriers. "Pharmaceutical acceptable" includes any excipient and/or additive which does not interfere with the effectiveness of the biological activity of the active ingredient and which is not toxic to the host to which it is administered. In a preferred embodiment, the pharmaceutical composition is a composition for subcutaneous administration, intramuscular administration or intravenous administration. These routes of administration are examples of preferred methods of administration for the treatment of the respective disorders/diseases described herein.

The pharmaceutical composition may contain other active ingredients than the PEGylated rhGH prodrug described herein.

Recombinant human growth hormone (rhGH)

Since recombinant human GH is identical in sequence to natural human GH, the term recombinant human growth hormone (rhGH) refers to the so-called equivalent biogenerics. Thus, the terms rhGH and hGH can be used interchangeably in this invention. The term "biogenerics" describes generic forms of biopharmaceutical substances; molecules obtained using biological processes, usually by methods of modern biotechnology. Generic pharmaceutical chemicals can be defined as molecules that, when compared to the original product, have substantially the same potency, are substantially chemically identical to their registered counterparts, are bioequivalent, are marketed through an abbreviated procedure after the expiration of the patent.

As one skilled in the art would know, it is now routine to make, for example, minor amino acid substitutions in target biologicals (here GR) without significantly affecting the activity of the biologicals.

In addition to recombinant human and biogenerics, the term recombinant human growth hormone (rhGH) refers to all possible rhGH polypeptides.

A precise description of possible rhGH polypeptides is given in WO-A 2005/079838 from Pharmacia Corporation, page 15, paragraph 0043 to paragraph 0053. The term "hGH polypeptide or hGH protein" in this specification encompasses all hGH polypeptides, preferably from mammals, more preferably from humans and mice, as well as variants, analogs, orthologues, homologues and derivatives thereof, and fragments thereof, which are characterized in that they promote growth in the growth phase and while maintaining normal physique, anabolism and fat metabolism.

The term "hGH polypeptide or protein" preferably refers to a 22 kDa hGH polypeptide having the sequence described by A.L. Grigorian et al., Protein Science (2005), 14, 902-913, as well as its variants, homologues and derivatives, which have almost the same biological activity (promoting growth in the growth phase and while maintaining normal physique, anabolism and fat metabolism) . More preferably, the term "polypeptide or hGH protein" refers to a polypeptide having exactly the above sequence.

hGH derivatives encompass in particular hGH prodrug conjugates containing permanently linked polymers such as PEG, i.e. a prodrug according to the invention may contain, in addition to one or more transient linker polymeric conjugates, other permanent linker polymeric conjugates.

The term "hGH polypeptide variants" as used herein refers to polypeptides from the same species but different from the reference hGH polypeptide. In general, the differences are limited so that the amino acid sequence of the reference and the variant is substantially the same, and, in most areas, identical.

Preferably, the hGH polypeptides are at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the reference polypeptide, preferably the hGH polypeptide has the sequence listed in A.L. Grigorian et al., Protein Science (2005), 14, 902-913. By a polypeptide having an amino acid sequence of at least, for example, 95% "identical" to the requested amino acid sequence, it is meant that the amino acid sequence of the polypeptide in question is identical to the requested sequence, except that the sequence of the polypeptide in question may include up to five amino acid substitutions for every 100 amino acids. the requested amino acid sequence. These reference sequence changes may occur at the amino or carboxy termini of the amino acid reference sequence, or anywhere between these terminal positions, inserted either separately between residues in the reference sequence, or as one or more adjacent groups in the reference sequence. The requested sequence may be the complete amino acid sequence, or any fragment defined as described herein.

Such hGH polypeptide variants may be natural variants, such as natural allelic variants encoded by one or more alternative forms of hGH occupying a given locus on the organism's chromosome, or isoforms encoded by natural splice variants derived from a single primary transcript. Alternatively, the hGH polypeptide variant may be a variant that is not known in nature, and which can be obtained using mutagenesis methods known in the art.

It is known in the art that one or more amino acids can be deleted from the N-terminus or C-terminus of a bioactive peptide or protein without significant loss of biological function (see, for example, Ron et al., (1993), Biol Chem., 268 2984-2988, the description of which is incorporated here by reference in its entirety). It will also be appreciated by one of ordinary skill in the art that certain amino acid sequences of hGH polypeptides can be altered without significantly affecting protein structure or function. Such mutations include deletions, insertions, inversions, repeats and substitutions, selected according to general rules known in the art to have minimal effect on activity. For example, instructions for making phenotypically latent amino acid substitutions are provided by Bowie et al. (1990), Science 247:1306-1310, incorporated herein by reference, where the authors indicate that there are two main approaches to study the resistance of an amino acid sequence to changes.

The first way refers to the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid substitutions at specific positions in cloned hGH, and selection or screening to identify sequences that retained functionality. These studies have shown that proteins are surprisingly resistant to amino acid substitutions. The authors also indicate that amino acid substitutions are likely to be permissive at certain positions in the protein. For example, the most masked amino acid residues require non-polar side chains, given that some characteristics of the surface side chains are usually retained. Other such phenotypically silent substitutions are described in Bowie et al., (1990) supra, referenced here.

Usually, as conservative substitutions, substitutions of aliphatic amino acids Ala, Val, Leu, and Phe with each other, interchange of hydroxyl residues Ser and Thr, substitution of acidic residues Asp and Glu, substitution between amide residues Asn and Gln, substitution of basic residues Lys and Arg, and substitutions among aromatic residues Phe, Tyr. In addition, the following groups of amino acids usually represent equivalent substitutions: (1) Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, lle, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His.

The term hGH polypeptide also encompasses all hGH polypeptides encoded by hGH analogues, orthologues and/or homologue species. As used herein, the term "hGH analogues" refers to hGHs from different and unrelated organisms that perform the same functions in each organism, but that do not originate from the ancestral structure that is common to the organisms' ancestors. Instead, similar hGHs occur separately and then evolve to perform the same function (or similar functions). In other words, similar hGH polypeptides are polypeptides with rather different amino acid sequences, but which have the same biological activity, namely, promote growth in the growth phase and maintain normal physique, anabolism and lipid metabolism. As used herein, the term "hGH orthologues" refers to hGH in two different species whose sequences are related to each other through a common homologous hGH in the ancestral species, but which have evolved to be distinct from each other. As used herein, the term "hGH homologues" refers to hGH from different organisms that perform the same functions in each organism and that are derived from an ancestral structure that is common to the organism's ancestors. In other words, hGH homologous polypeptides are polypeptides with similar amino acid sequences that perform the same biological function, namely promoting growth in the growth phase and maintaining normal physique, anabolism and lipid metabolism. Preferably, hGH polypeptide homologues can be defined as polypeptides having at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the reference hGH polypeptide, preferably the hGH polypeptide has the above sequence. Thus, an hGH polypeptide may be, for example: (i) one in which one or more amino acid residues are replaced by a conservative or non-conservative amino acid residue (preferably a conservative amino acid residue), and such substituted amino acid residue may or may not be encoded by the genetic code: or (ii) one in which one or more amino acid residues includes a substituent group; or (iii) one in which the hGH polypeptide is linked to another compound, such as a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol): or (iv) one in which additional amino acids are connected to the above form of the polypeptide, for example, a peptide fused to an IgG Fc region, or a leader or secretory sequence, or a sequence that is used to purify the above form of the polypeptide or proprotein sequence. hGH polypeptides may be monomers or multimers. Multimers can be dimers, trimers, tetramers, or multimers containing at least five monomeric polypeptide units. Multimers can also be homodimers or heterodimers. Multimers may result from hydrophobic, hydrophilic, ionic and/or covalent bonds and/or may be linked indirectly, for example, by the formation of liposomes. In one example, covalent bonds may be between heterologous sequences contained in a fusion protein containing an hGH polypeptide or fragment thereof (see, for example, US Pat. No. 5,478,925, the disclosure of which is incorporated here by reference in its entirety). In another example, an hGH polypeptide or fragment thereof is linked to one or more polypeptides, which may be either hGH polypeptides or heterologous polypeptides, via peptide linkers such as those described in US Pat. No. 5,073,627 (incorporated here by reference). Another way to obtain a multimer of hGH polypeptides is to use hGH polypeptides fused to a leucine zipper or isoleucine zipper polypeptide sequence, which are known to promote multimerization of the proteins in which they are found, using methods known to those skilled in the art, including the techniques set forth in WO 94/10308. In another example, hGH polypeptides can be linked by interactions between the Flag® polypeptide sequence contained in hGH fusion polypeptides containing the Flag® polypeptide sequence. hGH multimers can also be prepared using chemical methods known in the art, such as cross-linking using linker molecules and methods for optimizing the length of the linker molecule, known in the art (see, for example, US 5478925), known in the art. techniques of methods for obtaining one or more intermolecular bonds between cysteine residues located in the sequence of polypeptides, the presence of which is desirable in the multimer (see, for example, US 5478925, adding cysteine or biotin to the C-terminus or N-terminus of an hGH polypeptide and methods for obtaining trimers, containing one or more of these modified polypeptides (see, for example, US 5478925), or any of the 30 methods for obtaining liposomes containing hGH multimers (see, for example, US patent No. 5478925), the description of which is incorporated here by reference.

As used herein, the term “hGH polypeptide fragment” refers to any peptide or polypeptide comprising a contiguous range of a portion of the amino acid sequence of an hGH polypeptide, preferably a polypeptide having the above sequence.

PEGylated rhGH prodrug - preferred PEGs, polymer chains

As described above, the PEGylated rhGH prodrug described here should have relatively low activity.

Therefore, in a preferred embodiment, the total PEG load per growth hormone molecule is at least 25 kDa. In general, the total PEG load will be less than 1000 kDa. Preferably, the PEG load is at least 25 kDa and not more than 500 kDa, even more preferably at least 30 kDa and not more than 250 kDa, even more preferably at least 30 kDa and not more than 120 kDa, even more preferably at least 40 kDa and not more than 100 kDa, even more preferably at least 40 kDa and not more than 90 kDa.

The PEG may be attached to the hGH through one or more attachment points. With a single attachment point, the corresponding PEG in the PEG-hGH prodrug monoconjugate will be branched and contain at least 3 chains. Where there is more than one attachment point, such as in a bisconjugate, the corresponding PEG in the hGH PEG prodrug may be branched or linear. Bisconjugates may contain one or more temporary bonds, and the PEG may be linear or branched, or contain a mixture of one straight and one branched chain. If the bisconjugate contains one temporary bond and one linear and one branched chain, the temporary bond can be on any chain. If a branched PEG chain is used, there may be one or more branching entities.

A branched PEG is a PEG molecule consisting of a branch point connecting two or more PEG chains to form a molecule with a single attachment point for attachment to growth hormone. This may be two 20 kDa PEG chains combined to form one 40 kDa PEG molecule. If the molecule contains two or more branch points, the molecule is designated as 3- or 4-arm PEG, respectively.

Briefly, and subject to the above limitations, the PEG polymer is not limited to a particular structure, and may be linear, branched, or multi-armed (i.e., branched PEG or PEG attached to a polyol core), arborescent, or with cleavable linkers. Without being limited by theory, the goal of PEG loading is to provide a suitable molecular weight to obtain the required relatively low activity and avoid too high a PEG molecular weight, which can create other problems.

PEGylation of native human GH occurs at some lysine groups or at the N-terminal amine (F1), as detailed by Clark et al. (reference 2 here) on page 21973, table III. The F1 and LYS-140 positions are highly reactive. Moderately reactive positions are LYS-115, LYS-38 and LYS-70. Less reactive positions are LYS-172, LYS-41, LYS-158 and LYS-168.

In more general terms, the PEG used here in combination with a temporary linker can reduce the risk of lipoatrophy by appropriate selection of said polymer. However, the principles of this invention also apply to polymers other than PEG. Thus, the term PEG is used here only as an example for suitable polymers.

Thus, in a preferred embodiment, the hGH PEG prodrug is a monoconjugate conjugated with one of its primary amino groups to a self-cleaving ^L a functional group to an S ⁰ polymer chain. This S ⁰ polymer chain has a molecular weight of at least 5 kDa and contains at least one BS ¹ branching structure. Branching structure BS ¹ contains a second polymer chain S ¹ that has a molecular weight of at least 4 kDa.

As stated above, at least a third S ² polymer chain is required having a molecular weight of at least 4 kDa. The S ² polymer chain may be part of BS ¹ or may be another branch of S ⁰ or S ¹ , giving another BS ² branching structure that contains S ² .

If desired, more than 3 polymer chains are present in the prodrug conjugate of the invention, for example 4, 5, 6, 7 or 8. However, each other polymer chain has a molecular weight of at least 4 kDa. The total number of polymer chains is limited by the total weight of the prodrug conjugate, which should not exceed 1000 Da (without hGH-NH). Thus, a preferred embodiment of the present invention is a composition in which at least one of the branching structures BS ¹ , BS ² contains an additional fourth polymer chain S ³ having a molecular weight of at least 4 kDa, or one of S ⁰ , S ¹ , S ² contains a third branching structure BS ³ containing at least a fourth polymer chain S ³ having a molecular weight of at least 4 kDa. The self-cleavage group G ^a , which is necessary for the self-cleavage of L ^a , is contained in one of the branched structures or polymer chains.

Optionally, one of the branching structures serves as a group G ^a such that the branching structure consists of G ^a (instead of containing said group), which is also covered by the term "comprising". The production of a conjugate prodrug (AA) typically results in a mixture of conjugates where several hGH primary amino groups are conjugated to give different monoconjugates, different biconjugates, different triconjugates, and so on. prodrugs. The respective monoconjugated, bisconjugated or trisconjugated hGH PEG prodrugs can be separated by standard methods known in the art such as column chromatography and the like.

In monoconjugates of PEG hGH prodrugs, at least three polymer chains S ⁰ , S ¹ , S ² contain a "polymer fragment" that is characterized by one or more repeating structural units that can be distributed randomly, blocks or alternately. In addition, at least three polymer chains S ⁰ , S ¹ , S ² show an end group, which is usually a hydrogen atom or an alkyl group having from 1 to 6 carbon atoms, which may be branched or unbranched, for example, a methyl group , especially for PEG-based polymer chains, with the production of so-called mPEGs.

It is indicated that polymer fragments in at least three polymer chains S ⁰ , S ¹ , S ² may have additional chain-like substituents derived from repeating structural units and forming chains having a molecular weight of less than 4 kDa, which are not considered polymer chains S ⁰ , S ¹ , S ² etc. Preferably, at least three polymer chains S ⁰ , S ¹ , S ² have substituents with a molecular weight of less than 1000 Da.

A significant structural characteristic of S ⁰ is its critical distance. The critical distance defines the shortest distance between the point of attachment S ⁰ to L ^a and the first branching structure BS ¹ measured as connected atoms. The length of the critical distance affects the residual activity as described for compound 33. The critical distance is preferably less than 50, more preferably less than 20, most preferably less than 10.

At least typically, each of the three polymer chains S ⁰ , S ¹ and S ² contains a connecting fragment. G ^a is present in at least one of the connecting fragments. For polymer chains other than S ⁰ , the linking moiety is the building block linking the polymer moiety, eg S ¹ to BS ¹ , and the S ² polymer group to BS ² . For S ⁰ the connecting fragment is a structural element connecting L ^a and BS ¹ .

The connecting fragments may consist of C _1-50 alkyl chains, which are branched or unbranched, and which, if necessary, are interrupted or terminated by heteroatoms or functional groups selected from the group including -O-; -S-; N(R); C(O); C(O)N(R); N(R)C(O); one or more carbocycles or heterocycles, where R is a hydrogen atom or C _1-20 alkyl chains, which, if necessary, are interrupted or terminated by one or more of the above atoms or groups, which also have a hydrogen as a terminal atom; and where the carbocycle is phenyl; naphthyl; indenyl; indanyl; tetralinyl; C _3-10 cycloalkyl; and where the heterocycle is a 4-7 membered heterocyclyl; or 9-11 membered heterobicyclyl.

"C _3-10 cycloalkyl" or "C _3-10 cycloalkyl ring" means a cyclic alkyl chain having 3 to 10 carbon atoms, which may have carbon-carbon double bonds that are at least partially saturated, e.g. cyclopropyl, cyclobutyl , cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl. Each hydrogen atom of the cycloalkyl carbon may be substituted with a substituent. The term "C _3-10 cycloalkyl" or "C _3-10 cycloalkyl ring" also includes bridged bicycles such as norbonane or norbonene.

"4-7 membered heterocyclyl" or "4-7 membered heterocycle" means a ring with 4, 5, 6 or 7 ring atoms which may contain up to the maximum number of double bonds (aromatic or non-aromatic, which are wholly, partially or unsaturated), where at least one ring atom and up to 4 ring atoms are substituted with a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O) ₂ -), oxygen and nitrogen (including =N(O)-), and where the ring is connected to the rest of the molecule through a carbon or nitrogen atom. Examples of 4-7 membered heterocycles include azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazol , triazole, triazolidin, tetrazolidine, diazepane, azepine or homopiperazine.

"9-11 membered heterobicyclyl" or "9-11 membered heterobicycle" means a two-ring heterocyclic system with 9-11 ring atoms, where at least one ring atom is common to two rings, and which may contain up to up to the maximum number of double bonds (aromatic or non-aromatic ring that is fully saturated, partially saturated or unsaturated), where at least one ring atom, and up to 6 ring atoms are replaced by a heteroatom selected from the group consisting of sulfur (including -S (O)-, -S(O) ₂ -), oxygen and nitrogen (including =N(O)-), and where the ring is connected to the remaining molecule through a carbon or nitrogen atom. Examples of the 9-11 membered heterobicycle include indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazol, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinadoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydro isoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine or pteridine. The term 9-11-membered heterobicycle also includes two-ring spiro structures such as 1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocycles such as 8-azabicyclo[3.2.1]octane.

The carbocycle, heterocycle and heterobicycle may be substituted with C _1-20 alkyl optionally interrupted or terminated by heteroatoms or functional groups selected from the group consisting of —O—; -S-; N(R); C(O); C(O)N(R); N(R)C(O), where R is hydrogen or a C _1-10 alkyl chain, which is optionally interrupted or terminated by one or more of the above atoms or groups, which also have a hydrogen as a terminal atom.

A polymer fragment of at least three chains S ⁰ , S ¹ , S ² forms the main part of the chains, preferably at least 90% of the molecular weight of each chain, more preferably at least 95%, even more preferably at least 97.5% , even more preferably at least 99%. Thus, the main part of the chains is represented by a polymer fragment. Preferably, at least three S ⁰ , S ¹ , S ² chains are independently based on a polymer selected from the group consisting of polyalkyloxy polymers, hyaluronic acid and its derivatives, polyvinyl alcohols, polyoxazolines, polyanhydrides, poly(orthoesters), polycarbonates, polyurethanes, polyacrylic acids, polyacrylamides, polyacrylates, polymethacrylates, polyorganophosphazenes, polysiloxanes, polyvinylpyrrolidone, polycyanoacrylates and polyesters.

Preferably at least three chains S ⁰ , S ¹ , S ² are based on the same polymer. Preferably at least three S ⁰ , S ¹ , S ² chains are based on polyalkyloxy polymers. Even more preferably, at least three chains S ⁰ , S ¹ , S ² are based on polyethylene glycol.

The same applies to other chains S ³ , S ⁴ , S ⁵ , etc.

The chain S ⁰ contains a branching structure BS ¹ so that S ¹ is connected to S ⁰ . For the S ² link, a BS ¹ branching structure may be used, or another BS ² branching structure may be present, which may be part of S ⁰ or S ¹ .

Therefore, other branching structures may be present in which other chains are present. For example, if an S ³ chain is present, it may be linked to BS ¹ , BS ² or a BS ³ branching structure. Branching structure BS ³ if present, may be part of S ⁰ , S ¹ or S ² .

In general, any chemical that allows chain branching can be used. Preferably, the branching structures are independently selected from the group consisting of at least a 3-fold substituted carbocycle, at least a 3-fold substituted heterocycle, a tertiary carbon atom, a quaternary carbon atom, and a tertiary nitrogen atom, wherein the terms carbocycle and heterocycle are as defined above.

PEGylated rhGH prodrug - linker temporal structures, L ^a , G ^a

In publications from the field of self-cleavage, the groups that cause it are sometimes referred to as linkers to distinguish their structure from the carrier. However, it is often very difficult to clearly separate these structural characteristics. Therefore, in terms of the present invention, the self-cleaving group G ^a is considered to be part of the carrier S containing at least S ⁰ , S ¹ , S ² , BS ¹ and optionally BS ² . Changing the chemical nature of G ^a makes it possible to create self-cleavage properties of the corresponding carrier-bound prodrug to a large extent.

As described above, a PEGylated prodrug, where the drug is, for example, rhGH, as described in WO-A 2005/099768, and has release characteristics that are described here as a 1,6 cleavage system without the formation of toxic aromatics. This document broadly describes multiple, related to those described here, suitable structures of temporary linkers to obtain the appropriate target release profile. Other transient linker structures are generally/broadly described in, for example, other applications from Complex Biosystems GmbH applications such as WO-A 2005/034909, WO-A 2005/099768, WO-A 2006/003014 and WO-A 2006/136586 . More structures of temporary linkers are widely described, for example, in WO-A 99/30727 (Enzon Inc).

In order to address the present GH problems described above, the present inventors have chosen preferred transient linker structures to obtain the respective functional properties of the PEGylated rhGH prodrug described herein. Based on the detailed description of preferred linker structures provided herein, one skilled in the art will be able to prepare other suitable preferred transient linker structures that will yield a PEGylated rhGH prodrug with the appropriate functional properties described herein.

Suitable temporary linker structures which are self-hydrolyzable (self-cleaving) can preferably be selected for introduction into S ⁰ . Selectable linker structures are detailed below. Ideally, a conjugate of the invention has one or more of the following characteristics and/or advantages over current rhGH conjugates or formulations: can be easily synthesized in good yield, has a half-life in the preferred range, can be purified to produce homogeneous conjugate compositions, exhibits activity after self-cleavage, for example, in vitro and in vivo activity, and has pharmacodynamic effects superior to unmodified rhGH and previously described rhGH conjugates, and does not cause lipoatrophy. The structures described here demonstrate the release properties required here.

In general, carrier-bound prodrugs require the presence of cleavable functional groups linking the drug and the carrier. In the absence of self-hydrolysis-causing groups, functional groups that include drug-donated amino groups, such as aliphatic amide or ^La carbamate linkages, are generally very resistant to hydrolysis and the rate of amide bond cleavage is too low for therapeutic use of the prodrug system. If such stable bonds are used in carrier-bound prodrugs, cleavage of the functional group is not possible in a therapeutically useful period of time without biotransformation. In such cases, the linker exhibits a structural motif that is recognized as a substrate by the appropriate endogenous enzyme. In such a case, cleavage of the L ^a functional bond involves the complex containing the enzyme. Examples include peptide linkers that are recognized by endogenous proteases and cleaved by enzymes.

Enzyme levels can vary significantly between individuals, which causes biological variation in prodrug activation upon enzymatic cleavage. Enzyme levels can also be highly dependent on the site of administration. For example, when injected subcutaneously, certain areas of the body are known to provide a more predictable therapeutic effect than others. Such a high level of interindividual variation is undesirable. Moreover, it is difficult to correlate in vivo-in vitro pharmacokinetic properties for such prodrugs associated with an enzyme-dependent carrier. In the absence of a reliable in vivo-in vitro correlation, optimization of the release profile becomes a laborious task.

To avoid inter-individual variations and variability in the site of administration, it is desirable to use carrier-bound prodrugs that have cleavage kinetics in a therapeutically useful time period without the additional involvement of cleavage enzymes. Particularly for high molecular weight supports, more specifically for branched polymeric supports, access to the linking functional group La ^a may be limited for enzymes due to spatial grouping. Therefore, there is a need to create supported prodrugs that have self-cleaving properties.

The kinetics of self-cleavage, for example, can be measured in vitro by recording the rate of hydrolysis in a buffer solution without enzymes.

To introduce hydrolytic lability into functional groups L, such as amides or carbamates, it is necessary to create structural chemical components in the carrier in order for them to act, for example, as adjacent groups next to the functional groups. Such self-cleaving chemical structures that control the cleavage of the amide bond of the prodrug are referred to as self-cleaving G ^a groups. Self-cleaving groups can have a strong effect on the rate of hydrolysis of a given L ^a functional group.

Preferred ^La is selected from the group consisting of C(O)-O- and C(O)- which form, together with the hGH primary amino group, a carbamate or amide group. Thus, the composition according to the present invention is preferred wherein L ^a is selected from the group consisting of C(O)-O- and C(O)- which form, together with the hGH primary amino group, a carbamate or amide group to give the formula ( AA1) or (AA2)

The following section lists various structural components that can function as G ^a groups causing self-splitting. The group G ^a is a self-splitting group. G ^a may be present as such or as self-cleaving cascade groups which are opened for activation by an additional hydrolytic or enzymatic cleavage step. If G ^a is present as such, it drives the rate-limiting self-hydrolysis of L ^a .

G ^a examples:

A.J. Garman et al. (A.J. Garman, S.B. Kalindjan, FEBS Lett. 1987, 223 (2), 361-365 1987) use PEG5000-maleic anhydride to reversibly modify amino groups in tissue plasminogen activator and urokinase. Regeneration of the functional enzyme from the PEG-uPA conjugate by incubation at pH 7.4 in buffer by cleavage of maleamic acid bonds gives first order kinetics with a half-life of 6.1 h.

Simple aromatic moieties can cause lability in the attached carbamate bond (WO-A 01/47562). For example, a substituted or unsubstituted fluorenylmethyl group is used to labilize carbamate bonds in various bioactive agents when using a prodrug (Tsubery et al. J Biol Chem 279 (2004) 38118-24). Two PEG chains are attached to the fluorenyl group in WO-A 2007/075534.

Thus, G ^a is an aromatic ring or fluorenylmethyl directly attached to the carbamate functional group L ^a . Therefore, according to the present invention, a composition is preferred in which G ^a is an aromatic ring or fluorenyl methyl directly attached to a carbamate functional group formed by L ^a and the primary amino group of hGH. Alternatively, the transformation of G ^a can cause a molecular rearrangement in S ⁰ such as 1,4 or 1,6-cleavage. The rearrangement makes L ^a so much more labile that it is caused to split. The G ^a transformation is the rate-limiting step in the cascade mechanism. Ideally, the rate of cleavage of the temporary bond is identical to the desired release rate for the drug molecule in a given therapeutic scenario. In such a cascade system based on 1,6 cleavage, it is desirable that the cleavage of L ^a be substantially immediate after its lability has been caused by the transformation of G ^a . In addition, it is desirable that the rate-limiting cleavage kinetics take place in a therapeutically useful period of time without the need to involve additional enzymes to avoid the disadvantages associated with the predominantly enzymatic cleavage described above.

In R.B. Greenwald, A. Pendri, C.D. Conover, H. Zhao, Y.H. Choe, A. Martinez, K. Shum, S. Guan, J. Med. Chem., 1999, 42, 3657-3667 and PCT application WO-A 99/30727 describe a method for the synthesis of poly(ethylene glycol) prodrugs of amine-containing low molecular weight compounds based on the elimination of 1,4- or 1,6-benzyl. In this approach, the amino group of the drug molecule is linked through the carbamate group to the PEGylated benzyl moiety.

Poly(ethylene glycol) is attached to the benzyl group via an ester, carbonate, carbamate, or amide. The release of PEG from the drug molecule occurs through a combination of self-hydrolysis and enzymatic cleavage. The cleavage of the release-initiating masking group, in this approach, is followed by the classic and rapid cleavage of 1,4- or 1,6-benzyl. This linker system is also used for releasable poly(ethylene glycol) protein conjugates (S. Lee, R.B. Greenwald et al. Bioconj. Chem. 2001, 12 (2), 163-169). Lysozyme is used as a model protein because it loses activity if PEGylation occurs at the epsilon-amino group of lysine residues. Varying amounts of PEG linker are conjugated to the protein. Native protein regeneration from PEG conjugates takes place in rat plasma or in a non-physiological high pH buffer. See also F.M.H. DeGroot et al. (WO-A 2002/083180 and WO-A 2004/043493) and D. Shabat et al. (WO-A 2004/019993).

Thus, L ^a is a carbamate functional group, while the cleavage of this group is caused by the hydroxyl or amino group G ^a through the elimination of 1,4- or 1,6 benzyl S ⁰ , where G ^a contains ester, carbonate, carbamate or amide bonds that undergo rate-limiting transformation. In fact, G ^a can be cleaved off by hydrolysis.

Therefore, a composition according to the invention is preferred in which L ^a forms, together with the amino group of hGH, a carbamate functional group, the cleavage of said group is caused by the hydroxyl or amino group G ^a through the elimination of 1,4- or 1,6 benzyl S ⁰ , where G ^a contains ester, carbonate, carbamate or amide bonds that undergo rate-limiting conversion.

G ^a may contain a cascade splitting system that is triggered by components of G ^a that consist of a structural combination representing the above precursor. The G ^a precursor may contain additional temporary bonds such as an amide, ester or carbamate. The stability or susceptibility to hydrolysis of the temporary bond of the precursor (eg, carbamate) may be driven by self-hydrolytic properties or may require enzyme activity.

Antczak et al. (Bioorg Med Chem 9 (2001) 2843-48) describe a reagent that forms the basis for a macromolecular prodrug cascade system for amine-containing drug molecules. In this approach, the antibody serves as a carrier, the stable bond binds the antibody to an activating group bearing a cleavable masking group. Upon removal of the ester-bound masking group, ^La is cleaved off and releases the drug compound.

D. Shabat et al. (Chem. Eur. 3. 2004, 10, 2626-2634) describe a polymeric prodrug system based on the mandelic acid activating group. In this system, the masking group is linked to the activating group by a carbamate bond. The activating group is permanently conjugated to the polyacrylamide polymer via an amide bond. Upon activation of the masking group by the catalytic antibody, the masking group is cleaved off by cyclization and the drug is released. The activating group is still bound to the polyacrylamide polymer after drug release. M.-R. Lee et al. describe (Angew. Chem. 2004, 116, 1707-17 10) a similar prodrug system based on a mandelic acid activating group and an ester-linked masking group. However, in these linkers, the 1,6 cleavage step still produces a highly reactive aromatic intermediate. Even if the aromatic moiety remains permanently attached to the polymer carrier, side reactions with potentially toxic or immunogenic effects can be induced. Greenwald et al. published in 2000 a poly(ethylene glycol) drug delivery system for amine-containing prodrugs based on trimethyl blocking lactonization (RB Greenwald et al. J. Med. Chem. 2000, 43(3), 457-487; WO-A 02/089789) . In such a prodrug system, substituted o-hydroxyphenyldimethylpropionic acid is coupled to the amino groups of drug molecules by an amide bond. The hydroxy group is linked to the PEG by an ester, carbonate, or carbamate group. The rate-determining step in drug release is the enzymatic cleavage of these functional groups, followed by rapid amide cleavage via lactonization, releasing the aromatic by-product lactone. Recently, RB Greenwald et al. (Greenwald et al. J. Med. Chem. 2004, 47, 726-734) described a PEG prodrug system based on a bis(N-2-hydroxyethyl)glycinamide (bicinamide) linker. In this system, two PEG molecules are linked to a bicine molecule attached to the amino group of the drug molecule. The first two steps in prodrug activation involve enzymatic cleavage of both PEG molecules. Various relationships between PEG and bicine have been described, leading to different kinetics of prodrug activation. The main disadvantage of this system is the slow rate of hydrolysis of bicinamide conjugated to the drug molecule (t _1/2 =3 h in phosphate buffer), which results in the release of the bicine-modified prodrug intermediate, which may exhibit pharmacokinetic and pharmacodynamic properties that differ from those of the parent molecule. medicinal product.

More specifically, preferred L ^a and G ^a groups with specific spacer moieties for S ⁰ are described below.

The preferred structure according to WO-A 2005/099768 is selected from the general formulas (I) and (II):

where T is hGH-NH; X is a spacer fragment; Y ₁ and Y ₂ are each independently O, S or NR ₆ ; Y ₃ is O or S; Y ₄ is O, NR ₆ or -C(R ₇ )(R ₈ ); R ₃ is a moiety selected from the group consisting of hydrogen, substituted or unsubstituted linear, branched or cyclic alkyls or heteroalkyls, aryls, substituted aryls, substituted or unsubstituted heteroaryls, cyano groups, nitro groups, halogens, carboxy groups, carboxyalkyl groups, alkylcarbonyl groups or carboxamidoalkyl groups ; R ₄ is a moiety selected from the group consisting of hydrogen, substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryl, substituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted linear, branched or cyclic alkoxy, substituted or unsubstituted linear, branched or cyclic heteroalkyloxy, aryloxy or heteroaryloxy, cyano groups and halogens; R ₇ and R ₈ are each independently selected from the group consisting of hydrogen, substituted or unsubstituted linear, branched or cyclic alkyls or heteroalkyls, aryls, substituted aryls, substituted or unsubstituted heteroaryls, carboxyalkyl groups, alkylcarbonyl groups, carboxamidoalkyl groups, cyano groups and halogens; R ₆ is a group selected from hydrogen, substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryl, substituted aryl, substituted or unsubstituted heteroaryl; R ₁ is the remainder of S ⁰ ; W is a group selected from substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryl, substituted aryl, substituted or unsubstituted linear, branched or cyclic heteroalkyl, substituted or unsubstituted heteroaryl; Nu is a nucleophile; n is zero or a positive integer; and Ar is a multi-substituted aromatic hydrocarbon or a multi-substituted aromatic heterocycle.

In terms of this invention, the group L ^a is represented by Y ₃ -C(Y ₅ )NH- (together with the amino group hGH), G ^a is represented by Nu-WY ₄ -C(Y ₁ )Y ₂ and Ar(R ₄ ) _n -C( R ₃ )XR ₁ is S ⁰ , which also includes at least S ¹ , S ² , BS ¹ and optionally BS ² . In an alternative embodiment, S ¹ is attached through Ar or is R ₃ . Then the carbon atom adjacent to Y ₃ substituted with XR ¹ is a branching structure BS ¹ , S ¹ ends with Ar containing G ^a . Obviously, in this embodiment, the terms S ⁰ and S ¹ are interchangeable.

Preferably, in the formula (AA) or (AA1) S ⁰ is the formula (AAA1)

Where

G ^a has the above meaning;

S ⁰⁰ is CH ₂ ; or C(O);

S ^0A is an alkylene chain containing from 1 to 20 carbon atoms, which is optionally interrupted or terminated by one or more groups, cycles or heteroatoms selected from the group consisting of an optionally substituted heterocycle; ABOUT; S; C(O); and NH;

BS ¹ , BS ² , BS ³ are independently selected from the group consisting of N and CH.

S ^0B , S ^1A are independently an alkylene chain containing from 1 to 200 carbon atoms, which is optionally interrupted or terminated by one or more groups, cycles or heteroatoms selected from the group consisting of an optionally substituted heterocycle; ABOUT; S; C(O); and NH;

S ^0C , S ^1B are (C(O)) _n2 (CH ₂ ) _n1 (OCH ₂ CH ₂ ) _n OCH ₃ , where each n is independently an integer from 100 to 500, each n1 is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8 and n2 is 0 or 1.

S ² , S ³ are independently hydrogen or (C(O) _n2 (CH ₂ ) _n1 (OCH ₂ CH ₂ ) _n OCH ₃ , where each n is independently an integer from 100 to 500, each n1 is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and n2 is 0 or 1, provided that at least one of S ² , S ³ is other than hydrogen.

R ² , R ³ are defined as in formula (A) below.

The term heterocycle means a heterocycle as defined above. Optional substituents are, for example, oxo (=O), where the ring is at least partially saturated, branched or straight chain alkyl containing from one to 6 carbon atoms, or halogen. A preferred substituted heterocycle is succinimide.

Preferably, G ^a in formula (AAA1) is OC(O)-R and R is a partial structure of formula (I) shown below, where R1, R4, R5 and n are as defined below.

Another preferred embodiment is described in WO 06136586 A2. Therefore, the following structures are preferred:

where T means NH-hGH;

X is a spacer moiety such as R13-Y1;

Y1 is O, S, NR6, succinimide, maleimide, unsaturated carbon-carbon bonds, or any heteroatom containing a free electron pair, or absent;

R13 is selected from substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryls, substituted aryls, substituted or unsubstituted heteroaryls;

R2 and R3 are independently selected from hydrogen, acyl groups, or hydroxyl protecting groups;

R4-R12 is independently selected from hydrogen, X-R1, substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryls, substituted aryls, substituted or unsubstituted heteroaryls, cyano, nitro, halogen, carboxy, carboxamide;

R1 is the remainder of S ⁰ containing at least S ¹ , S ² , BS ¹ and optionally BS ² .

In this embodiment, L ^a is an amide group, and G ^a encompasses N-branched structures bearing OR ₂ /OR ₃ .

In yet another preferred embodiment, the preferred structure is a D-L prodrug conjugate wherein

- D is NH-hGH; And

- L is a biologically inactive linker fragment -L ¹ represented by formula (I),

where the dotted line shows the accession to the amino group of hGH through the formation of an amide bond;

X is C(R ⁴ R ^4a ); N( ^R4 ); ABOUT; C(R ⁴ R ^4a )-C(R ⁵ R ^5a ); C(R ⁵ R ^5a )-C(R ⁴ R ^4a ); C(R ⁴ R ^4a )-N(R ⁶ ); N(R ⁶ )-C(R ⁴ R ^4a ); C(R ⁴ R ^4a )-O; or OC(R ⁴ R ^4a );

X ¹ is C or S(O);

X ² is C(R ⁷ , R ^7a ) or C(R ⁷ , R ^7a )-C(R ⁸ , R ^8a );

R ¹ , R ^1a , R ² , R ^2a , R ³ , R ^3a , R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ⁷ , R ^7a , R ⁸ , R ^8a are independently selected from the group consisting of H and C _1-4 alkyl; or

if necessary, one or more of the pairs R ^1a /R ^4a , R ^1a /R ^5a , R ^4a /R ^5a , R ^4a /R ^5a , R ^7a /R ^8a form a chemical bond;

optionally, one or more of the pairs R ¹ /R ^1a , R ² /R ^2a , R ⁴ /R ^4a , R ⁵ /R ^5a , R ⁷ /R ^7a , R ⁸ /R ^8a together with the atom to which they attached, form C _3-7 cycloalkyl;

or 4-7 membered heterocyclyl;

optionally, one or more of the pairs R ¹ /R ⁴ , R ¹ /R ⁵ , R ¹ /R ⁶ , R ⁴ /R ⁵ , R ⁷ /R ⁸ , R ² /R ³ together with the atom to which they attached, form ring A;

if necessary, R ³ /R ^3a together with the nitrogen atom to which they are attached, form a 4-7-membered heterocycle;

A is selected from the group consisting of phenyl; naphthyl; indenyl; indanyl; tetralinyl; C _3-10 cycloalkyl; 4-7 membered heterocyclyl; and 9-11-membered heterobicyclyl; And

where L ¹ is substituted with one group L ² -Z and, if necessary, substituted additionally, provided that the hydrogen marked with an asterisk in formula (I) is not substituted by a substituent; Where

L ² is a single chemical bond or spacer; And

Z is the remainder of S ⁰ containing at least S ¹ , S ² , BS ¹ and optionally BS ² .

In this embodiment, L ^a is an amide group and G ^a is N(H*)X ¹ (O) and a chain linked to N, including N substituents.

Prodrug conjugates of this type are described in European Patent Application No. 08150973.9

Therefore, a composition according to the invention is preferred wherein L ^a -S ⁰ is represented by the formula (AAA2),

where the dotted line shows the attachment to the primary amino group hGH so that L ^a and the amino group form an amide bond;

X is C(R ⁴ R ^4a ); N( ^R4 ); ABOUT; C(R ⁴ R ^4a )-C(R ⁵ R ^5a ); C(R ⁵ R ^5a )-C(R ⁴ R ^4a ); C(R ⁴ R ^4a )-N(R ⁶ ); N(R ⁶ )-C(R ⁴ R ^4a ); C(R ⁴ R ^4a )-O; or OC(R ⁴ R ^4a );

X ¹ is C or S(O);

X ² is C(R ⁷ , R ^7a ) or C(R ⁷ , R ^7a )-C(R ⁸ , R ^8a );

R ¹ , R ^1a , R ² , R ^2a , R ³ , R ^3a , R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ⁷ , R ^7a , R ⁸ , R ^8a are independently selected from the group consisting of H and C _1-4 alkyl; or

if necessary, one or more of the pairs R ^1a /R ^4a , R ^1a /R ^5a , R ^4a /R ^5a , R ^4a /R ^5a , R ^7a /R ^8a form a chemical bond;

if necessary, one or more of the pairs R ¹ /R ^1a , R ² /R ^2a , R ⁴ /R ^4a , R ⁵ /R ^5a , R ⁷ /R ^7a , R ⁸ /R ^8a combined with the atom to which they attached to give C _3-7 cycloalkyl or 4-7 membered heterocyclyl;

if necessary, one or more of the pairs R ¹ /R ⁴ , R ¹ /R ⁵ , R ¹ /R ⁶ , R ⁴ /R ⁵ , R ⁷ /R ⁸ , R ² /R ³ combined with the atom to which they attached to form ring A;

optionally, R ³ /R ^3a are combined together with the nitrogen atom to which they are attached to form a 4-7 membered heterocycle;

A is selected from the group consisting of phenyl; naphthyl; indenyl; indanyl; tetralinyl; C _3-10 cycloalkyl; 4-7 membered heterocyclyl; and 9-11-membered heterobicyclyl; And

where S ⁰ is substituted with one group L ² -Z and, if necessary, substituted additionally, provided that the hydrogen marked with an asterisk in formula (I) is not substituted by a substituent; Where

L ² is a single chemical bond or spacer; And

Z is the formula (AAA2a)

where S ⁰⁰ , S ^0A , S ^0B , S ^0C , S ^{1A , S 1B ,} S ² , S ³ , BS ¹ , BS ² and BS ³ are as defined for formula (AAA1) above.

"Alkyl" means a straight or branched hydrocarbon chain. Each alkyl hydrogen may be substituted with a substituent.

"C _1-4 alkyl" means a chain containing 1-4 carbon atoms, for example, if present at the end of the molecule: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl tert-butyl, or, for example, -CH ₂ -, -CH ₂ -CH ₂ -, -CH (CH ₃ ) -, -CH ₂ -CH ₂ -CH ₂ -, -CH (C ₂ H ₅ ) -, -C (CH ₃ ) ₂ - when two fragments of the molecule are connected by an alkyl group. Each C _1-4 alkyl hydrogen may be substituted with a substituent.

"C _1-6 alkyl" means an alkyl chain containing 1-6 carbon atoms, for example, if present at the end of the molecule: C _1-4 alkyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec- butyl; tert-butyl, n-pentyl, n-hexyl, or, for example, -CH ₂ -, -CH ₂ -CH ₂ -, -CH (CH ₃ )-, -CH ₂ -CH ₂ -CH ₂ -, -CH (C ₂ H ₅ )-, -C(CH ₃ ) ₂ -, when two fragments of the molecule are linked by an alkyl group. Each C _1-6 alkyl hydrogen may be substituted with a substituent.

Therefore, "C _1-18 alkyl" means an alkyl chain containing 1-18 carbon atoms, and "C _8-18 alkyl" means an alkyl chain containing 8-18 carbon atoms. Therefore, "C _1-50 alkyl" means an alkyl chain containing 1-50 carbon atoms.

"C _2-50 alkenyl" means a branched or unbranched alkenyl chain containing from 2 to 50 carbon atoms, for example, if present at the end of the molecule: -CH=CH ₂ , -CH=CH-CH ₃ , -CH ₂ -CH =CH ₂ , -CH=CH-CH ₂ -CH ₃ , -CH=CH-CH=CH ₂ , or, for example, -CH=CH-, when two fragments of the molecule are linked by an alkenyl group. Each C _2-50 hydrogen atom of the alkenyl may be substituted with a substituent as defined below. Therefore, the term "alkenyl" refers to a carbon chain with at least one carbon-carbon double bond. One or more triple bonds may be present, if desired.

"C _2-50 alkynyl" means a branched or straight chain alkynyl chain containing from 2 to 50 carbon atoms, for example, if present at the end of the molecule: -C≡CH, -CH ₂ -C≡CH, CH ₂ -CH ₂ -C ≡CH, CH ₂ -C≡C-CH ₃ , or, for example, -C≡C- when two fragments of the molecule are linked by an alkynyl group. Each C _2-50 hydrogen atom of the alkynyl may be substituted with a substituent as defined below. Therefore, the term "alkynyl" refers to a carbon chain with at least one carbon-carbon triple bond. One or more double bonds may be present, if desired.

"C _3-7 cycloalkyl" or "C _3-7 cycloalkyl ring" means a cyclic alkyl chain containing from 3 to 7 carbon atoms, which may contain carbon-carbon double bonds that are at least partially saturated, e.g. cyclopropyl, cyclobutyl , cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl. Each cycloalkyl hydrogen may be substituted with a substituent. The term "C _3-7 cycloalkyl" or "C _3-7 cycloalkyl ring" also includes bridged bicycles such as norbornane or norbornene. Therefore, "C _3-5 cycloalkyl" means cycloalkyl containing 3 to 5 carbon atoms.

Therefore, "C _3-10 cycloalkyl" means cyclic alkyl containing from 3 to 10 carbon atoms, for example, C _3-7 cycloalkyl; cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl. The term "C _3-10 cycloalkyl" also includes at least partially saturated carbomono and bicycles.

"Halogen" means fluorine, chlorine, bromine or iodine. It is generally preferred that the halogen is fluorine or chlorine.

"4-7 membered heterocycle yl" or "4-7 membered heterocycle" means a ring with 4, 5, 6 or 7 ring atoms which may contain up to the maximum number of double bonds (aromatic or non-aromatic ring which is completely saturated, partially saturated or unsaturated), where at least one atom to up to 4 ring atoms are substituted with a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O) ₂ -), oxygen and nitrogen (including =N(O)-), and where the ring is connected to the rest of the molecule through a carbon or nitrogen atom. Examples of 4-7 membered heterocycles include azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazol , triazole, triazolidin, tetrazolidine, diazepane, azepine or homopiperazine.

"9-11 membered heterobicyclyl" or "9-11 membered heterobicycle" means a two-ring heterocyclic system with 9-11 ring atoms, where at least one ring atom is common to both rings, and which may contain up to up to the maximum number of double bonds (aromatic or non-aromatic ring that is fully saturated, partially saturated or unsaturated), where at least one ring atom up to 6 ring atoms are substituted with heteroatoms selected from the group consisting of sulfur (including -S( O)-, -S(O) ₂ -), oxygen and nitrogen (including =N(O)-), and where the ring is connected to the rest of the molecule through a carbon or nitrogen atom. Examples of the 9-11 membered heterobicycle include indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazol, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydro isoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, poutine or pteridine.

The term 9-11-membered heterobicycle also includes two-ring spiro structures such as 1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocycles such as 8-azabicyclo[3.2.1]octane.

Preferably, L ^a -S ⁰ is selected from the group consisting of

where R is H or C _1-4 alkyl; Y is NH, O or S; and R ¹ , R ^1a , R ² , R ^2a , R ³ , R ^3a , R ⁴ , X, X ¹ , X ² are as defined above.

Even more preferably, L ^a -S ⁰ is selected from the group consisting of

where R is as defined above.

At least one (up to four) hydrogen atoms is substituted with an L ² -Z group. If more than one L ² -Z group is present, each L ² and each Z may be independently selected. Preferably, only one L ² -Z group is present.

In general, S ⁰ may be substituted with L ² -Z at any position except for the replacement of the hydrogen atom marked with an asterisk in the above formula. Preferably, one to four hydrogen atoms in R, R ¹ -R ⁸ directly, or as a C _1-4 alkyl hydrogen atom or other groups given in the definition of R and R ¹ -R ⁸ , are substituted with L ² -Z.

In addition, S ⁰ can be additionally substituted if necessary. In general, any substituent may be used which does not affect the cleavage principle.

Preferably, one or more other optional substituents are independently selected from the group consisting of halogen; CN; COOR ⁹ ; ^OR9 ; C(O)R ⁹ ; C(O)N(R ⁹ R ^9a ); S(O) ₂ N(R ⁹ R ^9a ); S(O)N(R ⁹ R ^9a ); S(O) ₂ R ⁹ ; S(O)R ⁹ ; N(R ⁹ )S(O) ₂ N(R ^9a R ^9b ); ^SR9 ; N(R ⁹ R ^9a ); _NO2 ; OC(O)R ⁹ ; N(R ⁹ )C(O)R ^9a ; N(R ⁹ )S(O) ₂ R ^9a ; N(R ⁹ )S(O)R ^9a ; N(R ⁹ )C(O)OR ^9a ; N(R ⁹ )C(O)N(R ^9a R ^9b ); OC(O)N(R ⁹ R ^9a ); T; C _1-50 alkyl; C _2-50 alkenyl; or C _2-50 alkynyl, where T; C _1-50 alkyl; C _2-50 alkenyl; and C _2-50 alkynyl optionally substituted with one or more R ¹⁰ that are the same or different, and where C _1-50 alkyl; C _2-50 alkenyl; and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of T, -C(O)O-; -ABOUT-; -C(O)-; -C(O)N(R ¹¹ )-; -S(O) ₂ N(R ¹¹ )-; -S(O)N(R ¹¹ )-; -S(O) ₂ -; -S(O)-; -N(R ¹¹ )S(O) ₂ N(R ^11a )-; -S-; -N(R ¹¹ )-; -OC(O)R ¹¹ ; -N(R ¹¹ )C(O)-; -N(R ¹¹ )S(O) ₂ -; -N(R ¹¹ )S(O)-; -N(R ¹¹ )C(O)O-; -N(R ¹¹ )C(O)N(R ^11a )- and -OC(O)N(R ¹¹ R ^11a );

R ⁹ , R ^9a , R ^9b are independently selected from the group consisting of H; T; and C _1-50 alkyl; C _2-50 alkenyl; or C _2-50 alkynyl, where T; C _1-50 alkyl; C _2-50 alkenyl; and C _2-50 alkynyl optionally substituted with one or more R ¹⁰ that are the same or different, and where C _1-50 alkyl; C _2-50 alkenyl; and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of T, -C(O)O-; -ABOUT-; -C(O)-; -C(O)N(R ¹¹ )-; -S(O) ₂ N(R ¹¹ )-; -S(O)N(R ¹¹ )-; -S(O) ₂ -; -S(O)-; -N(R ¹¹ )S(O) ₂ N(R ^11a )-; -S-; -N(R ¹¹ )-; -OC(O)R ¹¹ ; -N(R ¹¹ )C(O)-; -N(R ¹¹ )S(O) ₂ -; -N(R ¹¹ )S(O)-; -N(R ¹¹ )C(O)O-; -N(R ¹¹ )C(O)N(R ^11a )- and -OC(O)N(R ¹¹ R ^11a );

T is selected from the group consisting of phenyl; naphthyl; indenyl; indanyl; tetralinyl; C _3-10 cycloalkyl; 4-7 membered heterocyclyl; or 9-11-membered heterobicyclyl, where T is optionally substituted with one or more R ¹⁰ that are the same or different;

R ¹⁰ is halogen; CN; oxo (=O); COOR ¹² ; ^OR12 ; C(O)R ¹² ; C(O)N(R ¹² R ^12a ); S(O) ₂ N(R ¹² R ^12a ); S(O)N(R ¹² R ^12a ); S(O) ₂ R ¹² ; S(O)R ¹² ; N(R ¹² )S(O) ₂ N(R ^12a R ^12b ); ^SR12 ; N(R ¹² R ^12a ); _NO2 ; OC(O)R ¹² ; N(R ¹² )C(O)R ^12a ; N(R ¹² )S(O) ₂ R ^12a ; N(R ¹² )S(O)R ^12a ; N(R ¹² )C(O)OR ^12a ; N(R ¹² )C(O)N(R ^12a R ^12b ); OC(O)N(R ¹² R ^12a ) or C _1-6 alkyl, where C _1-6 alkyl is optionally substituted with one or more halogens which are the same or different;

R ¹¹ , R ^11a , R ¹² , R ^12a , R ^12b are independently selected from the group consisting of H; or C _1-6 alkyl, where C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different.

The term "interrupted" means that the group is inserted between two carbon atoms or at the end of a carbon chain between carbon and hydrogen atoms.

L ² is a single chemical bond or spacer. If L ² is a spacer, it is preferably defined as one or more of the optional substituents defined above, provided that L ² is substituted with Z.

Therefore, if L ² is other than a single chemical bond, L ² -Z is COOR ⁹ ; ^OR9 ; C(O)R ⁹ ; C(O)N(R ⁹ R ^9a ); S(O) ₂ N(R ⁹ R ^9a ); S(O)N(R ⁹ R ^9a ); S(O) ₂ R ⁹ ; S(O)R ⁹ ; N(R ⁹ )S(O) ₂ N(R ^9a R ^9b ); ^SR9 ; N(R ⁹ R ^9a ); OC(O)R ⁹ ; N(R ⁹ )C(O)R ^9a ; N(R ⁹ )S(O) ₂ R ^9a ; N(R ⁹ )S(O)R ^9a ; N(R ⁹ )C(O)OR ^9a ; N(R ⁹ )C(O)N(R ^9a R ^9b ); OC(O)N(R ⁹ R ^9a ); T; With _1-50 alkyl; C _2-50 alkenyl or C _2-50 alkynyl, where T; C _1-50 alkyl; C _2-50 alkenyl and C _2-50 alkynyl are optionally substituted with one or more R ¹⁰ that are the same or different, and where C _1-50 alkyl; C _2-50 alkenyl; and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-; -ABOUT-; -C(O)-; -C(O)N(R ¹¹ )-; -S(O) ₂ N(R ¹¹ )-; -S(O)N(R ¹¹ )-; -S(O) ₂ -; -S(O)-; -N(R ¹¹ )S(O) ₂ N(R ^11a )-; -S-; -N(R ¹¹ )-; -OC(O)R ¹¹ ; -N(R ¹¹ )C(O)-; -N(R ¹¹ )S(O) ₂ -; -N(R ¹¹ )S(O)-; -N(R ¹¹ )C(O)O-; -N(R ¹¹ )C(O)N(R ^11a )- and -OC(O)N(R ¹¹ R ^11a );

R ⁹ , R ^9a , R ^9b are independently selected from the group consisting of H; Z; T; and C _1-50 alkyl; C _2-50 alkenyl; or C _2-50 alkynyl, where T; C _1-50 alkyl; C _2-50 alkenyl; and C _2-50 alkynyl optionally substituted with one or more R ¹⁰ that are the same or different, and where C _1-50 alkyl; C _2-50 alkenyl; and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of T, -C(O)O-; -ABOUT-; -C(O)-; -C(O)N(R ¹¹ )-; -S(O) ₂ N(R ¹¹ )-; -S(O)N(R ¹¹ )-; -S(O) ₂ -; -S(O)-; -N(R ¹¹ )S(O) ₂ N(R ^11a )-; -S-; -N(R ¹¹ )-; -OC(O)R ¹¹ ; -N(R ¹¹ )C(O)-; -N(R ¹¹ )S(O) ₂ -; -N(R ¹¹ )S(O)-; -N(R ¹¹ )C(O)O-; -N(R ¹¹ )C(O)N(R ^11a )- and -OC(O)N(R ¹¹ R ^11a );

T is selected from the group consisting of phenyl; naphthyl; indenyl; indanyl; tetralinyl; C _3-10 cycloalkyl; 4-7 membered heterocyclyl; or 9-11-membered heterobicyclyl, where t is optionally substituted with one or more R ¹⁰ that are the same or different;

R ¹⁰ is Z; halogen; CN; oxo (=O); COOR ¹² ; ^OR12 ; C(O)R ¹² ; C(O)N(R ¹² R ^12a ); S(O) ₂ N(R ¹² R ^12a ); S(O)N(R ¹² R ^12a ); S(O) ₂ R ¹² ; S(O)R ¹² ; N(R ¹² )S(O) ₂ N(R ^12a R ^12b ); ^SR12 ; N(R ¹² R ^12a ); _NO2 ; OC(O)R ¹² , N(R ¹² )C(O)R ^12a ; N(R ¹² )S(O) ₂ R ^12a ; N(R ¹² )S(O)R ^12a ; N(R ¹² )C(O)OR ^12a ; N(R ¹² )C(O)N(R ^12a R ^12b ); OC(O)N(R ¹² R ^12a ) or C _1-6 alkyl, where C _1-6 alkyl is optionally substituted with one or more halogens which are the same or different.

R ¹¹ , R ^11a , R ¹² , R1 ^2a , R ^12b are independently selected from the group consisting of H, Z or C _1-6 alkyl, where C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different,

provided that one of R ⁹ , R ^9a , R ^9b , R ¹⁰ , R ¹¹ , R ^11a , R ¹² , R ^12a , R ^12b is Z.

Even more preferred general aromatic structures are listed below.

Where

NH-rhGH is an rhNR residue attached to a temporary linker;

R1, R2, R3, R4, R5 are independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl,

PEG is a PEGylation moiety attached to a temporary linker and n=1 or 2, and

X is selected from C1-C8 alkyl or C1-C12 heteroalkyl.

The term "C1-C12 heteroalkyl" means an alkyl chain containing from 1 to 12 carbon atoms, which are optionally interrupted by heteroatoms, functional groups, carbocycles or heterocycles, as defined above.

In a preferred variPEG embodiment in formula (A), L ^a is a carbamate group attached to rhGH, G ^a is an aromatic oxygen group, a carbonyl attached thereto, and a substituent attached to the carbonyl, as shown in formula I.

More preferred structures are described by general formula I, which is part of structure (A) in the general aromatic linker structure above:

and where the preferred examples of formula I contain:

More preferred are aromatic structures of formula II which are part of structure (A) in the overall aromatic linker structure above:

Formula II

and where preferred examples of formula II contain:

More preferred structures of formula III are part of structure (A) in the overall aromatic linker structure above, wherein PEG-X is

Formula III

and PEG-W includes the following substituent groups:

Some examples of preferred prodrug conjugates are shown below:

R is selected from hydrogen, methyl, ethyl, propyl and butyl,

X is selected from C1-C8 alkyl or C1-C12 heteroalkyl.

Also, in the preferred and more preferred embodiments, the implementation of the PEG means, preferably, the residue S ⁰ containing at least S ¹ , S ² , BS ¹ and optionally BS ² .

In a preferred embodiment, the prodrugs of this invention are selected from the group consisting of

where m is an integer from 200 to 250 and n is an integer from 100 to 125;

where n is an integer from 400 to 500;

where n is an integer from 400 to 500; And

where n is an integer between 400 and 500.

methods known in the art. However, especially for compounds of formula (AA1), it is preferred to construct the prodrug molecule by convergent synthesis, providing a first precursor molecule containing one or more thiol groups and an activated carbonate group and a second precursor molecule containing a maleimide group to react in an addition reaction to form a thiosuccinimide group. , and to react such a combined precursor molecule with hGH to give a compound of formula (AA1).

Therefore, another aspect of the present invention is a process for the preparation of a compound of the formula hGH-NH-C(O)O-S ⁰ (AA1) wherein S ⁰ is as defined above and contains at least one group

where the method contains the following stages;

(a) reacting a compound of formula ROC(O)OS ⁰ -SH (AA1') with a compound of formula

(AA2'), where R is a suitable residue for an activated carbonate group, and where S ^0' and S ^0'' are chosen to obtain S ⁰ containing at least one group

to give a compound of formula ROC(O)OS ⁰ , and

(b) reacting a compound of formula ROC(O)OS ⁰ with hGH-NH ₂ , where hGH-NH ₂ is hGH with one of its primary amino groups, to give a compound of formula (AA1).

Suitable R groups for carbonate functional groups include substituted alkyl or carbocyclic or heterocyclic groups such as aryl or cycloalkyl groups, groups such as pentafluorophenyl or NHS.

Assays to determine the functional property of rhGH PEGylated prodrug

Activity and half-life of the conjugate

In order to determine the potency and half-life of the prodrug conjugate described here, it is necessary to synthesize a "permanent" conjugate that does not undergo self-hydrolysis—that is, a permanent conjugate.

This is achieved by synthesizing a molecule identical to the rhGH PEGylated prodrug, in addition to modifying the portion of the linker structure that initiates self-cleavage. Such an appropriate compound will have a residual activity and a circulating half-life of the conjugate identical to the rhGH PEGylated prodrug. In general, for any prodrugs conjugated to a self-hydrolyzable (self-cleaving) transient linker, one can expect the synthesis of a molecule identical to the prodrug, except for the ability to self-cleave, by making minor modifications to the linker structure.

The reason for using the appropriate conjugates is that if the self-hydrolyzable (self-cleaving) transient linker described here is used in the assay of Example 1, it is clear that the conjugate will immediately start to release the unmodified drug, which will affect the results of the assay. In other words, it is not possible to measure the residual activity without obtaining a permanent conjugate, since the unchanged native drug, such as rhGH, will be released and participate in the measured activity. This is obvious to a person skilled in the art. For this reason, explained above, the residual activity of prodrugs conjugated to a self-hydrolyzable (self-cleaving) temporary linker is expressed as the activity of the corresponding permanent conjugates.

Permanent conjugates are prepared in the same manner as prodrugs conjugated to a self-hydrolyzable (self-cleaving) temporary linker (Examples 20-23), but with slight modification of the linker structure so that the linker can no longer self-cleave. The preparation of permanent conjugates is described in Example 10 - Example 19.

According to one embodiment of the present invention, the PEGylated rhGH prodrugs described herein are characterized in that:

(1): if PEG is bound to rhGH in the prodrug conjugate, the prodrug has less than 5% GH activity relative to native growth hormone without PEG to avoid injection site lipoatrophy; And

(2): PEG is linked to rhGH via a self-hydrolyzable (self-cleavable) transient linker, where the rate of self-hydrolysis of the linker is such that the in vivo half-life is between 10 hours and 600 hours.

Analysis to determine property (1) is detailed in working example 1 below.

Based on these detailed instructions, one of ordinary skill in the art will easily measure this residual prodrug activity.

In a preferred embodiment, the residual activity of property (1) is less than 5%, more preferably less than 3%, even more preferably less than 1%, and most preferably practically absent.

The analysis to determine property (2) is detailed in working example 2 below. Based on these detailed instructions, one of ordinary skill in the art will easily measure the rate of self-cleavage of the temporary prodrug linker. In a preferred embodiment, the in vivo self-cleavage rate is such that the in vivo half-life is from 20 hours to 300 hours, more preferably from 20 hours to 150 hours, even more preferably from 30 hours to 150 hours, even more preferably from 30 hours to 100 hours, even more preferably 40 hours to 100 hours, even more preferably 50 to 75 hours, and even more preferably 30 to 75 hours.

In vivo and in vitro correlation

It is known from the above patent applications from Ascendis Pharma (Complex Biosystems Company) that there is a good correlation between in vitro and in vivo linker cleavage rates. The in vivo release kinetics can be easily predicted from in vitro experimental data.

Lipoatrophy

As described above, lipoatrophy is lipolysis occurring near the injection site. Therefore, in vitro measurement of growth hormone lipolysis and growth hormone conjugates can be used to assess the lipoatrophic effect of the conjugates.

To determine the lipolytic effect of the prodrug conjugate described here, it is necessary to synthesize a "permanent" conjugate that is not subject to self-hydrolysis - that is, a permanent conjugate.

The assay for determining lipoatrophy is described here in detail in Working Example 3. Based on these detailed instructions, a person skilled in the art will easily measure lipoatrophy.

In a preferred embodiment, the PEGylated rhGH prodrug conjugate described herein has a lipoatrophic effect that is comparable to human growth hormone measured in the lipoatrophy assay of Example 3 and other identical dosing regimen conditions.

GH related diseases

The term "GH-associated" diseases from the second aspect refers to diseases and conditions in which a person can benefit from GH.

These include, but are not limited to, growth hormone deficiency, adult-onset growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, short bowel syndrome, chronic renal failure, low weight for gestational age (LGA), HIV syndrome malnutrition, anti-aging, rheumatoid arthritis, idiopathic short stature, homeobox gene, and short stature somatopause. Other conditions characterized by short stature are also included, which include Noonan's syndrome, skeletal dysplasia, Down's syndrome, short stature associated with prolonged use of steroids, Aarskog's syndrome, among others. Also included are chronic kidney disease, juvenile rheumatoid arthritis; cystic fibrosis, HIV infection in children treated with HART (highly active antiretroviral therapy) (HIV/VASL (HIV-associated lipodystrophy syndrome) children); short stature in babies born at very low birth weight (VLBW) but low birth weight for a given gestational age (SGA); skeletal dysplasia; hypochondroplasia; achondroplasia; idiopathic short stature (ISS); GHD (growth hormone deficiency) in adults; fragility in or long bones such as the tibia, fibula, femur, humerus, radius, ulna, collarbone, metacarpus, metatarsus, and fingers; fragility in and spongy bones such as the skull, base of the arm and base of the leg; patients after surgery on tendons or ligaments in, for example, hands, knee or shoulder; distraction osteogenesis; disorders arising from joint or disc replacement, meniscus repair, spinal fusion, or prosthesis fixation, such as in the knee, hip, shoulder, elbow, wrist, or jaw; disorders arising from the fixation of osteosynthetic material such as nails, screws and plates; non-connection or improper connection in fractures; disorders arising from osteotomy, for example, in the tibia or 1st finger; disorders arising from graft implantation; articular cartilage degeneration in the knee caused by injury or arthritis; osteoporosis in patients with Turner syndrome; osteoporosis in men; adult patients on chronic dialysis (ACD); malnutrition-associated cardiovascular disease in VPHD; elimination of cachexia in VPHD; cancer in PVHD; chronic obstructive pulmonary disease in PVCD; HIV in PVHD; aging against the background of HPLC; chronic liver disease in VPHD, fatigue syndrome in VPHD; Crohn's disease; deterioration of liver function; men with HIV infections; short bowel syndrome; general obesity; HIV-associated lipodystrophy syndrome (WASL); male infertility; patients after elective surgery, alcohol/drug detox, or neurological injury; aging; frail elderly; osteoarthritis; traumatic cartilage injury; erectile disfunction; fibromyalgia; memory disorders; depression; traumatic brain injury; subarachnoid hemorrhage; very low birth weight; metabolic syndrome; glucocorticoid myopathy; and short stature due to glucocorticoid treatment in children.

Blueprints

The figures show the following.

On FIG. 1 shows SDS-PAGE analysis of purified permanent PEG-hGH conjugates, where lane 1: HiMark™ pre-stained high molecular weight protein standard; lane 2: connection 23; lane 3: compound 23; lane 4: compound 25; lane 5: compound 26, lane 6: compound 33; lane 7: connection 32; lane 8: connection 28; lane 9: compound 28; lane 10: compound 28; lane 11: compound 30; lane 12: compound 34; lane 13: compound 34; lane 14: compound 27; lane 15: connection 29.

On FIG. Figure 2 shows the size exclusion chromatogram of the quenched reaction solution during the synthesis of conjugate 28 and the size exclusion chromatogram of purified 28.

On FIG. 3 shows cation exchange chromatography purification of conjugate 35 and size exclusion chromatogram of purified 35.

On FIG. 4 shows cation exchange chromatography purification of conjugate 36 and size exclusion chromatogram of purified 36.

On FIG. 5 shows the purification by cation exchange chromatography of conjugate 37 and the size exclusion chromatogram of purified 37.

On FIG. 6 shows size exclusion chromatograms of conjugate 35 samples incubated in buffer at pH 7.4 and 37°C at various time points.

On FIG. 7-10 show preferred prodrug conjugates of the present invention with S ⁰ , S ¹ , S ² , L ^a , G ^a , BS ¹ , BS ² , BS ³ and critical distance.

On FIG. 11 shows the pharmacodynamic response curves for conjugate 36.

In detail, in Fig. 7 and 8 show exemplary structures of type 35 and 38 (AA1, AAA1), where at least 5 kDa polymer chain S ⁰ containing G ^a and BS ¹ and BS ² is denoted as S ⁰ ; the carbamate group derived from L ^a and the primary amino group of hGH is designated L ^a ; BS ¹ contains at least 4 kDa polymer chain, designated as S ¹ where S ¹ contains BS ³ that contains at least 4 kDa polymer chain, designated as S ³ . BS ² contains at least 4 kDa polymer chain, designated as S ² . Critical distances are given by 18 atoms for FIG. 7 and 4 atoms for FIG. 8.

On FIG. 9 and 10 are structures of the type (AA2, AAA2), where the amide group derived from L ^a and the hGH primary amino group is designated as L ^a , and the residue attached to L ^a is designated as S ⁰ containing G ^a and "PEG" , is an S ⁰ residue containing at least BS ¹ , S ¹ and S ² (all not shown).

EXAMPLES

Ways

Analytical preparative RP-HPLC

Analytical RP-HPLC/ESI-MS was performed on Waters equipment, consisting of a 2695 sample management system, 2487 Double Absorption Detector and a ZQ 4000 ESI instrument equipped with 5 µm Reprosil Pur 300 columns. ODS-3 (75×1.5 mm) (Dr. Maisch, Ammerbuch, Germany; flow rate: 350 µl/min, typical gradient: 10-90% acetonitrile in water, 0.05% TFA over 5 min). Preparative RP-HPLC uses a Waters 600 controller and a 2487 double absorption detector equipped with the following columns (Reprosil Pur 300 ODS-3)

A): 100×20 mm, flow rate 10 ml/min, typical gradient: 10-90% acetonitrile in water, 0.1% TFA for 11 min

or

B): 100×40 mm (10 µm particles), flow rate 40 ml/min, typical gradient: 10-90% acetonitrile in water, 0.1% TFA over 11 min.

Cation exchange chromatography

Purification of conjugates by cation exchange chromatography is carried out using the system Explorer (GE Healthcare) equipped with a Macrocap SP column. The appropriate conjugate in 20 mM sodium acetate buffer pH 4 is applied to a column which has been pre-equilibrated in 20 mM sodium acetate buffer pH 4 (buffer A). The column is washed with three column volumes of buffer A to remove unreacted PEG reagent. The conjugates are eluted using a gradient of 10-60% buffer B (20 mM sodium acetate, 1 M sodium chloride, pH 4.5) in 20 column volumes, or 0-40% buffer B in 20 column volumes and then 40-80 % B in the amount of three column volumes. The flow rate is 7 ml/min and the eluent is monitored by detection at 280 nm.

Anion exchange chromatography

Purification of conjugates by anion exchange chromatography is carried out using the system Explorer system (GE Healthcare) equipped with a Source Q column. The appropriate conjugate in 20 mM Tris/HCl buffer pH 7.5 (Buffer C) is applied to the column, which is pre-equilibrated in Buffer C. The column is washed with three column volumes of Buffer C to remove unreacted reagent PEG. The conjugates are eluted using a gradient of 0-20% buffer D (20 mM Tris/HCl, 1 M sodium chloride, pH 7.5) in 25 column volumes. The flow rate is 5 ml/min and the eluent is monitored by UV detection at 280 nm. Alternatively, a buffer system of 20 mM bis-Tris/HCl, pH 6.5 (buffer E) and 20 mM bis-Tris/HCl, 1 M sodium chloride, pH 6.5 (buffer F) is used.

Analytical size exclusion chromatography

Analytical size exclusion chromatography is carried out on the system Explorer (GE Healthcare). Samples are analyzed using a Superdex 200 or Sepharose 6 column (10 x 300 mm) and 20 mM sodium phosphate, 135 mM sodium chloride, pH 7.4 is used as the mobile phase. The flow rate is 0.75 ml/min and the eluted hGH and polymer-hGH conjugates are measured at 215 nm and 280 nm.

Determination of activity of pff-activated mPEG-linker reagents

A certain amount of pff-activated mPEG-linker reagent (3-5 mg) is dissolved in 100 μl of water. Add 10 μl of 0.5 M NaOH, and the reaction mixture is subjected to interaction for 60 min at 40°C. Add 1.5 µl of TFA and 10% of this mixture is analyzed by analytical RP-HPLC. Chromatograms are recorded at 260 and 280 nm. The peak corresponding to pentafluorophenol (pff) is integrated. The determined values are compared with a suitable calibration curve obtained by analyzing certain amounts of PFP using analytical RP-HPLC and integrating chromatograms recorded at 260 and 280 nm.

SDS-PAGE analysis

Permanent mPEG-hGH conjugates are analyzed using NuPAGE® Novex Tris-Acetate gels (1.5 mm thick, 15 lanes), NuPAGE Tris-Acetate SDS-Running Buffer, HiMark™ prestained high molecular weight protein standard, and Simply Blue stain ™ SafeStain (Invitrogen). 0.2-0.6 μg of the conjugate is applied to each lane and electrophoresis and subsequent staining is performed according to the supplier's protocol.

Example 1 Assay to Measure hGH PEGylated Prodrug and hGH Activity

The biological activity of hGH is measured using standard assays known to those skilled in the art. As described in EP 1715887 B1 and above, the biological activity associated with native or modified hGH (e.g. PEGylated hGH) can be measured using the standard FDC-P1 cell proliferation assay, (Clark et al., Journal of Biological Chemistry 271: 21969-21977) or receptor binding assay (US 5057417). Line 8 (page 14) of EP 1715887 B1 describes that the preferred in vitro activity should be as high as possible, most preferably the modified hGH has equal or improved in vitro biological activity. In the present invention, the biological activity should be as low as possible compared to natural hGH. The authors of this invention do exactly the opposite of what is described in EP 1715887 B1.

In Vitro Assay

The in vitro activity of the permanent PEG-hGH conjugate described in the examples below is determined using one or more standard in vitro biological activity assays. Standard assays that can be used include cell proliferation assays using, for example, FDC-PI cells (see, for example, Clark et al., Journal of Biological Chemistry, 271: 21969-21977, 1996), or Ba/F3 cells -hGHRs that express receptors for hGH, hGH delta 135-146, or Nb2 rat lymphoma cells that proliferate in response to hGH via lactogenic receptors (see, e.g., Alam, K.S., et al., J. Biotech 2000, Feb. 28, 78(1), 49-59). Receptor binding assays can also be used (see, for example, US Pat. No. 5,057,417).

Nb2-11 is a clone of Nb-2 rat lymphoma colony, which is derived from a lymphoma graft that develops in the thymus/lymph node of a male Noble (Nb) rat after prolonged estrogen treatment. The cells are derived from pre-T cells and are dependent on mammalian lactogens such as prolactin for proliferation. Nb2-11 can also be mutogenetically stimulated by IL-2. Injection of Nb2 cells into Nb rats causes an increase in malignant tumors, which are extremely sensitive to treatment with vinca alkaloids. Karyotypic analysis showed that the cell colony had only five well-developed chromosome anomalies. Cells do not express surface immunoglobulin, and their dependence on lactogens is confirmed. Protocols for the use of Nb2-11 cells in bioassays are available from ECACC upon request.

As described in WO 2006102659 at page 74, paragraph 0240, example 7, the biological activity of hGH and the conjugates described herein can be assessed in vitro using the NB2-11 rat lymphoma cell proliferation assay. Briefly, rat lymphoma-derived NB2-11 cells are incubated with hGH, which results in the binding of the hGH molecule to its receptor on the cell surface. Receptor binding induces a signal transduction cascade that results in cell proliferation. The results of the analysis are based on the determined protein content and 100% bioactivity of unmodified hGH.

Conclusion:

Based on the detailed instructions of this example 1, the person skilled in the art will easily determine the residual activity of the prodrug.

Example 2: Assay to measure the rate of self-cleavage of a temporary prodrug linker

Determination of in vitro half-life

To determine in vitro linker cleavage rates of PEG-linker-hGH conjugates, compounds are dissolved in buffer at pH 7.4 (e.g., 10 mM sodium phosphate, 140 mM NaCl, 3 mM EDTA) and the solution is filtered through a 0.22 µm filter and incubated at 37°C. Samples are taken at specific time intervals and analyzed by RP-HPLC or size exclusion chromatography at 215 nm. Peaks corresponding to released hGH are integrated and plotted against incubation time. Curve fitting software is used to determine first order splitting rates.

Determination of the correlation between in vivo half-life and in vitrolin vivo half-life

In vivo linker cleavage rates are determined by comparing the pharmacokinetics of the permanent PEG-hGH conjugates with the corresponding transient PEG-linker-hGH conjugate having the same PEG fragment after intravenous injection in a rat. First, a permanent PEG-hGH conjugate is administered intravenously to rats, and blood samples are taken at certain time intervals, plasma is obtained and analyzed for hGH using ELISA (enzyme-linked immunosorbent assay).

Then, a temporary PEG-hGH conjugate is administered intravenously to rats, and blood samples are taken at certain time intervals, plasma is obtained and analyzed for hGH using ELISA.

The in vivo half-life is calculated from the ratio of the hGH concentration of the transient conjugate divided by the determined concentration of the hGH permanent conjugate at the appropriate time points and plotted. The data are compared with in vitro half-life measurements.

Conclusion

Based on the detailed instructions of this example 2, one skilled in the art will easily determine the in vivo half-life of the hGH-PEGylated prodrug.

Example 3 Assay to Measure Lipoatrophy

As stated above, the compound PHA-794428 is PEGylated-hGH and is described in EP 1715887 from Pharmacia. According to www.clinicaltrials.gov, the analysis ended on December 10, 2007. The decision of Pfizer (Pharmacia) to end the program was due to cases of injection site lipoatrophy, which was described in clinical phase 2 studies after a single injection of PHA 794428. Lipoatrophy is a term describing localized loss of adipose tissue and is visible on a person as pits in the skin (visible to the eye).

Analysis

There are several in vitro methods described in the art for measuring lipoatrophy. One of the proposals is described in the publication J. Anim. Sci (1972), 35: 794-800 (L.J. Machlin) at page 795. Others are described in Int. J. Cancer; 80, 444-447 (1999).

In general, lipoatrophy can be measured as suggested below.

The lipolytic effect can be determined using an in vitro assay involving isolated mammalian adipocytes, preferably murine adipocytes. The test samples are incubated under physiologically relevant conditions with a predetermined number of adipocytes in Krebs-Ringer bicarbonate buffer containing the appropriate nutrients for up to 6 hours. The concentration of isolated glycerol is determined by standard methods, for example, enzymatic or radiometric determination. Control samples containing only adipocytes are analyzed to determine the spontaneous release of glycerol.

The lipolytic effect of native unmodified recombinant human growth hormone and permanently PEGylated recombinant human growth hormone was compared to that of transiently PEGylated recombinant human growth hormone.

Conclusion

Based on the detailed instructions of this example 3, the person skilled in the art will easily determine the lipoatrophic effect.

Example 4: Synthesis of permanent linker reagent 12a and temporary linker reagents 12b and 12c

Synthesis of compound 6

6-Aminohexan-1-ol (2.85 g, 24.3 mmol) was dissolved in aq. HBr (48%, 10 ml, 89 mmol) and stirred at 80° C. for 3 hours. Excess HBr is evaporated at 50-65° C. and 15 Torr and the residue is dried in vacuo.

1: Yield 6.07 g (96%)

MS [M+H] ⁺ = 180.3 g/mol (MW+H calc = 180.0 g/mol)

DBU (3.5 ml, 23.2 mmol) is added to a suspension of 6-bromhexylamine hydrobromide 1 (3.03 g, 11.6 mmol) and triphenylmethanethiol (2.14 g, 7.74 mmol) in DCM (25 ml) is added ) and DMSO (13 ml). The reaction mixture was stirred for 30 minutes at room temperature and diluted with water (150 ml).

The aqueous layer is extracted with ether and the combined organic phase is evaporated. 2 purified by RP-HPLC.

2: Yield 1.17 g (40%)

MS [M+H] ⁺ = 376.7 g/mol (MM+H calc = 376.2 g/mol)

DBU (4.56 ml, 30.0 mmol) was added to 6-bromohexanoic acid (3.90 g, 20.0 mmol) and triphenylmethanethiol (11.1 g, 40.0 mmol) in DCM (40 ml). After stirring at room temperature for 1 h, ice-cold 1 M H ₂ SO ₄ (50 ml) was added and the mixture was extracted with DCM. The combined organic phase is dried over Na ₂ SO ₄ and concentrated in vacuo. Compound 3 was purified by silica gel column chromatography (200 ml) using heptane/ethyl acetate (4/1, R _f =0.2) as mobile phase.

3: Yield 5.83 g (75%)

DMAP (37 mg, 0.31 mmol) is added to 6-tritylsulfanylhexanoic acid 3 (5.83 g, 14.9 mmol), thiazolidin-2-one (3.56 g, 29.9 mmol) and dicyclohexylcarbodiimide (3, 08, 14.9 mmol) in DCM (100 ml). After stirring at room temperature for 1 h, 1 M HCl (0.6 ml) was added and the mixture was filtered. The filtrate is concentrated in vacuo and compound 4 is purified by column chromatography on silica gel (180 ml) using heptane/ethyl acetate (1/1) as mobile phase.

4: Yield 7.15 g (97%) as yellow oil

A solution of 1-(2-thioxothiazolidin-3-yl)-6-tritylsulfanylhexan-1-one 4 (1.53 g, 3.11 mmol) in THF (13 mL) was added over 2 min to 6-tritylsulfanylhexylamine 2 (1 .17 g, 3.11 mmol) in DMSO (1 ml) and THF (5 ml). After adding triethylamine (435 μl, 3.11 mmol), the reaction mixture was stirred for 90 minutes at room temperature. Ether (200 ml) and water (100 ml) are added and the phases are separated. After extraction of the aqueous phase with ether, the combined organic phases are dried over Na ₂ SO ₄ and concentrated in vacuo. Compound 5 was purified by silica gel column chromatography (150 ml) using heptane/ethyl acetate (2/1, R _f =0.1) as mobile phase.

5: Yield 1.23 g (53%)

MS [M+Na] ⁺ = 770.6 g/mol (MM+Na calc = 770.4 g/mol)

Under nitrogen atmosphere, a 1 M solution of LiAlH ₄ in THF (1.2 ml, 4.8 mmol) was placed in a dry flask and a solution of compound 5 (509 mg, 0.680 mmol) in 10 ml of THF was added over 4 min. The mixture is stirred at reflux for 2 hours until complete conversion of the starting material, shown by thin layer chromatography (heptane/ethyl acetate 1:1). The reaction mixture was carefully quenched with a 10:1 suspension of water in diethyl ether until gas evolution ceased. The mixture is poured into 50 ml of a saturated potassium sodium tartrate solution and stirred for 90 minutes. 90 ml of ethyl acetate are added and the phases are separated. The aqueous phase is extracted with ethyl acetate (4×20 ml), and the combined organic phase is washed with saturated salt solution (30 ml), dried over MgSO ₄ , filtered and concentrated to obtain a clear oil. Compound 6 was adsorbed onto silica and purified by flash chromatography (30 g silica, CH ₂ Cl ₂ /MeOH 20:1 (v/v) + 0.1% NEt ₃ ). The product is obtained as an off-white viscous oil.

6: Yield 270 mg (54%)

MS [M+H] ⁺ = 734.4 g/mol (MM+H calc = 734.4 g/mol)

R _f =0.28 (CH ₂ Cl ₂ /MeOH 19:1)

Synthesis of compound 9

AlCl ₃ (23.0 g, 172.3 mmol) is added to glutaric anhydride (10.0 g, 86.2 mmol) in anisole (85 ml, 781 mmol). The reaction mixture is heated to 110° C. for 2 hours, cooled to room temperature, poured into 3 N HCl/ice, and extracted with dichloromethane. The solid phase is extracted with dichloromethane (4×20 ml) and the combined organic layers are washed with brine (30 ml), dried over MgSO ₄ , filtered and concentrated to give a red oil which is recrystallized from toluene. Product 7 is obtained as an off-white solid.

7: Yield 5.2 g (48%)

MS [M+Na] ⁺ =245.8 (MM+Na calc=245.1 g/mol)

AlCl ₃ (9.0 mg, 68 mmol) is added to compound 7 (5.0 g, 23 mmol) in 1,2-dichloroethane. The reaction mixture is stirred for 14 hours at 85°C and then cooled to room temperature. Ice-cold 1N HCl (50 ml) was added and the mixture was extracted with ethyl acetate (4×30 ml). The combined organic layers are dried over Na ₂ SO ₄ , filtered and concentrated in vacuo to give a light red solid which is used in the next step without further purification.

8: Yield 3 g (62%)

MS [M+H] ⁺ = 209.1 (MM+H calc = 209.1 g/mol)

Dicyclohexylcarbodiimide (532 mg, 2.6 mmol), acid 8 (403 mg, 1.9 mmol), HOSu (297 mg, 2.6 mmol) and collidine (1.0 ml, 7.8 mmol) in DCM (10 ml) was stirred for 90 min at room temperature. After removal of the dicyclohexylurea by filtration, amine 6 (947 mg, 1.3 mmol) in DCM (5 ml) and DIEA (450 μl, 2.6 mmol) are added to the filtrate and the mixture is reacted for 14 hours at room temperature. Add 1 n H ₂ SO ₄ (2×50 ml) and the phases are separated. The aqueous phase is extracted with ethyl acetate (4×20 ml), and the combined organic phases are washed with saturated salt solution (30 ml), dried over MgSO ₄ , filtered and concentrated in vacuo. The residues are purified by column chromatography on silica gel (150 ml) using heptane/ethyl acetate (1/2, R _f =0.66) as mobile phase.

9: Yield 819 mg (69%)

MS [M+Na] ⁺ =946.4 (MM+Na calculated = 946.4 g/mol)

Synthesis of permanent linker reagent 12a and temporary linker reagent 12b and 12c

Compound 9 (1 eq., 175 mg, 0.19 mmol) was dissolved in dry THF (1.5 ml), p-nitrophenyl chloroformate (1.1 eq., 42 mg, 0.21 mmol) and DIPEA (2 eq. , 66 μl, 0.38 mmol) and the mixture was stirred for 60 minutes at room temperature. Diethylamine (R1=R2=Et, 2 copies, 39 µl, 0.38 mmol) is added and stirring is continued for 15 minutes. The solvent was removed in vacuo, 100 μl AcOH was added and compound 10a was purified by RP-HPLC.

MS [M+Na] ⁺ =1045.9 (MM+Na calculated = 1045.5 g/mol)

NaBH ₄ (5 eq., 37 mg, 0.05 mmol) is added to compound 10a containing the HPLC fraction (acetonitrile/H20 ~ 3/1 (v/v) + 0.1% TFA) and the mixture reacts for 10 min at room temperature. An additional portion of NaBH ₄ (5 equiv., 37 mg, 0.95 mmol) is added and the reaction mixture is stirred until complete conversion of the starting material, confirmed by LC/MS analysis (10 min at room temperature). Compound 11a was purified by RP-HPLC and lyophilized, 11a: Yield 95 mg (49% with respect to compound 9)

MS [M+Na+H] ⁺ =1047.7 (MM+Na calc = 1047.5 g/mol)

Compound 9 (1 eq., 175 mg, 0.19 mmol) was dissolved in dry THF (1.5 ml), p-nitrophenyl chloroformate (1.1 eq., 42 mg, 0.21 mmol) and DIPEA (2 eq. ., 66 μl, 0.38 mmol) and the mixture was stirred for 60 minutes at room temperature.

N,N,N'-triethylethane-1,2-diamine (R1=Et, R2=2-(diethylamino)ethyl, 2 eq, 68 µl, 0.38 mmol) is added and stirring is continued for 15 minutes. 100 μl AcOH is added, the solvent is removed in vacuo and compound 10b is purified by RP-HPLC and lyophilized.

10b: Yield 147 mg as TFA salt (65%)

MS [M+Na] ⁺ =1116.4 (MM+Na calc = 1116.6 g/mol)

Compound 10c was synthesized as described above using N,N,N'-trimethylpropane-1,3-diamine (R1=Me, R2=3-(dimethylamino)propyl, 56 μl, 0.38 mmol) as the diamine.

10s: Yield 134 mg as TFA salt (59%)

MS [M+Na] ⁺ = 1088.4 (MM+Na calc = 1088.6 g/mol)

NaBH ₄ (46 mg, 1.2 mmol) is added to compound 10b (147 mg, 0.12 mmol) in MeOH/water = 95:5 (v/v) (3 ml) in two doses and the mixture is stirred for 1 h at room temperature. After addition of AcOH (300 μl) and concentration, product 11b was purified by RP-HPLC and lyophilized.

11b: Yield 107 mg as TFA salt (73%)

MS [M+Na] ⁺ = 1118.4 (MM+Na calc = 1118.6 g/mol)

Compound 11c was synthesized according to the same protocol.

11c: Yield of 65 mg as TFA salt (54%) from 134 mg of starting material

MS [M+Na] ⁺ = 1090.4 (MM+Na calc = 1090.6 g/mol)

Under a nitrogen atmosphere, bis-pentafluorophenyl carbonate (2.5 eq., 25 mg, 63 µmol), DMAP (1 mg) and DIEA (5 eq., 22 µl, 127 µmol) are added to compound 11a (1 eq., 26 mg, 26 µmol) in dry acetonitrile (0.5 ml). The reaction mixture was stirred for 30 min at room temperature, cooled to 0°C and acidified with AcOH (200 μl). Product 12a is purified by RP-HPLC and lyophilized.

12a: Yield 13 mg (42%)

MS [M+Na] ⁺ = 1258.2 (MM+Na calc = 1257.5 g/mol)

Compounds 12b and 12c were prepared according to the procedures for the preparation of compound 11b (56 mg, 48 μmol) and compound 11c (88 mg, 73 μmol), respectively.

12b: Yield 63 mg as TFA salt (93%)

MS [M+H] ⁺ = 1306.3 (MM+H calc = 1306.6 g/mol)

12s: Yield 41 mg as TFA salt (41%)

MS [M+H] ⁺ = 1278.4 (MM+Na calc = 1278.5 g/mol)

Example 5 Synthesis of Permanent Lettering Reagent 14a and Interim Linker Reagent 14c

Compounds 13a and 13c were synthesized as described in WO 2005/099768 A2.

Under a nitrogen atmosphere, bispentafluorophenyl carbonate (631 mg, 1.6 mmol), DMAP (20 mg, 0.16 mmol) and DIEA (556 μl, 3.2 mmol) are added to compound 13a (364 mg, 0.64 mmol) in dry acetonitrile (5 ml). The reaction mixture was stirred for 15 min at room temperature, cooled to 0°C and acidified with acetic acid (1 ml). Product 14a was purified by RP-HPLC and lyophilized.

14a: Yield 379 mg (77%)

MS [M+Na] ⁺ = 800.4 (MM+Na calculated = 800.3 g/mol)

Compound 14c was prepared according to the procedure for preparing compound 13c (97 mg, 130 μmol).

14s: Yield 114 mg as TFA salt (94%)

MS [M+H] ⁺ = 821.5 (MM+H calculated = 821.3 g/mol)

Example 6 Synthesis of Permanent Lettering Reagent 15

Glutaric anhydride (0.41 mmol), amine 6 (200 mg, 0.27 mmol), DIPEA (72 μl, 0.41 mmol) and DMAP (11 mg, 0.09 mmol) are stirred in acetonitrile (3 ml) in for 2 h at 80°C. The mixture is cooled to room temperature and acetic acid (200 μl) is added. Product 15 is purified by RP-HPLC and lyophilized.

15: Yield 130 mg (57%)

MS [M+Na] ⁺ = 870.2 (MM+Na calculated = 870.4 g/mol)

Example 7: Synthesis of activated mPEG linker reagents mPEG maleimide starting materials:

mPEG residues after interaction with the thiol group (R3 in the structures below):

The vertical dotted line indicates the site of attachment of the thiol group in the corresponding structure

Synthesis of permanent pff-activated mPEG linker reagents 17aa, 17ab, 17ac, 17ad and temporary pff-activated mPEG linker reagents 17b, 17ca, and 17cb

Carbonate 12a (13 mg, 10 µmol) was stirred in 10 µl AcOH, 700 µl HFIP, 1 µl TFA and 2 µl TES for 10 at room temperature. The volatiles are removed under nitrogen flow and compound 16a is purified by RP-HPLC.

16a: Yield 3.8 mg (5 µmol)

MS [M+H] ⁺ =751.3 (MM+H calc=751.3 g/mol)

Compounds 16b and 16c are prepared, respectively, from compounds 12b (7.7 mg, 5.4 µmol) and 12c (2 mg, 1.5 µmol) in that order,

16b: Yield 2.5 mg (2.7 µmol)

MS [M+Na] ⁺ = 845.1 (MM+Na calc = 844.3 g/mol)

16s: Yield 0.5 mg (0.6 µmol)

MS [M+Na] ⁺ = 816.6 (MM+Na calc = 816.3 g/mol)

mPEG-maleimide 1B (NOF, Japan) (521 mg, 12.7 µmol) is added to 3.5 mg (3.9 µmol) of compound 16c in 4 ml 3/1 (v/v) acetonitrile/water + 0 .1% TFA.

200 µl of 0.5 M phosphate buffer pH 7.4 is added and the mixture reacts for 10 min at room temperature. Add 1 μl (13 μmol) mercaptoethanol, and the reaction mixture is acidified to pH 4-5 with the addition of TFA. Compound 17ca was purified by RP-HPLC and lyophilized.

17ca: Yield 220 mg (pff-carbonate activity 82%)

Compound 17cb was synthesized as described for compound 17ca using compound 16c (3.5 mg, 3.9 µmol) and mPEG maleimide 2B (656 mg, 16 µmol).

17cb: Yield 130 mg (pff-carbonate activity 85%)

184 mg (8.8 µmol) mPEG-maleimide 1A (NOF, Japan) are added to compound 16a (2.0 mg, 2.7 µmol) in 4 ml 1/1 (v/v) acetonitrile/water + 0 .1% TFA. 200 µl of 0.5 M phosphate buffer pH 7.4 is added and the mixture reacts for 10 min at room temperature. Add 0.2 μl (1.6 μmol) mercaptoethanol, and the reaction mixture is acidified to pH 2-3 with the addition of TFA. Compound 17aa is separated from unreacted PEG using RP-HPLC and lyophilized.

17aa: Yield 90 mg (pff-carbonate activity 88%)

Compound 17ab was synthesized as described above using compound 16a (3.8 mg, 5.0 µmol) and 680 mg (16 µmol) of mPEG-maleimide 1B (NOF, Japan).

17ab: Yield 250 mg (pff-carbonate activity 83%)

Compound 17ac was synthesized as described above using compound 16a (2.5 mg, 3.3 μmol) and 200 mg (9.5 μmol) of mPEG-maleimide 2A (Jenkem, PR China).

17ac: Yield 80 mg (pff-carbonate activity 80%)

Compound 17ad was synthesized as described above using compound 16a and mPEG-maleimide 2B.

Compound 17b was synthesized as described for compound 17cb using compound 16b and mPEG-maleimide 1B.

Example 8: Synthesis of pff-activated permanent mPEG linker reagents 19aa and 19ab and transient permanent mPEG linker reagent 19c

Carbonate 14c (20 mg, 21 µmol) was stirred in 10 µl AcOH, 400 µl HFIP, and 5 µl TES for 10 min at room temperature and cooled to 0°C.

Ice-cold acetonitrile/water = 9/1 (v/v) is added and compound 18c is separated by RP-HPLC and lyophilized.

18s: Yield 5.0 mg as TFA salt (7.2 µmol)

MS [M+H] ⁺ =579.6 (MM+H calc. = 579.2 g/mol)

Compound 18a was synthesized as described above using carbonate 14a (24 mg, 31 μmol).

18a: Yield 8.0 mg (15 µmol)

MS [M+H] ⁺ =536.2 (MM+H calc=536.5 g/mol)

205 mg (5 µmol) mPEG-maleimide 3A (NOF, Japan) is added to compound 18a (4.0 mg, 7.5 µmol) in 2 ml 1/1 (v/v) acetonitrile/water + 0.1% TFA . Add 100 μl of 0.5 M phosphate buffer (pH 7.4) and the mixture is subjected to interaction for 10 min at room temperature. The reaction mixture was acidified to pH 2-3 with TFA, and compound 19aa was separated from unreacted PEG using RP-HPLC and lyophilized.

19aa: Yield 125 mg (pff-carbonate activity 85%)

Compound 19ab was prepared respectively from 410 mg (5 μmol) of mPEG-maleimide 3B (NOF, Japan) and compound 18a (4.0 mg, 7.5 μmol).

19ab: Yield 265 mg (pff-carbonate activity 87%)

Compound 19c was prepared from 205 mg of mPEG-maleimide 3A and compound 18c (5 mg, 7.2 µmol) respectively

19s: Yield 120 mg (pff-carbonate activity 88%)

Example 9: Synthesis of permanent 4-branched 80 kDa mPEG-NHS ester derivative 22

Acid 15 (12 mg, 14 µmol) was stirred in 1 ml TFA, 1 ml DCM and 10 µl TES for 10 min at room temperature. The volatiles are removed under nitrogen flow and the dithiol 20 is purified by RP-HPLC.

20: Yield 2.9 mg (8 µmol)

MS [M+Na] ⁺ =386.8 (MM+Na calc=386.2 g/mol)

Compound 20 (1 mg, 2.8 µmol) in 170 µl acetonitrile is added to mPEG-maleimide 1B (NOF, Japan) (380 mg, 9.2 µmol) in 4 ml 1/1 (v/v) acetonitrile/water + 0.1% TFA, Add 200 µl of 0.5 M phosphate buffer pH 7.4 and the mixture reacts for 10 min at room temperature. Add 0.6 μl (7.8 μmol) of mercaptoethanol, and the reaction mixture is acidified to pH 4-5 with the addition of TFA. The buffer is changed to 0.005% HCl (HiPREP Desalting column, 26/10 GE Healthcare) and Compound 21 is lyophilized without further purification.

21: Yield 320mg

Compound 21 was dissolved in 50 ml of toluene and the polymer solution was azeotropically dried for two hours at reflux using a Dean-Stark trap. The polymer solution is then cooled to room temperature. The dried mPEG linker reagent 21 was precipitated by the addition of chilled ether (60 ml).

Dicyclohexylcarbodiimide (1.2 mg, 6 µmol) in DCM was added to a solution of compound 21 (240 mg, 3 µmol) and N-hydroxysuccinimide (0.7 mg, 6 µmol) in DCM (3 ml). The reaction mixture was stirred for 14 hours at room temperature and compound 22 was precipitated by the addition of cold ether (20 ml). Product 22 is dried in vacuo.

22: Yield 200mg

Example 10: Synthesis of permanent amide-linked mPEG-hGH monoconjugate 23 and mPEG ₂ -hGH bisconjugate 24 using a linear 40 kDa derivative of mPEG-succinimidylhexanoate

In hGH, the buffer is changed to 50 mM sodium borate pH 8.5 (alternatively, sodium borate pH 8 or sodium borate pH 9 can be used). The hGH concentration is approximately 2.5 mg/ml. A threefold molar excess of 40 kDa mPEG-succinimidylhexanoate derivative (NOF, Japan) relative to the amount of hGH is dissolved in water to form a 20% (w/v) reagent solution (a fourfold or fivefold molar excess may alternatively be used). The reagent solution is added to the hGH solution and mixed. The reaction mixture is incubated for 2 hours at room temperature and quenched with hydroxylamine at room temperature and pH 7 for two hours. The quenched reaction mixture was analyzed by size exclusion chromatography. Monoconjugate 23 and bisconjugate 24 are purified by cation exchange chromatography. Alternatively, anion exchange chromatography may be used for purification. The purified conjugates are analyzed by SDS-PAGE (FIG. 1).

Example 11: Synthesis of permanent amide-linked mPEG-hGH monoconjugate 25 and mPEG-hGH bisconjugate 26 using a branched 40 kD mPEG-NHS ester derivative

Permanent mPEG-hGH monoconjugate 25 and bisconjugate 26 were synthesized as described in Example 10 using a branched 40 kDa mPEG-NHS ester (NOF, Japan). The purified conjugates are analyzed by SDS-PAGE (Figure 1).

Example 12: Synthesis of permanent amide-linked mPEG-hGH monoconjugate 27 using a 4-branched 80 kDa mPEG-NHS ester

Permanent mPEG-hGH monoconjugate 27 was prepared as described in Example 10 using the 4-branched 80 kDa mPEG-NHS ester 22.

Purified compound 27 was analyzed by SDS-PAGE (FIG. 1).

Example 13: Synthesis of permanent carbamate-linked mPEG-hGH monoconjugate 28 using 40 kDa 4-branched mPEG-pentafluorophenyl carbonate derivative 17aa

In hGH, buffer is changed to 50 mM sodium borate pH 9 (sodium borate pH 8.5 or sodium borate pH 8 can be used alternatively). The hGH concentration is approximately 2.5 mg/ml. A fourfold molar excess of the constant 4-branched 40 kDa mPEG linker reagent 17aa relative to the amount of hGH was dissolved in water to give a 20% (w/v) reagent solution. The reagent solution is added to the hGH solution and mixed. The reaction mixture is incubated for 1.5 h at room temperature and quenched by incubation in 100 mm hydroxylamine at pH 7 and room temperature for 2 hours. The quenched reaction mixture was analyzed by size exclusion chromatography (FIG. 2 top). Permanent mPEG-linker-hGH monoconjugate 28 was purified by anion exchange chromatography at pH 7.5 and analyzed by SDS-PAGE (FIG. 1) and size exclusion chromatography (FIG. 2 bottom).

Example 14: Synthesis of permanent carbamate-linked mPEG-hGH monoconjugate 29 using a 4-branched 80 kD derivative of mPEG-pentafluorophenyl carbonate

The permanent carbamate-linked mPEG-hGH monoconjugate 29 was synthesized according to Example 13 using the 4-branched 80 kDa derivative of mPEG-pentafluorophenyl carbonate 17ab. Purified compound 29 was analyzed by SDS-PAGE (FIG. 1).

Example 15: Synthesis of permanent carbamate-linked mPEG-hGH monoconjugate 30 using 40-kDa 40-kD mPEG-pentafluorophenyl carbonate derivative 17ac

Permanent hPEG-hGH monoconjugate 30 was synthesized as described in Example 13 using the 40-kDa 40-kDa derivative of mPEG-pentafluorophenyl carbonate 17ac. Purified compound 30 was analyzed by SDS-PAGE (FIG. 1).

Example 16: Synthesis of permanent mPEG-hGH monoconjugate 31 using 4-p-branched derivative of 80 kDa mPEG-pentafluorophenyl carbonate

The permanent mPEG-hGH monoconjugate 31 can be synthesized according to Example 13 using the 4-branched 80 kD mPEG pentafluorophenyl carbonate derivative 17ad.

Example 17: Synthesis of permanent carbamate-linked mPEG-hGH monoconjugate 32 using a 4-branched 40 kD derivative of mPEG-pentafluorophenyl carbonate

The permanent carbamate-linked mPEG-hGH monoconjugate 32 was synthesized as described in Example 13 using the 40-kDa 4-branched derivative of mPEG-pentafluorophenyl carbonate 19aa. Purified compound 32 was analyzed by SDS-PAGE (FIG. 1).

Example 18: Synthesis of permanent carbamate-linked mPEG-hGH monoconjugate 33 using a 4-branched 80 kD derivative of mPEG-pentafluorophenyl carbonate

Permanent mPEG-hGH monoconjugate 33 was synthesized as described in Example 13 using the 4-branched 80 kDa derivative of mPEG-pentafluorophenyl carbonate 19ab. Purified compound 33 was analyzed by SDS-PAGE (FIG. 1).

Example 19: Synthesis of permanent amine-linked mPEG-hGH monoconjugate 34 using a branched derivative of 40 kD mPEG-propionaldehyde

In hGH, the buffer is changed to 50 mM MES buffer H 6 (HEPES buffer pH 7 may alternatively be used) and the hGH concentration is adjusted to 1.5 mg/ml. A threefold molar excess of 40 kDa mPEG-propionaldehyde (GL2-400AL3, NOF, Japan) relative to the amount of hGH was dissolved in water to give a 25% (w/v) reagent solution. The reagent solution is added to the hGH solution and mixed. An aliquot of a 1 M stock solution of sodium cyanoborohydride in water is added to give a final concentration of 25 mM in the reaction mixture. The solution is incubated for 18 hours at room temperature in the dark. The reaction is quenched by adding Tris buffer. The reaction mixture was analyzed by size exclusion chromatography. Conjugate 34 was purified by cation exchange chromatography. The purified mPEG-hGH monoconjugate 34 was analyzed by SDS-PAGE (FIG. 1).

Example 20: Synthesis of transient carbamate-linked mPEG-hGH monoconjugate 35 using transient 40 kD mPEG-pentafluorophenyl carbonate 4-branched derivative 19c

In hGH, the buffer is changed to 50 mM sodium borate pH 9 (sodium borate pH 8.5 or sodium borate pH 8 can be used alternatively) and the hGH concentration is adjusted to 2.5 mg/ml. Four times the temporary mPEG linker reagent 19c relative to the amount of hGH was dissolved in water to obtain a 20% (w/v) reagent solution. The reagent solution is added to the hGH solution and mixed. The reaction mixture was incubated for 1 h at room temperature and quenched by incubation in 100 mM hydroxylamine at pH 7 and room temperature for 2 h. mPEG-linker-hGH monoconjugate was purified by anion exchange chromatography at pH 6.5 (Fig. 3 top) and analyzed size exclusion chromatography (Fig. 3 below).

Example 21: Synthesis of transient mPEG-linker-hGH monoconjugate 36 using a 4-branched 80 kDa derivative of hPEG-pentafluorophenyl carbonate

The hGH is buffered with 100 mM sodium borate pH 9 (alternatively sodium borate pH 8.5 or sodium borate pH 8 can be used) and the hGH concentration is adjusted to 10 mg/ml. A fourfold excess of the temporary 4-branched 80 kDa mPEG linker reagent 17ca relative to the amount of hGH was dissolved in water to make a 25% (w/v) reagent solution. The reagent solution is added to the hGH solution and mixed. The reaction mixture was incubated for 45 min at room temperature and quenched by incubation in 100 mM hydroxylamine at pH 7 and room temperature for 2 h. .4 below).

Example 22: Synthesis of transient mPEG-hGH monoconjugate 37 using 4-branched 80 kD mPEG-pentafluorophenyl carbonate derivative 17cb

mPEG-linker-hGH conjugate 37 was synthesized as described in Example 21 using activated mPEG-linker reagent 17cb.

The cation exchange chromatogram and analytical size exclusion chromatogram are shown in FIG. 5 above and below, respectively.

Example 23: Synthesis of transient carbamate-linked mPEG-hGH monoconjugate 38 using 4-branched 80 kD mPEG-pentafluorophenyl carbonate derivative 17b

The transient carbamate-linked mPEG linker-hGH conjugate 33 can be synthesized as described in Example 21 using the transient 4-branched 80 kDa mPEG linker reagent 17b.

Example 24 Assay to measure the activity of PEGylated hGH product and hGH

One skilled in the art will readily determine the residual activity of a polymeric prodrug, expressed as the activity of the corresponding permanent polymeric conjugate, using the standard assays described in Example 1.

More specifically, NB2-11 cells are grown in serum-free medium supplemented with 100 ng/ml hGH. For the in vitro proliferation assay, a cell suspension containing 2×10 ⁵ cells/ml is washed twice with medium free of serum and hGH and dispensed into a 96-well flat-bottomed microtiter plate (10 ⁴ cells/well). Compounds are tested in triplicate using a series of titration steps (9 steps, using a 3-fold dilution between steps). Cells with compound solutions are incubated for 48 hours followed by incubation for 2.5 hours with cell proliferation reagent WST-1. Proliferation of NB2-11 was determined via absorbance read using an ELISA reader and the response plotted as a function of concentration and EC50 values were determined. Results are shown as % residual in vitro biological activity relative to unmodified hGH in Table 1.

In the in vitro experiments described above, native hGH (source: Novo Nordisk, Denmark) is used as a reference compound. This method of obtaining hGH is used for the synthesis of permanent PEG-hGH conjugates.

Table 1: In vitro bioactivity of permanently PEGylated hGH conjugates compared to native hGH (Norditropin, Novo Nordisk, Denmark)

Conclusion:

As can be seen from Table 1, by conjugating an appropriate PEG molecule to hGH in vitro, the activity of PEGylated hGH can be reduced to less than 5% of that of native unconjugated hGH. For example, conjugation of 4×20 kD branched PEG with hGH reduces the residual activity to 0.7% of the unconjugated standard.

Further, from these results, it was also unexpectedly found that the residual activity of PEGylated growth hormone is related not only to the size of the attached PEG, but also to the degree of branching and the distance between hGH and branch points in the PEG structure.

Linear PEG

More specifically, the attachment of 40 kDa linear PEG to hGH gives an in vitro activity of 10.3% (compound 23) compared to native hGH.

Branched PEG

When branched 2×20 kDa PEG (compound 25) is attached in vitro, the activity is further reduced to 4.4% compared to native hGH.

Further, when 4×20 kDa PEG with a short distance between hGH and branch points in the PEG reagent (compound 27 and 29) is attached, the in vitro activity is further reduced to 0.7%, respectively, compared to native hGH. Surprisingly, by attaching a 4x20 kDa PEG with a relatively long spacer between human growth hormone and the first exact branch in the PEG reagent (e.g. Compound 33), in vitro activity is reduced to a lesser extent (2.2%), indicating the importance of the spacer. between the hGH functional group and the first branch point in the branched PEG reagent.

Conjugation of more than one PEG moiety to hGH to produce bisPEG conjugates reduces in vitro activity to less than 0.5%. (for example, connection 24 and 26).

Example 25 In Vitro Determination of the Self-Cleavage Rate of Conjugate 35, 36, 37 and 38

The self-cleavage rate of conjugate 35, 36 and 37 at pH 7.4 and 37°C was determined as described in Example 2. For these conjugates, a self-cleavage half-life of approximately 75 hours was determined. FIG. 6 shows size exclusion chromatograms of samples of incubated 35 analyzed at 0 h, 8 h, 47 h, 97 h and 168 h showing slow release of hGH from conjugate 35 over time. The rate of self-cleavage of conjugate 38 can be determined accordingly and gives half-lives of approximately 50 hours.

Example 26: Assay to measure the terminal in vivo half-life of PEGylated hGH prodrugs, which is expressed in terms of the in vivo half-life of the corresponding permanent conjugate

The pharmacokinetics of the permanent conjugates are determined after intravenous injection of 0.25 mg (hGH equivalents) in rats. In order to select a conjugate suitable for weekly human injections, a plasma half-life of more than 10 hours in rats is desirable.

A single dose of 0.25 mg hGH or 0.25 mg constant PEG-hGH conjugate (dose based on hGH) was administered intravenously per rat to male Wistar rats (200-250 g). Two groups of two animals each are used for each compound. 200-300 µl of whole blood is taken sublingually to obtain 100 µl of Ca-Heparin plasma per animal and time point. Samples are collected at 0.5, 3, 24, 48, 72 and 96 hours for group 1 and at 5, 8, 32, 56, 80 and 168 hours for group 2. Plasma samples are stored at -80°C until analysis.

The hGH and PEG-hGH conjugate concentrations are measured using the hGH ELISA kit (DSL). Plasma concentrations are calculated from the calibration curve of the appropriate conjugate or hGH and plotted against time, and the terminal half-life (t _1/2 ) is calculated using a single chamber model. The results of determining the half-life are presented in table 2.

To select a conjugate suitable for weekly administration to humans, conduct pharmacokinetic studies in rats. Since the half-life of PEGylated conjugates is 5 times faster in rats than in humans, the half-life of PEGylated hGH in rats should be about 10 hours or more. To obtain an estimate of the half-life of the conjugated hGH PEGylated prodrug without linker cleavage, the permanently conjugated corresponding conjugate was administered to rats. The results of the determination of the in vivo half-life are presented in table 2.

Table 2: Half-life of permanent PEG-hGH conjugates in rats

Conclusion:

From table 1 and table 2 it is obvious that the residual activity is inversely correlated with the half-life, for example, a high degree of residual activity causes faster elimination. This is typical of conjugates cleared by receptor-mediated clearance mechanisms. Moreover, in order to obtain an hGH PEGylated prodrug that can be administered once a week to a human and with low residual activity, a PEG molecule with one or more branch points and a molecular weight of 40 kDa or higher is preferred. Alternatively, conjugation of PEGs with more than one hGH site to produce bisPEG-hGH conjugates results in a long terminal half-life.

Example 27 Pharmacodynamic study of transient carbamate-linked mPEG-linker-hGH conjugate 36 and human growth hormone in cynomolgus monkeys

The object of this analysis is to compare the pharmacodynamic response in cynomolgus monkeys of a single dose of transient carbamate-linked mPEG-linker-hGH conjugate 36 with daily dosing of growth hormone for one week.

The following dosage groups are being investigated:

Since the transient carbamate-linked mPEG-linker-hGH conjugate 36 is transiently PEGylated using the 80 kD PEG group, the amounts of hGH in the 5 and 10 mg/kg dosing groups of the transient carbamate-linked mPEG-linker-hGH conjugate 36 are approximately 1 and 2 mg /kg, respectively. Therefore, the amount of hGH in a 10 mg/kg group of temporary carbamate-linked mPEG-linker-hGH conjugate 36 is equivalent to a daily dose of 0.3 mg/kg hGH. Each test compound is administered subcutaneously to 2 cynomolgus monkeys (1 male, 1 female) at a dose of 1 mg/kg. The age and weight of the animals are 2.5-3 years and 2.0-2.5 kg, respectively.

Blood samples are collected from the femoral artery/vein to determine serum concentrations of IGF-1, a pharmacodynamic marker of human growth hormone. Blood samples were taken at the following time points: 0 (before drug use), 3, 6, 12, 24, 36, 48, 72, 96, 120 and 144 hours after dosing on Day 1.

Blood samples are collected, clotting and then stored on an ice block or wet ice until centrifuged. After centrifugation, serum samples are aliquoted into pre-labeled tubes and tightly capped. The tubes are stored at -70° C. after being aliquoted into the tubes. IGF-1 levels in serum samples are measured using the Quantikine Human IGF-1 ELISA kit (R&D systems), which has been adapted and validated for use in the determination of IGF-1 levels in cynomolgus monkey serum. The pharmacodynamic response of the test substances is shown in FIG. 11. Daily administration of hGH and a single administration of transient carbamate-linked mPEG-linker-hGH conjugate 36 increased IGF-1 levels compared to levels measured in the vehicle group. A single dose of 5 mg/kg transient carbamate-linked mPEG-linker-hGH conjugate 36 is equivalent to daily hGH, while a single dose of 10 mg/kg of transient carbamate-linked hPEG-linker-hGH conjugate 36 is superior to daily hGH. This clearly shows that a weekly dose of transient carbamate-linked mPEG-linker-hGH conjugate 36 is superior to an equivalent hGH dose.

Abbreviations:

DBU - 1,3-diazabicyclo[5.4.0]undecene

DXM - dichloromethane

DIEA - diisopropylethylamine

DMAP - dimethylaminopyridine

DMF - N,N-dimethylformamide

DMSO - dimethyl sulfoxide

equiv. - stoichiometric equivalent

fmoc - 9-fluorenylmethoxycarbonyl

HFIP - hexafluoroisopropanol

HOSu - N-hydroxysuccinimide

LCMS - liquid chromatography combined with mass spectrometry

Mal - maleimidopropionyl

MS - mass spectrum

MM - molecular weight

PEG - polyethylene glycol

RP-HPLC - Reversed Phase High Performance Liquid Chromatography

R _f - retention factor

room pace. - room temperature

SEC - size exclusion chromatography

Suc - succinimidopropionyl

TES - triethylsilane

TFA - trifluoroacetic acid

THF - tetrahydrofuran

Trt - trityl

LINKS

1. et al. J. Pediatr. Endocrinol. Metab. 1999 Jan-Feb; 12(1): 95-7.

2. Clark et al., 1996, Journal of Biological Chemistry 271: 21969-21977.

3. Girard, J. Mehls, O., J. Clin Invest. March 1994; 93(3): 1163-1171.

4. Philip Harris et al. Horm. Res. 2006; 65 (suppl. 4): 1-213, CF1-98 GH/IGF Treatment with title "First in-human study of PEGylated recombinant human growth factor".

5 Veronese, F.M. "Enzymes for Human Therapy: Surface Structure Modifications," Chimica Oggi, 7: 53-56 (1989).

Claims (30)

1. Human growth hormone (hGH) prodrug conjugate of formula (I): Where T is hGH-NH; X is a spacer fragment; fragment: selected from the group consisting of R ₁ is a polymeric carrier having: first chain; second chain; And the third chain where the first chain contains the first branching structure BS ¹ selected from the group consisting of a tertiary carbon atom, a quaternary carbon atom and a tertiary nitrogen atom; And where the critical distance is less than 50 atoms, where the critical distance is the shortest distance between the first branching structures BS ¹ and the site of attachment of the fragment to the rest of the prodrug conjugate, measured as bonded atoms; fragment selected from the group consisting of And where the molecular weight of the prodrug without hGH-NH is at least 30 kDa and a maximum of 120 kDa. 2. Prodrug according to claim 1, wherein the critical distance is less than 20 atoms. 3. A prodrug according to claim 1 or 2, wherein the critical distance is less than 10 atoms. 4. A prodrug according to claim 1 or 2, wherein the molecular weight of the prodrug without hGH-NH is at least 40 kDa and a maximum of 100 kDa. 5. The prodrug according to claim 1 or 2, wherein the molecular weight of the prodrug without hGH-NH is at least 40 kDa and a maximum of 90 kDa. 6. Prodrug according to claim 1 or 2, wherein the first, second and third chains are independently based on a polymer selected from the group consisting of polyalkyloxy polymers, hyaluronic acid and its derivatives, polyvinyl alcohols, polyoxazolines, polyanhydrides, poly(orthoesters), polycarbonates , polyurethanes, polyacrylic acids, polyacrylamides, polyacrylates, polymethacrylates, polyorganophosphazenes, polysiloxanes, polyvinylpyrrolidones, polycyanoacrylates and polyesters. 7. Prodrug according to claim 1 or 2, wherein the first, second and third chains are based on a polyalkoxy polymer. 8. A prodrug according to claim 1 or 2, wherein the first, second and third chains are based on polyethylene glycol. 9. A method of treating a growth hormone-associated disease in a human, comprising administering to a human a human growth hormone prodrug conjugate according to any one of paragraphs. 1-8 in a clinically effective amount.

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