MXPA06008888A - N-terminally monopegylated human growth hormone conjugates, process for their preparation, and methods of use thereof - Google Patents

Abstract

The present invention provides a chemically modified human Growth Hormone (hGH) prepared by attaching a polyethylene glycol butyraldehyde moiety to the N-terminal phenylalanine of the protein. The chemically modified protein according to the present invention may have a much longer lasting hGH activity than that of the un-modified hGH, enabling reduced dose and scheduling opportunities. The present invention also includes methods of use for the treatment and/or prevention of diseases or disorders in which use of growth hormone isbeneficial.

Description

CONJUGATES OF THE HUMAN GROWTH HORMONE MONOPEGILADOS IN THE EXTREME N, PROCEDURE FOR ITS PREPARATION AND METHODS TO USE THEM The present application claims the priority of the application for USA No. 10/771895, filed on February 4, 2004, which is incorporated as a reference in its entirety as if it were written in this report.

FIELD OF THE INVENTION The present invention relates to a chemical modification, including PEGylation, of human growth hormone (hGH) and its agonist variants, by means of which the chemical and / or physiological properties of hGH can be changed. PEGylated hGH may have a longer residence time in plasma, lower clearance rate, better stability, less antigenicity, less heterogeneity of PEGylation or one of its combinations. The present invention also relates to methods for modifying hGH. In addition, the present invention relates to pharmaceutical compositions comprising the modified hGH. An additional modality is the use of modified hGH for the treatment of growth and developmental disorders.

BACKGROUND OF THE INVENTION Human growth hormone (hGH) is a protein comprising a single chain of 191 amino acids crosslinked by two disulfide bridges, and the monomeric form has a molecular weight of 22 kDa. Human GH is secreted by the pituitary gland, and can also be produced by recombinant genetic engineering. The hGH will produce growth in all body tissues that can grow. HGH plays an important role, not only in the promotion of growth in the growth phase of human beings, but also in the maintenance of body composition, anabolism, and normal lipid metabolism (K. Barneis. And U. Keller , Baillieres Clin Endocrinol, Metab.10: 337 (1996)). Recombinant hGH has been commercially available for several years. Two types of therapeutically useful recombinant hGH preparations are found in the market: the authentic one, e.g. ex. Genotropin® or Nutropin®, and an analogue with an additional methionine moiety at the N-terminal end, e.g. ex. Somatonorm®. HGH is used to stimulate linear growth in a patient with pituitary dwarfism also called growth hormone deficiency (GHD) or Turner syndrome, but other indications have also been suggested, including the long-term treatment of growth retardation in children They were born small for gestational age, for the treatment of patients with Prader-Willi syndrome (PWS), chronic renal failure (CRF), AIDS wasting, and aging. Patients with GH deficiency in adults (DHCa) have different problems, such as characteristic changes in body composition including increased fat mass, decreased lean body mass and extracellular fluid, and reduced bone mineral density, metabolic abnormalities of lipids and cardiovascular dysfunction. Many of these problems improve with substitution therapy with hGH (J. Verhelst J and R., Abs. Drugs, 62: 2399 (2002)). A major biological effect of growth hormone (GH) is to promote growth in young mammals and maintain tissues in older mammals. Among the systems of affected organs include the skeleton, connective tissue, muscles, and viscera such as liver, intestine, and kidneys. Growth hormones exert their effect by interacting with specific receptors on the membrane of the target cell. HGH is a member of the family of homologous hormones that includes placental lactogens, prolactins, and other species or genetic variants, or growth hormone (Nicoll, C. S., et al. (1986) Endocrine Reviews 7: 169). HGH is uncommon within these, in that it has a broad species specificity and binds to the somatogenic receptor (Leung, DW, et al. [1987] Nature 330; 537) or cloned prolactin (Boutin, JM, et al. al. [1988] Cell; 53: 69). The cloned gene for hGH has been expressed in a secreted form in Escherichia coli (Chang, CN, et al. [1987] Gene 55: 189), and its DNA and amino acid sequence have been described (Goeddel, et al. ([1979] Nature 281: 544; Gray, et al. [1985] Gene 39: 247).Human growth hormone (hGH) participates in much of the regulation of normal human growth and development. This pituitary hormone has a multitude of biological effects including linear growth (somatogenesis), lactation, activation of macrophages, insulin-like and diabetogenic effects among others (Chawla, R., K. (1983) Ann. Rev. Med. 34, 519; Edwards, CK et al. (1988) Science 239, 769; Thomer, MO, et al. (1988) J. Clin. Invest. 81: 745). Growth hormone deficiency in children leads to dwarfism, which has been satisfactorily treated for more than a decade by exogenous administration of hGH. In adults, as well as in children, hGH maintains a normal body composition increasing nitrogen retention and stimulation of musculoskeletal growth, and mobilizing body fat. Visceral adipose tissue is particularly sensitive to hGH. In addition to enhancing lipolysis, hGH decreases the uptake of triglycerides in body fat stores. The concentrations of IGF-I (insulin-like growth factor I), and IGFBP3 (insulin-like growth factor-binding protein 3) are increased by hGH. Human growth hormone (hGH) is a single chain polypeptide consisting of 191 amino acids (molecular weight 21, 500). Disulfide bonds link positions 53 and 165, and positions 182 and 189. Niall, Nature, New Biology, 230: 90 (1971). HGH is a potent anabolic agent, especially due to the retention of nitrogen, phosphorus, potassium and calcium. The treatment of hypophysectomized rats with GH can restore at least a part of the growth rate of the rats. Moore et al., Endocrinology 122: 2920-2926 (1988). Among its most striking effects in subjects with hypopituitarism (GH deficiency) is the accelerated linear growth of the bone plate cartilage, resulting in an increase in height. Kaplan, Growth Disorders ¡n Children and Adolescents (Springfield, IL: Charles C. Thomas, 1964). HGH produces a variety of physiological and metabolic effects in different animal models including linear bone growth, lactation, activation of macrophages, insulin-like and diabetogenic effects, and others (RK Chawla et al., Annu Rev. Med. 34: 519 (1983); OGP Isaksson et al., Annu., Rev. Physiol., 47, 483 (1985); CK Edwards et al., Science 239, 769 (1988); MO Thomer and ML Vanee, J. Clin. Invest. : 745 (1988), JP Hughes and HG Friesen, Ann. Rev. Physiol. 47: 469 (1985)). It has been described that, especially in women after menopause, GH secretion decreases with age. Millard et al., Neurobiol. Aging, 11: 229-235 (1990); Takahashi et al., Neuroendocrinology M, L6-137-142 (1987). See also, Rudman et al., J. Clin. Invest., 67: 1361-1369 (1981) and Blackman, Endocrinology and Aging, 16: 981 (1987). In addition, according to a study some manifestations of aging, including the decrease in lean body mass, expansion of adipose tissue mass, and thinning of the skin, can be reduced with GH treatment three times a week. See, for example, Rudman et al., N. Eng. J.

Med., 323: 1-6 (1990) and the attached article in the same journal by Dr. Vanee (pp. 52-54). These biological effects derive from the interaction between hGH and specific cellular receptors. Two different human receptors have been cloned, the hepatic receptor of hGH (DW Leung et al., Nature 330: 537 (1987)) and the human prolactin receptor (JM Boutin et al., Mol. Endocrinology 3: 1455 (1989)). However, there are likely to be others including the human placental lactogen receptor (M. Freemark, M. Comer, G. Komer, and S. Handwerger, Endocrinol 120: 1865 (1987)). These homologous receptors contain a glycosylated extracellular domain of hormone binding, a single transmembrane domain, and a cytoplasmic domain, which differ considerably in sequence and size. It is assumed that one or more receptors play a determining role in the physiological response to hGH. It is generally observed that physiologically active proteins administered in a body can show their pharmacological activity only for a short period of time due to their high rate of clearance in the body. further, the relative hydrophobicity of these proteins may limit their stability and / or solubility. In order to decrease the rate of clearance, improve stability or suppress the antigenicity of therapeutic proteins, some methods have been proposed in which proteins are chemically modified with water-soluble polymers. Chemical modification of this type can effectively block the physical contact of a proteolytic enzyme with the main chain of the protein, thus preventing degradation. The chemical bonding of some water-soluble polymers can effectively reduce renal clearance due to the greater hydrodynamic volume of the molecule. Additional advantages include, in some circumstances, increased stability and time in the circulation of the therapeutic protein, increased solubility, and decreased immunogenicity. Poly (alkylene oxide), particularly polyethylene glycol (PEG), is one such chemical portion that can be used to prepare therapeutic protein products (the verb "pegylate" means to bind at least one molecule of PEG). It has been shown that the binding of polyethylene glycol protects against proteolysis, Sada, et al., J. Fermentation Bioengineering 71: 137-139 (1991), and methods for joining some polyethylene glycol portions are available. See U.S. Pat. No. 4,179,337, Davis et al., "Non-immunogenic Polypeptides," filed December 18, 1979; and U.S. Pat. No. 4,002,531, Royer, "Modifying Enzymes with Polyethylene Glycol and Product Produced Thereby," filed on January 11, 1977. For a review, see Abuchowski et al., In Enzymes as Drugs. (J. S. Holcerberg and J. Roberts, eds., Pp. 367-383 (1981)). Other water-soluble polymers have been used, such as copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly (1,3-dioxolane), poly (1, 3,6-trioxane), ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers).

A number of examples of therapeutic pegylated proteins have been described. ADAGEN®, a pegylated formulation of adenosine deaminase, is approved to treat severe combined immunodeficiency disease. OCASPAR®, a pegylated L-asparaginase has been approved to treat all patients with hypersensitivity. Pegylated superoxide dismutase has been used in clinical trials to treat head injuries. Pegylated α-interferon (US 5,738,846, 5,382,657) has been approved to treat hepatitis; It is reported that pegylated glucocerebrosidases and pegylated hemoglobin have been used in preclinical trials. Another example is pegylated IL-6, document EF 0442724, entitled, "Modified hIL-6," which describes the addition of polyethylene glycol molecules to IL-6. Another specific therapeutic protein, which has been chemically modified, is the granulocyte colony stimulating factor, (G-CSF). G-CSF induces the proliferation and rapid release of neutrophil granulocytes into the bloodstream, and thus provides the therapeutic effect of fighting infection. European Patent Publication EP 0401384, published December 12, 1990, entitled, "Chemically Modified Granulocyte Colony Stimulating Factor," describes materials and methods for preparing G-CSF to which polyethylene glycol molecules bind. Modified G-CSFs and their analogs are also disclosed in EP 0473268, published March 4, 1992, entitled "Continuous Relase Pharmaceutical Compositions Comprising to Polypeptide Covalently Conjugated To Water Soluble Polymer", which describe the use of different G- CSF and derivatives covalently conjugated to a water soluble polymer, such as polyethylene glycol. A modified polypeptide having human granulocyte colony stimulating factor activity is disclosed in EP 0335423 published October 4, 1989. In US Pat. 5,824,784 methods for modifying proteins at the N-terminus or their analogs, and resulting compositions, including chemically modified G-CSF compositions at the N-terminus are described. 5,824,778 describes chemically modified G-CSF. For polyethylene glycol, a variety of means have been used to bind polyethylene glycol molecules to the protein. In general, polyethylene glycol molecules are connected to the protein by a reactive group of the molecule. For such binding, amino groups, such as those of the lysine or N-terminal portions, are convenient. For example, Royer (U.S. Patent No. 4,002,531, see above) discloses that reductive alkylation was used to bind molecules of polyethylene glycol to an enzyme. Chamow et al., Bioconjugate Chem. 5: 133-140 (1994) describe the modification of CD4 immunoadhesin with monomethoxypolyethylene glycol aldehyde by reducing alkylation. The authors describe that 50% of CD4-Ig were modified with MePEG under conditions that allowed control in the degree of pegylation. See above on page 137. The authors also describe that the in vitro binding capacity of this modified CD4-lg (to gp 120 protein) decreases at a rate that correlates with the degree of MePEGylation. Ibid. U.S. Pat. No. 4,904,584, Shaw, filed February 27, 1990, refers to the modification of the number of portions of lysine in proteins, for the binding of polyethylene glycol molecules to reactive amino groups. WO 93/00109 relates to a method for stimulating a tissue sensitive to mammalian or avian GH, which comprises maintaining a concentration of GH in the plasma effective and continuous for a period of 3 or more days. It is disclosed that one way to achieve such concentration in the plasma is through the use of GH coupled to a macromolecular substance such as PEG (polyethylene glycol). It is disclosed that coupling to a macromolecular substance results in a better half-life. PEGylated human growth hormone has been described in WO 93/00109 using mPEG-aldehyde-5000 and mPEG- (N-hydroxysuccinimidyl ester) (mPEG-NHS-5000). The use of mPEG-NHS resulted in heterogeneous mixtures of multiple forms of pegylated hGH. WO 93/00109 also describes the use of mPEG-malelmide for Pegilar variants of hGH with cysteine. Document 99/03887 describes a variant with cysteine of growth hormone that is PEGylated. Called BT-005, it is assumed that this conjugate is more effective in stimulating weight gain in rats with growth hormone deficiency, and that they have a longer half-life than hGH.

Clark et al. Have also described PEGylated human growth hormone using carboxymethylated PEG succinimidyl ester. { Journal of Biological Chemistry 271: 21969-21977, 1996). Clark et al. describe hGH derivatives of increasing sizes using mPEG-NHS-5000, which is selectively conjugated with primary amines. Increasing levels of modification with PEG reduced affinity for their receptor and increased CE5o in a cell-based assay up to 1500 times. Olson et al., Polymer Preprints 38: 568-569, 1997, describe the use of N-hydroxysuccinimide (NHS) -PEG and succinimidyl propionate (SPA) PEG to achieve PEGylated hGH species. WO 94/20069 prophetically discloses PEGylated hGH as part of a formulation for pulmonary delivery. US 4,179,337 discloses methods of PEGylation of enzymes and hormones to obtain water-soluble, non-immunogenic and physiologically active polypeptide conjugates. GH is mentioned as an example of a hormone for PEGylation. EP 458064 A2 describes the PEGylation of natural cysteine portions or that have been introduced into the somatotropin. EP 458064 A2 further mentions the incorporation of two portions of cysteine in a loop called omega loop, which is described to be located in portions 102-112 in wild bovine somatotropin, more specifically EP 458064 A2 describes the replacement of portions 102 to 112 of the bovine somatotropin of Ser a Cys and of Tyr to Cys respectively. Document 95/11987 suggests the binding of PEG in the thio group of a portion of cysteine that is present in the parent molecule or introduced by site-directed mutagenesis. WO 95/11987 refers to the PEGylation of the nexin-1 protease, however the General PEGylation of hGH and other proteins. WO 99/03887 describes, for example, growth hormone modified by insertion of 25 cys serine portions and the binding of PEG to the introduced cysteine portions. WO 00/42175 relates to a method for preparing proteins containing free cysteine portions for the binding of PEG. WO 00/42175 describes the following muteins of hGH: t3C, S144C and T148C and their PEGylation in cysteine. WO 97/11178 (as well as US 5,849,535, US 6,004,931, and US 6,022,711) refers to the use of GH variants as hGH agonists or antagonists. WO 97/11178 also describes PEGylation of hGH, including PEGylation of lysine and introduction or substitution of lysine (eg, K168A, K172R). WO 9711178 also describes the G120K substitution. WO 03/044056 describes a variety of pegylated hGH species including a PEG (branched 40K) -aldehyde and hGH conjugate. Previous descriptions of PEGylated hGH require the binding of multiple PEGs, which results in undesirable heterogeneity of the product, to achieve a greater hydrodynamic volume than the 70K molecular weight cut-off of renal filtration (Knauf, MJ et al, J. Biol. Chem. 263: 15064-15070,1988). Currently, the administration of rhGH is daily for a long period of time, and therefore a less frequent administration would be convenient. A molecule of hGH with a longer half-life in the circulation would decrease the number of administrations needed and potentially provide optimal therapeutic hGH levels with the simultaneous enhanced therapeutic effect. Despite a series of attempts to PEGylate hGH, a PEGylated hGH molecule with the appropriate properties is still necessary to be a viable commercial product. The present invention provides PEG-hGH conjugates having a single PEG predominantly linked to the N-terminal phenylalanine of hGH, which provides advantages over other PEG-hGH conjugates. The union of multiple low molecular weight PEG (5 kd) at the a- or e-amino sites (N-terminus and nine pots in hGH) has been described using mPEG-aldehyde-5000 or mPEG- (N-hydroxysuccinimidyl ester) ) (mPEG-NHS-5000) in WO 93/00109, Clark et al. (Journal of Biological Chemistry 271: 21969-21977, 1996), and Olson et al. (Polymer Preprints 38: 568-569, 1997). This results in a heterogeneous population. As an illustration, a hGH with nine plants can have some molecules that have ten PEGs attached, some with nine, some with eight, some with seven, some with six, some with five, some with four, some with three, some with two, some with one and some without any. And among the molecules that have several PEGs, the PEG may not be bound at the same sites in the different molecules. This resulting heterogeneity is disadvantageous when a therapeutic product is developed, making the conjugation, purification and characterization difficult, expensive and highly irreproducible. Another method has been (WO 00/42175) the use of hGH variants containing free cysteine portions for the binding of PEG. However, this strategy can lead to incorrect folding of the protein with incorrectly matched diesulfide bonds and result in a heterogeneous PEGylated product having the PEGs attached to some or all of the cysteines. Having multiple PEGs bound to multiple sites can lead to molecules that have fewer stable bonds between the PEG and the different sites, which can be dissociated at different rates. This makes it difficult to accurately predict the pharmacokinetics of the product resulting in incorrect dosing. A heterogeneous product also poses unwanted problems to obtain regulatory approval for the therapeutic product. Therefore, it would be convenient to have a hGH molecule PEGylated having only one PEG attached in a single site. The present invention addresses this need in a number of ways.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to chemically modified hGH and its agonist variants, which have at least one improved chemical or physiological property selected, but not limited, with a lower clearance rate, longer duration of residence in the plasma, greater stability, greater solubility, and less antigenicity. Therefore, as described below in more detail, the present invention has a number of aspects that relate to chemically modifying polypeptides, including but not limited to hGH and its agonist variants, as well as specific modifications using a polyethylene glycol moiety. -butiraldehyde. The present invention also relates to methods for producing chemically modified hGH and its agonist variants. Particularly, the present invention relates to a method for producing a chemically modified hGH using butyraldehyde, which results in a higher N-terminal selectivity of the binding. The present invention also relates to the use of the chemically modified hGH and its agonist variant of the present invention, alone or in combination with another therapeutic agent. The present invention also relates to the use of the chemically modified hGH and its agonist variant of the present invention, alone or in combination with another therapeutic agent for preventing and / or treating disorders and / or diseases in which GH treatment is useful. .

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A, 1B and 1C are an HPLC plotting a tryptic map analysis of the reaction of hGH and PEG (branched 40K) butyraldehyde-hGH or PEG (branched 40K) aldehyde-hGH. Figure 1A is the triptych map of the PEG (branched 40K) butyraldehyde-hGH. Figure 1B is the triptych map of the PEG (branched 40K) aldehyde-hGH. Figure 1C is the tryptic map of hGH without PEGylar. T1 is the N-terminal tryptic fragment. Figure 2 shows the amino acid sequence of human growth hormone. (SEQ ID NO: 1). Figure 3 shows the efficacy of PEG (branched 40K) butyraldehyde-hGH in the rat weight gain test. Female hypophysectomized Sprague-Dawley rats of 4-5 weeks of age (100-125 g) were purchased from Harlan Labs. After introducing them into the animal facilities, the animals were kept at a constant room temperature of 26.7 ° C and were kept at a constant temperature of 26.7 ° C. Weighed daily for 4-10 days in order to establish the initial growth rates. Beginning on day 0, the rats (-100 g) of the control groups received a daily subcutaneous injection of -0.3 mg / kg hGH (closed circles), or PBS (open circles), for eleven consecutive days. The PEG (branched 40K) butyraldehyde-hGH (solid squares) test group received single doses of 1.8 mg / kg of PHA-794428 on days 0 and 6. There were 8-10 animals per group. The average +/- ETM growth is plotted. Figure 4 shows the effects of promoting the dose-sensitive growth of PEG (branched 40K) butyraldehyde-hGH in rats. This efficacy study was carried out in a manner similar to that described in figure 3, except that a single varied dose of the PEG (branched 4ÓK) butyric aldehyde-hGH (day 0, only) was administered and the study was carried out for 6 days. The control groups received daily injections of hGH 0.3 mg / kg (black circles), or PBS vehicle (white circles) for six consecutive days. PEG (branched 40K) butyraldehyde-hGH was administered at a dose of 1.8 mg / kg (black squares), 0.6 mg / kg (white squares), 0.2 mg / kg (black triangles) or 0.067 mg / kg (white triangles) . There were 8 animals per group. Figure 5 shows the growth of the tibia in response to PEG (branched 40K) butyraldehyde-hGH. Hypofisectomized rats were treated as described in figure 3. On day 11 the animals were sacrificed, the left tibias were removed and an X-ray analysis was performed and the lengths of the bones were measured using a caliper. The average length +/- ETM is plotted. The asterisks indicate significant differences with the control group (p <; 0.05). Figure 6 shows the levels of IGF-1 in the plasma during the 6-day efficacy study. The animals were treated as described in Figure 4. Blood samples were taken at different time points, and serum IGF-1 levels were determined by ELISA. The means +/- ETM are represented graphically.

DETAILED DESCRIPTION OF THE INVENTION HGH and its agonist variants are members of a family of recombinant proteins, described in US 4,658,021 (methionyl-human growth hormone-Met-1-191-hGH) and US 5,633,352. Its recombinant production and methods of use are detailed in US 4,342,832. 4,601, 980; US 4,898,830; US 5,424,199; and US 5,795,745. Any purified and isolated hGH or its agonist variant can be used, which is produced by host cells such as E. coli and transformed or transfected animal cells using recombinant genetic techniques. Additional hGH variants are described in US 6,143,523 and WO 92/09690 published June 11, 1992. Among them, hGH or its agonist variant which is produced by transformed E. coli is particularly preferred. Said hGH or its agonist variant can be obtained in large quantities with high purity and homogeneity. For example, hGH or its agonist variant above, can be prepared according to a method described in US 4,342,832, 4; 601, 980; US 4,898,830; US 5,424,199; and US 5,795,745. The expression "substantially has the following amino acid sequence" means that the above amino acid sequence may include one or more amino acid changes (deletion, addition, insertion or substitution) with the proviso that said changes do not produce any disadvantage of non-similarity in the function of hGH or its agonist variant. It is more preferable to use hGH or its agonist variant having substantially an amino acid sequence, in which at least one lysine, aspartic acid, glutamic acid, unpaired cysteine portions, a free N-terminal a-amino group or a free C-terminal carboxyl group. The term "hGH polypeptide or hGH protein", when used herein, encompasses all hGH polypeptides, preferably of mammalian species, more preferably of human and murine species, as well as their variants, analogs, orthologs, homologues , and derivatives, and their fragments, which are characterized by promoting growth in the growth phase and maintaining the body composition, anabolism and metabolism of normal lipids. Preferably, the term "hGH polypeptide or protein" refers to the hGH polypeptide of SEQ ID NO: 1, as well as to its variants, homologs and derivatives that exhibit essentially the same biological activity (promotion of growth in the growth phase and maintenance of body composition, anabolism and normal lipid metabolism). More preferably, the term "hGH polypeptide or protein" refers to the polypeptide of SEQ ID NO: 1. The term "hGH polypeptide variants", as used herein, refers to polypeptides thereof. species but which differ from a reference hGH polypeptide. In general, the differences are limited, so that the amino acid sequences of the reference polypeptide and the variant are very similar together, and in many regions they are identical. Preferably, the hGH polypeptides are at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference hGH polypeptide, preferably the hGH polypeptide of SEQ ID No. .: 1. For a polypeptide having an amino acid sequence that is at least, for example, 95% "identical" to a given amino acid sequence, it is intended that the amino acid sequence of the target polypeptide be identical to the given sequence , except that the target polypeptide sequence may include up to five amino acid alterations per 100 amino acids of the given amino acid sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or elsewhere between those terminal positions, interspersed individually between portions in the reference sequence or in one or more contiguous groups in the reference sequence. The given sequence may be a whole sequence of amino acids of the reference sequence or any specified fragment as described herein. Said variants of the hGH polypeptide can be natural variants, such as natural allelic variants encoded by one or more alternative forms of a hGH, occupying a given locus on a chromosome of an organism, or isoforms encoded by natural splice variants that originate from a single primary transcript. Alternatively, a variant of the hGH polypeptide may be a variant that is known to be natural, and which may be made using mutagenesis techniques known in the art. It is known in the art that one or more amino acids can be removed from the N or C-terminus of a bioactive peptide or protein without substantial loss of biological function (see, for example, Ron et al., (1993), Biol. Chem. ., 268 2984-2988; the description of which is hereby incorporated by reference in its entirety). One skilled in the art will also recognize that some amino acid sequences of hGH polypeptides can be varied without significant effect of the structure or function of the protein. Said mutations include deletions, insertions, inversions, repetitions and substitutions selected according to general rules known in the art, so that they have a small effect on the activity. For example, Bowie et al. (1990), Science 247: 1306-1310, hereby incorporated in its entirety as a reference, provide guidance regarding how to make substitutions for phenotypically silent amino acids, and the authors indicate that there are two main methods for studying the tolerance of a sequence. of amino acids to change. The first method is based on the process of evolution, in which mutations are accepted or rejected by natural selection. The second method uses genetic engineering to introduce amino acid changes at specific positions of a cloned hGH and selections or screens to identify sequences that maintain functionality. These studies have shown that proteins are surprisingly tolerant to amino acid substitutions. The authors also indicate that amino acid changes are likely to be allowed in a certain position of the protein. For example, the most buried portions of amino acids require non-polar side chains, while in general few features of surface side chains are retained. Other phenotypically silent substitutions are described by Bowie et al., (1990), see above, and references cited therein. Typically conservative substitutions are considered substitutions, from one amino acid for another, between the aliphatic amino acids, Ala, Val, Leu and Phe; Exchange of the hydroxyl portions Ser and Thr, exchange of the Asp and Glu acid portions, substitution between the amide portions Asn and Gln, exchange of the basic portions Lys and Arg, and substitutions between the aromatic portions Phe and Tyr. In addition, the following amino acid groups in general represent equivalent changes: (1) Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, He, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His. The hGH polypeptide expression also encompasses all hGH polypeptides encoded by analogs, orthologs and / or homologous species of hGH. As used herein, the term "hGH analogues" refers to hGH from different and unrelated organisms that perform the same functions in each organism, but that do not come from an ancestral structure that the ancestors of the organisms had in common. Instead, analog hGHs arise separately and later evolve to perform the same function (or similar functions). In other words, the analogous hGH polypeptides are polypeptides with quite different amino acid sequences but which perform the same biological activity, particularly promoting growth in the growth phase and maintaining normal body composition, anabolism and lipid metabolism. As used herein, the term "orthologs of hGH" refers to hGH of two different species, whose sequences are related to each other by an homologous hGH in an ancestral species, but which have evolved to become different from each other. As used herein, the term "homologues of hGH" refers to hGH of different organisms that perform the same function in each organism, and that come from an ancestral structure that the ancestors of organisms had in common. In other words, homologous hGH polypeptides are polypeptides with fairly similar amino acid sequences that perform the same biological activity, particularly promoting growth in the growth phase and maintaining body composition., anabolism and metabolism of normal lipids. Preferably, the homologous hGH polypeptides can be defined as polypeptides that are at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference hGH polypeptide, preferably the hGH polypeptide of SEQ ID NO: 1. Thus, a hGH polypeptide according to the invention can be, for example: (i) one in which one or more of the amino acid portions are replaced by a conserved or non-conserved amino acid portion (preferably a conserved amino acid portion) and said substituted amino acid portion may or may not be encoded by the genetic code; or (ii) one in which one or more of the amino acid portions includes a substituent group; or (iii) one in which the hGH polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (eg, polyethylene glycol); or (v) one in which additional amino acids are fused to the previous form of the polypeptide, such as a peptide from the IgG Fe fusion region or leader or secretory sequence or a sequence that is used to purify the previous form of the polypeptide or a proprotein sequence. The hGH polypeptides can be monomers or multimers. The multimers may be dimers, trimers, tetramers or multimers comprising at least five monomer polypeptide units. The multimers can also be homodimers or heterodimers. The multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and / or covalent associations and / or may be linked indirectly, for example, by the formation of liposomes. In one example, the covalent associations are between the heterologous sequences contained in a fusion protein containing a hGH polypeptide or its fragment (see for example, U.S. Patent No. 5,478,925, the disclosure of which is incorporated herein by reference). its entirety as a reference). In another example, a hGH polypeptide or its fragment binds to one or more polypeptides that can be hGH polypeptides or heterologous polypeptides, by peptide linkers such as those described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Another method for preparing hGH multimeric polypeptides involves the use of hGH polypeptides fused to a leucine zipper polypeptide or isoleucine zipper sequence that is known to promote multimerization of the proteins in which they are found, using techniques known to the skilled artisan. in the art, including the teachings of WO 94/10308. In another example, the hGH polypeptides may be associated by interactions between the Flag® polypeptide sequences contained in fusion hGH polypeptides containing the Flag® polypeptide sequence. HGH multimers can also be generated using chemical techniques known in the art, such as cross-links using linker molecules and techniques for optimizing the length of the linker molecule (see, eg, US 5,478,925), techniques known in the art. the technique for forming one or more cross-links between molecules between the cysteine portions located within the sequence of the polypeptides that are to be contained in the multimer (see, e.g., US 5,478,925), addition of cysteine or biotin at the C-terminus or N-terminus of the hGH polypeptide and techniques for generating multimers containing one or more of these modified polypeptides (see, e.g., US 5,478,925), or any of the techniques for generating liposomes containing hGH multimers (see, for example, U.S. Patent No. 5,478,925), the descriptions of which are incorporated by reference in their entirety. As used herein, the term "hGH polypeptide fragment" refers to any peptide or polypeptide comprising a contiguous stretch of a portion of the amino acid sequence of a hGH polypeptide, preferably the polypeptide of SEQ ID No. .: 1. More specifically, a hGH polypeptide fragment comprises at least 6, preferably at least 8 to 10, more preferably 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 125, 150, 175, 191 consecutive amino acids of a hGH polypeptide according to the present invention. The hGH polypeptide fragment can be further described as a subgenus of hGH polypeptides comprising at least 6 amino acids, wherein "at least 6" is defined as any integer between 6 and the integer representing amino acid C -terminal of a hGH polypeptide, including the polypeptide of SEQ ID No. 1. Also included are species of hGH polypeptide fragments of at least 6 amino acids in length, as described above, which are further specified in terms of their N-terminal and C-positions. terminal. Also encompassed by the term "hGH polypeptide fragment" as individual species are all hGH polypeptide fragments, of at least 6 amino acids in length, as described above, which can be specified in particular by an N-terminal position and C-terminal. That is, each combination of an N-terminal and C-terminal position that can occupy a fragment of at least 6 contiguous amino acid portions in length, in any given amino acid sequence of the sequence listing of the present invention, is included in the present invention. It should be noted that the former polypeptide fragment species of the present invention can alternatively be described by the formula "a a b"; where "a" is equal to the position of the N-terminal amino acid, and "b" is equal to the C-terminal amino acid position of the polynucleotide; and further where "a" is equal to an integer between 1 and the number of amino acids of a polypeptide sequence of hGH minus 6, and where "b" is equal to an integer between 7 and the number of amino acids in the sequence of hGH polypeptide; and where "a" is an integer less than "b" in at least 6. The fragments of the above hGH polypeptide can be predicted immediately using the above description and therefore are not listed individually only for the purpose of not unnecessarily lengthening the descriptive memory. In addition, the above fragments need not have a biological activity of hGH, although polypeptides having these activities are preferred embodiments of the invention, since they would be useful, for example, in immunoassays, in epitope mapping, epitope tagging, as vaccines, and as molecular weight markers. The above fragments can also be used to generate antibodies against a particular part of the polypeptide. Also included in the expression "hGH polypeptide fragment" are the domains of the hGH polypeptides. Said domains may finally comprise linear or structural patterns or identifiers including, but not limited to, leucine zippers, helix-turn-helix patterns, post-translational modification sites such as glycosylation sites, ubiquitination sites, alpha helices, beta sheets, peptides signal encoding signal sequences that direct the secretion of encoded proteins, sequences involved in the regulation of transcription such as homosequences, acid extenders, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites. Said domains can exhibit a particular biological activity such as DNA or RNA binding, protein secretion, transcription regulation, enzymatic activity, substrate binding activity, etc. A domain has a size generally comprised between 3 and 191 amino acids. In a preferred embodiment, the domains comprise a number of amino acids which is any integer between 6 and 191. The domains can be synthesized using any methods known to those skilled in the art, including those described herein for preparing hGH polypeptides. to produce anti-hGH antibodies. Among the methods for determining the amino acids that make up a domain with a particular biological activity, studies and mutagenesis assays are included to determine the biological activity to be tested. Particularly preferred fragments in the context of the present invention are hGH polypeptides that substantially retain the biological activity, particularly the promotion of growth in the growth phase, and the maintenance of body composition, anabolism and normal lipid metabolism. Alternatively, the patterns, domains and / or identifiers of polypeptides of the invention can be searched in databases using any computer method known to those skilled in the art. Search databases include Prosite (Hofmann et al., (1999) Nucí Acids Res. 27: 215-219; Bucher and Bairoch (1994) Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman et al. , Eds., Pp53-61, AAAlPress, Menlo Park), Pfam (Sonnhammer et al., (1997) Proteins 28 (3): 405-20, Henikoff et al., (2000) Nucleic Acids Res. 28 (1) : 228-30, Bateman et al., (2000) Nucleic Acids Res. 28 (1): 263-6), Blocks (Henikoff et al., (2000) Electrophoresis 21 (9): 1700-6), Print ( Attwood et al., (1996) Nucleic Acids Res. 24 (1): 182-8), Prodom (Sonnhammer and Kahn (1994) Protein Sci. 3 (3): 482-92; Corpet et al. (2000) Nucleic Acids Res. 28 (1): 267-9), Sbase (Pongor et al. (1993) Protein Eng. 6 (4): 391-5; Murvaí et al., (2000) Nucleic Acids Res. 28 (1): 260-2), Smart (Schuitz et al., (1998) Proc. Nati. Acad. Sci. USA 95, 5857-5864), Dali / FSSP (Holm and Sander (1996J Nucleic Acids Res. 24 (l): 206-9; Holm and Sander (1997) Nucleic Acids Res. 25 (l): 231-4; Holm and Sander (1999) Nucleic Acids Res. 27 (1): 244-7), HSSP (Sander and Schneider (1991) Proteins 9 (1): 56-68.), CATH (Orengo et al., (1997) Structure 5 (8): 1093-108; Pearl et al., (2000) Biochem. Soc. Trans. 28 (2): 269-75), SCOP (Murzin et al., (1995) J. Mol. Biol. 247 (4): 536-40; et al., (2000) Nucleic Acids Res. 28 (1): 257-9), COG (Tatusov et al., (1997) Science 278, 631: 637; Tatusov et al., (2000) Nucleic Acids Res. 28 (I): 33-6), databases of specific families and their derivatives (Nevill-Manning et al., (1998) Proc. Nati, Acad. Sci. USA, 95, 5865-5871; Yona et al. , (1999) Proteins 37 (3): 360-78; Attwood et al., (2000) Nucleic Acids Res. 28 (1): 225-7), each of whose descriptions is hereby incorporated by reference. In its entirety as a reference. For a review of available databases, see number 1 of volume 28 of Nucleic Acid Research (2000), the description of which is incorporated herein by reference. The term "hGH polypeptide fragment" also encompasses fragments carrying epitopes. These epitopes can be antigenic epitopes or both an antigenic epitope and an immunogenic epitope. An immunogen epitope is defined as a part of a protein that elicits an antibody response in vivo when the polypeptide is the immunogen. On the other hand, a region of the polypeptide to which an antibody binds is defined as an antigenic epitope. An epitope can comprise as few as 3 amino acids in a spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least 6 of said amino acids, and more often at least 8-10 of said amino acids. An epitope-bearing fragment of hGH according to the invention can be any fragment whose length is between 6 amino acids and the full-length sequence of a hGH polypeptide, preferably a fragment between 6 and 50 amino acids. The epitope-bearing fragments can be specified by the number of contiguous amino acid portions (as a subgenus) or by the N-terminal and C-terminal positions (as a species) as described above. Fragments that function as epitopes can be prepared by any conventional means (see, e.g., Houghten (1985), Proc. Nati, Acad. Sci. USA 82: 5131-5135 and U.S. Patent 4,631, 21 , the descriptions of which are hereby incorporated by reference in their entirety). Methods for determining the amino acids that make up an epitope include X-ray crystallography, two-dimensional nuclear magnetic resonance, and epitope mapping, e.g. eg, the Pepscan method described by Geysen et al., (1984), Proc. Nati Acad. Sci. U.S.A. 81: 3998-4002; PCT publications WO 84/03564 and WO 84/03506, the disclosures of which are hereby incorporated by reference in their entirety. Another example is the algorithm of Jameson and Wolf, (1988), Comp. Appl. Biosci. 4: 181-186 (said reference is incorporated in its entirety as a reference). The Jameson-Wolf antigenic analysis can be carried out, for example, using the PROTEAN computer program, using the default parameters (Version 4.0 Windows, DNSTAR, Inc.) The present invention also provides for the exclusion of any species of fragment of hGH specified by the N-terminal and C-terminal positions, or of any fragment specified by the subgenus by the size of amino acid portions as described above. Any series of fragments specified by the N-terminal and C-terminal positions or by the size of the amino acid portions as described above can be excluded as an individual species. The present invention also provides for the exclusion of any hGH domain or epitope-bearing fragment in the same way. The hGH polypeptides of the present invention can be prepared in any suitable manner. Said hGH polypeptides and their fragments can be purified from natural sources, can be chemically synthesized, produced by recombinant techniques including in vitro translation techniques or expression in a recombinant cell capable of expressing hGH cDNA, or a combination of these methods, using techniques known to those skilled in the art (see, for example, "Methods in Enzymology, Academic Press, 1993" for a variety of methods to purify proteins; Creighton, (1983) Proteins: Structures and Molec Principies, W.H. Freeman & Co. 2nd Ed., T. E., New York; and Hunkapiller et al., (1984) Nature, 310 (5973): 105-11, for the chemical synthesis of proteins, and Davis et al. (1986) Basic Methods in Molec Biology, ed., Elsevier Press, NY, for recombinant techniques, the descriptions of which are incorporated by reference in their entirety). The polypeptides of the present invention are preferably provided in an isolated form, and may be partially or preferably substantially purified.

The terms "polynucleotides that are at least x% identical to a reference polynucleotides" and "polypeptides that are x% identical to a reference polypeptide" encompass polynucleotides or polypeptides whose sequence of portions (nucleotides or amino acids respectively) has a percentage of identity, as defined below, equal to or greater than x, compared to said reference polynucleotide or polypeptide, respectively. Percentage identity is determined after optimal alignment of two polynucleotide or polypeptide sequences in a comparison window, wherein the portions of the polynucleotide or polypeptide sequences in the comparison window may comprise additions or deletion of one or more portions in order to optimize the alignment of the sequence. The comparison window contains a certain number of positions (either a portion or a gap corresponding to an insertion / deletion of a portion), said number of positions corresponding to the size of the window. Each position of the window can present one of the following situations: 1) There is a portion (nucleotide or amino acid) in this position in the first aligned sequence, and a different portion in the same position in the second aligned sequence, in other words the second sequence has a substituted portion in this position compared to the first sequence. 2o) There is a portion (nucleotide or amino acid) in this position in the first aligned sequence, and the same portion in the same position in the second aligned sequence. 3o) There is a portion (nucleotide or amino acid) in this position in the first aligned sequence, and there is no portion in the same position in the second aligned sequence, in other words, the second sequence presents a deletion in this position compared to the first sequence. The number of positions within the comparison window that belong to the first category defined above is called R1. The number of positions within the comparison window that belong to the second category defined above is called R2. The number of positions within the comparison window that belong to the third category defined above is called R3. The identity percentage (% id) can be calculated by any of the following formulas:% id = R2 / (R1 + R2 + R3) x 100, or% id = (R2 + R3) / (RI + R2 + R3) x 100 The sequence alignment for comparison can be carried out using any of a variety of algorithms and sequence comparison programs known in the art. Such programs and algorithms include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, FASTDB, WU-BLAST, Gapped-BLAST, PSI-BLAST (Pearson and Lipman, (1988), Proc. Nati. Acad. Sci. USA 85: 2444-2448; Altschul et al., (1990), J. Mol. Biol. 215: 403-410; Altschul et al., (1993), Nature hGHtics 3: 266-272; Altschul et al., (1997), Nuc. Acids Res. 25: 3389-3402; Thompson et al., (1994), Nuc. Acids Res., 22: 4673-4680; Higgins et al., (1996), Meth. Enzymol. 266: 383-402; Brutlag et al. (1990) Comp. App. Biosci. 6: 237-245; Jones and Swindells, (2002) Trends Biochem. Sci. 27: 161-4; Olsen et al. (1999) Pac. Symp. Biocomput; 302-13), whose descriptions are incorporated in their entirety as a reference. In a particular embodiment, the Smith-Waterman method is used, with a scoring matrix such as PAM, PAM 250 or preferably with BLOSUM matrices such as BLOSUM60 or BLOSUM62 and with the default parameters (penalty for gap opening = 10 and penalty for the extension of a gap = 1) or with parameters specified by the user, preferably higher than the default parameters. In another particular embodiment, the sequences of the proteins and nucleic acids are aligned using the Basic Local Alignment Search Tool ("BLAST") programs with the default parameters, or with modified parameters provided by the user. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., (1992), Science 256: 1443-1445; Henikoff and Henikoff, (1993), Proteins 17: 49-61, whose descriptions are hereby incorporated by reference in their entirety). Less preferably, the PAM or PAM250 matrices can also be used (see, eg, Schwartz and Dayhoff, (1978), eds., Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation , the description of which is hereby incorporated by reference in its entirety). In yet another particular embodiment, the polynucleotide or polypeptide sequences are aligned using the FASTDB computer program based on the algorithm of Brutlag et al. (1990), see above. The preferred parameters used in a FASTDB alignment of DNA sequences are: Matrix = Unitary, k-tuple = 4, mismatch penalty = 1, joint penalty = 20, randomized group length = 0, cut-off score = 1 , gap penalty = 5, penalty for gap size = 0.05, window size = 500 or the length of the present nucleotide sequence, provided it is shorter. The preferred parameters used in the alignment of amino acids in FASTDB are: matrix = PAM, k-tuple = 2, penalty for mismatch = 1, penalty for union = 20, length of randomized group = 0, cut score = 1, penalty per gap = 5, penalty for gap size = 0.05, window size = 500 or the length of the present amino acid sequence, provided it is shorter. In accordance with the present invention, polyethylene glycol is covalently linked by amino acid portions of hGH or its agonist variants. A variety of activated polyethylene glycols having a number of different functional groups, linkers, configurations, and molecular weights are known to those skilled in the art, which can be used to create conjugates of PEG-hGH or conjugates of PEG-hGH agonist (for reviews see Roberts MJ et al., Adv. Drug Del. Rev. 54: 459-476, 2002), Harris JM et al., Drug Delivery Sytems 40: 538-551, 2001). The present invention relates to a method that uses the aldehyde chemistry to selectively direct the PEG portion to the N-terminus, using a butyrylaldehyde connecting portion. The butyrylaldehyde linker results in greater N-terminal specificity compared to the acetaldehyde linker (Table 1 and Figures 1A, 1 B and 1C). One embodiment of the present invention is a human growth hormone-PEG conjugate having the structure of formula I or formula II Formula I mPEG-0 < CH2CH2O) n (CH2) mCHrNH-R Formula II wherein n is an integer between 1 and 10; m is an integer between 1 and 10; R is the human growth hormone, methionyl-growth hormone, or a variant of growth hormone. In a particular embodiment n is between 1 and 5 and m is between 1 and 5. In a particular embodiment of formula I: n is 1 and m is 1; n is 1 and m is 2; n is 1 and m is 3; n is 1 and m is 4; n is 1 and m is 5; n is 1 and m is 6; n is 1 and m is 7, n is 1 and m is 8; n is 1 and m is 9; n is 1 and m is 10; n is 2 and m is 1; n is 2 and m is 2; n is 2 and m is 3; n is 2 and m is 4; n is 2 and m is 5; n is 2 and m is 6; n is 2 and m is 7; n is 2 and m is 8; n is 2 and m is 9; n is 2 and m is 10; n is 3 and m is 1; n is 3 and m is 2; n is 3 and m is 3; n is 3 and m is 4; n is 3 and m is 5; n is 3 and m is 6; n is 3 and m is 7; n is 3 and m is 8; n is 3 and m is 9; n is 3 and m is 10; n is 4 and m is 1; n is 4 and m is 2; n is 4 and m is 3, n is 4 and m is 4, n is 4 and m is 5; n is 4 and m is 6; n is 4 and m is 7; n is 4 and m is 8; n is 4 and m is 9; n is 4 and m is 10; n is 5 and m is 1; n is 5 and m is 2; n is 5 and m is 3; n is 5 and m is 4; n is 5 and m is 5, n is 5 and m is 6, n is 5 and m is 7; n is 5 and m is 8; n is 5 and m is 9; n is 5 and m is 10; n is 6 and m is 1; n is 6 and m is 2; n is 6 and m is 3, n is 6 and m is 4; n is 6 and m is 5; n is 6 and m is 6; n is 6 and m is 7; n is 6 and m is 8, n is 6 and m is 9; n is 7 and m is 10; n is 7 and m is 1; n is 7 and m is 2; n is 7 and m is 3; n es7 and m is 4; n is 7 and m is 5; n is 7 and m is 6; n is 7 and m is 7; n is 7 and m is 8; n is 7 and m is 9; n is 7 and m is 10; n is 8 and m is 1; n is 8 and m is 2; n is 8 and m is 3; n is 8 and m is 4; n is 8 and m is 5; n is 8 and m is 6; n is 8 and m is 7; n is 8 and m is 8; n is 8 and m is 9; n is 8 and m is 10; n is 9 and m is 1; n is 9 and m is 2; n is 9 and m is 3; n is 9 and m is 4; n is 9 and m is 5; n is 9 and m is 6; n is 9 and m is 7; n is 9 and m is 8; n is 9 and m is 9; n is 9 and m is 10; n is 10 and m is 1; n is 10 and m is 2; n is 10 and m is 3; n is 10 and m is 4; n is 10 and m is 5; n is 10 and m is 6; n is 10 and m is 7; n is 10 and m is 8; n is 10 and m is 9; n is 10 and m is 10. A specific modality is a conjugate of human growth hormone-PEG that has the structure mPEG-O (CH2CH2O) 4CH2CH2CH2CHrNH-R wherein R is human growth hormone, methionyl-human growth hormone or a variant of human growth hormone. Another specific embodiment of the present invention is the human growth hormone-PEG conjugate wherein the human growth hormone comprises or consists of the amino acid sequence of SEQ ID NO: 1. A specific embodiment of the present invention is a human growth hormone-PEG conjugate wherein more than 80%, more preferably 81%, more preferably 82%, more preferably 83%, more preferably 84%, more preferably 85%, more preferably 86%, more preferably 87 %, more preferably 88%, more preferably 89%, more preferably 90%, more preferably 91%, more preferably 92%, more preferably 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97 %, and more preferably 98%, of the polyethylene glycol is conjugated to the amino terminal phenylalanine of the amino acid sequence of SEQ ID NO: 1. Another specific embodiment of the present invention n is a human growth hormone-PEG conjugate in which more than 90% of the polyethylene glycol is conjugated to the amino terminal phenylalanine of the amino acid sequence of SEQ ID NO: 1. Another specific embodiment of the present invention is a conjugate of human growth hormone-PEG in which more than 95% of the polyethylene glycol is conjugated to the amino terminal phenylalanine of the amino acid sequence of SEQ ID NO: 1. Another specific embodiment of the present invention is a conjugate of human growth hormone -PEG in which more than 98% of the polyethylene glycol is conjugated to the amino terminal phenylalanine of the amino acid sequence of SEQ ID NO: 1. The polyethylene glycol used in the present invention is not restricted to any particular form or molecular weight range. The molecular weight of the polyethylene glycol can be between about 500 and about 100,000 Daltons. The term "approximately" indicates that in polyethylene glycol preparations, some molecules will weigh more, and some less, that the molecular weight exposed, and the molecular weight exposed refers to the average molecular weight. It is understood that there is some degree of polydispersity associated with polymers such as polyethylene glycol. It is preferable to use PEG with low polydispersity. Typically, a PEG with a molecular weight of about 500 to about 60,000 is used. A specific PEG molecular weight range of the present invention is from about 1,000 to about 40,000. In another specific embodiment the molecular weight of the PEG is greater than about 5,000 to about 40,000. In another specific embodiment, the molecular weight of the PEG is from about 20,000 to about 40,000. Other sizes may be used, depending on the desired therapeutic profile (eg, duration of desired sustained release, effects, if any on biological activity, degree or lack of antigenicity and other known effects of polyethylene on a protein. therapy). For example, polyethylene glycol can have an average molecular weight of about 200, 500, 1, 000, 1, 500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000 , 8,500, 9,000, 9,500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500 , 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 Dalton.

In another embodiment, the polyethylene glycol is a branched PEG having more than one PEG bound portion (see US 5,932,462, US 5,342,940, US 5,643,575, US 5,919,455, US 6,113,906, US 5,183,660, Kodera Y., Bioconjugate Chemistry 5 : 283-288 (1994), and WO 02/09766.In a preferred embodiment, the molecular weight of each polyethylene glycol of the branched PEG is about 5,000-20,000.In a specific embodiment, the molecular weight of each polyethylene glycol of the branched PEG is About 20,000 Poly (alkylene oxides), particularly polyethylene glycols, bind to hGH or its agonist variant by a reactive end group, which may or may not leave a connecting (spacing) portion between the PEG and the protein. In order to form the conjugates of the hGH or its agonist variant of the present invention, the polymers such as the poii (alkylene oxide) are converted to activated forms, as they are understood to mean those skilled in the art. The reactive group, for example, is a reactive terminal group, which mediates a bond between the chemical portions in the protein and the polyethylene glycol. Typically, one or both terminal hydroxyl groups of the polymer ends (ie, the alpha and omega terminal hydroxyl groups) are converted to reactive functional groups, which allow covalent conjugation. This process is often referred to as "activation" and the polyethylene glycol product having the reactive group is referred to as "an activated polyethylene glycol". In a specific embodiment, one or more terminal hydroxyl groups of the polymer are converted or capped with a non-reactive group. In a specific embodiment one of the terminal hydroxyl groups of the polymer is converted or capped with a methyl group. As used herein, the term "mPEG" refers to a PEG, which is capped at one end with a methyl group. MPEG can be structurally represented as CH30- (CH2CH2?) N-H Polymers containing both a and e linking groups are referred to as "bis-activated poly (alkylene oxides)". Polymers containing the same reactive group in the terminal hydroxyls a and e are sometimes referred to as "homobifunctional" or "homobis-activated". Polymers containing different reactive groups in the terminal hydroxyl a and e are sometimes referred to as "heterobifunctional" (see for example WO 01/26692) or "heterobis-activated". Polymers containing a single reactive group are referred to as "mono-activated" or "mono-functional" poly (alkylene oxides). Other substantially non-antigenic polymers are "activated" or "functionalized" in the same way. Therefore, the activated polymers are suitable for mediating a bond between the chemical moieties in the protein, such as a- or e-amino, carboxyl or thiol groups, and polyethylene glycol. The bis-activated polymers can thus react with two protein molecules or one protein molecule and one small reactive molecule in another embodiment to efficiently form protein-polymer or protein-small molecule cross-linked conjugates.

In a preferred embodiment of the invention, secondary amine or amide linkages are formed using the α-amino terminal group or e-amino groups of the lysine of hGH or its agonist variant and activated PEG. In another preferred aspect of the invention, a secondary amine linkage is formed between the N-terminal primary a-amino or e-amino group of the hGH or its agonist variant and the PEG-aldehyde chain alone or branched by reductive alkylation with a suitable reducing agent such as NaCNBH3, NaBH3, pyridine-borane, etc. as described by Chamow et al., Bioconjugate Chem. 5: 133-140 (1994), patent of E.U.A. n °. 4,002,531, WO 90/05534, and patent of E.U.A. n °. 5,824,784. In a preferred embodiment, at least 70%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, and more preferably at least 98% of the polyethylene glycol it is in the a-amino-terminal group. The conjugation reactions, so-called pegylation reactions, have historically been carried out in solution with a molar excess of polymer and without taking into account where the polymer would bind to the protein. However, such general techniques have typically been shown to be unsuitable for conjugating bioactive proteins to non-antigenic polymers and that sufficient bioactivity is retained. One way to maintain the bioactivity of the hGH or its agonist variant is to substantially avoid conjugation of the reactive groups of the hGH or its agonist variant associated with the receptor binding site (s), in the method of coupling the polymer. Another aspect of the present invention is to provide a process for conjugating polyethylene glycol to hGH or its agonist variant while maintaining high levels of retained activity. The chemical modification by a covalent bond can be carried out under any suitable conditions generally adopted in a reaction of a biologically active substance with the activated polyethylene glycol. The conjugation reaction is carried out under relatively mild conditions to prevent the inactivation of hGH or its agonist variant. Soft conditions include maintaining the pH of the reaction solution in the range of 3 to 10 and the reaction temperatures in the range of about 0-37 ° C. In cases where the portions of reactive amino acids in the hGH or its agonist variant have free amino groups, the above modification is preferably carried out in a non-limited list of suitable pH regulators (pH 3 to 10), including phosphates , MES, citrate, acetate, succinate or HEPES, for 1-48 hours at 4 ° -37 ° C. When the target is N-terminal amino groups with reagents such as PEG-aldehydes, pH 4-7 is preferably maintained. The activated polyethylene glycol can be used in an amount of about 0.01-100 times, preferably about 0.01-2.5 times, the molar amount of the number of free amino groups of the hGH or its agonist variant. On the other hand, when the portions of reactive amino acids in the hGH or its agonist variant have free carboxyl groups, the above modification is preferably carried out at a pH of about 3.5 to about 5.5, for example, the modification with polyoxyethylene diamine is brought to in the presence of carbodiimide (pH 4-5) for 1-24 hours at 4 ° -37 ° C. The activated polyethylene glycol can be used in an amount of 0.01-300 times the molar amount of the number of free carboxyl groups of the hGH or its agonist variant. In separate embodiments, the upper limit for the amount of polymer included in the conjugation reactions exceeds approximately 1: 1 the reactive amount of activated polymer and hGH or its agonist variant without forming a substantial amount of high molecular weight species, is say, more than about 20% of the conjugates containing more than about one polymer chain per molecule of hGH or its agonist variant. For example, in this aspect of the invention, it is contemplated that ratios of up to about 6: 1 can be used to form significant amounts of the desired conjugates which can then be isolated from any high molecular weight species. In another aspect of this invention, bifunctional activated PEG derivatives can be used to generate molecules of PEG-hGH or its polymeric agonist variant., in which multiple molecules of hGH or its agonist variant are crosslinked by PEG. Although the reaction conditions described herein can yield significant amounts of hGH or its unmodified agonist variant, hGH or its unmodified agonist variant can be easily recycled in subsequent batches for further conjugation reactions. The methods of the present invention surprisingly generate very little, i.e., less than about 30%, and more preferably, less than 10% of high molecular weight species and species that contain more than one polymer chain per hGH or its variant agonist These reaction conditions contrast with those typically used for polymer conjugation reactions, in which the activated polymer is present with a molar excess of several times with respect to the target. In other aspects of the invention, the polymer is present in amounts of about 0.1 to about 50 equivalents per equivalent of hGH or its agonist variant. In other aspects of the invention, the polymer is present in amounts of about 1 to about 10 equivalents per equivalent of hGH or its agonist variant. The conjugation reactions of the present invention initially provide a mixture or reaction set containing conjugates of mono- and di-PEG-hGH, unreacted hGH, unreacted polymer, and typically less than about 20% of high molecular weight species . High molecular weight species include conjugates containing more than one polymer chain, and / or species of PEG-hGH or its polymerized agonist variant. After separating the unreacted species and high molecular weight species, the compositions containing mono- and di-polymer-hGH conjugates or their agonist variant are recovered. Since most of the conjugates include a single polymer chain, the conjugates are substantially homogeneous. These hGH or its modified agonist variant have at least about 0.1% of the in vitro biological activity associated with hGH or its native or unmodified agonist variant, measured using FDC-P1 cell proliferation assays (Clark et al. Biological Chemistry 271: 21969-21977, 1996), receptor binding assay (US 5,057,417), or hypophysectomized rat growth (Clark et al., Journal of Biological Chemistry 271: 21969-21977, 1996). However, in preferred aspects of the invention, hGH or its modified agonist variant has approximately 25% of the biological activity in vitro, more preferably, hGH or its modified agonist variant has approximately 50% of the biological activity in vitro, more preferably, hGH or its modified agonist variant has approximately 75% biological activity in vivo, and more preferably hGH or its agonist variant has equivalent or better in vivo activity. The methods of the present invention preferably include ratios of polymer to hGH or its agonist variant rather limited. Thus, it has been found that conjugates of hGH or its agonist variant are predominantly limited to species that contain only one polymer chain. In addition, the binding of the polymer to the reactive groups of the hGH or its agonist variant is substantially less random than when larger molar excesses of the polymeric linker are used. The hGH or its unmodified agonist variant present in the reaction mixture, after inactivating the conjugation reaction, can be recycled to subsequent reactions using ion exchange or size exclusion chromatography, or similar separation techniques. An hGH or its modified agonist variant with polyethylene glycol, particularly a chemically modified protein according to the present invention, can be purified from a reaction mixture by conventional methods that are used to purify proteins., such as dialysis, precipitation by addition of salt, ultrafiltration, ion exchange chromatography, hydrophobic interaction chromatography (HIC), gel chromatography and electrophoresis. Ion exchange chromatography is particularly effective for separating unreacted polyethylene glycol and hGH or its agonist variant. In a further embodiment of the invention, the mono- and di-polymer-hGH species or their agonist variant are isolated from the reaction mixture to separate the high molecular weight species, and the hGH or its unmodified agonist variant. The separation is carried out by placing the mixed species in a pH buffer solution containing approximately 0.5-10 mg / ml of the hGH conjugates or their agonist-polymer variant. Suitable solutions have a pH of about 4 to about 10. The solutions preferably contain one or more pH freezing salts selected from KCl, NaCl, K2HP04, KH2P04, Na2HP04, NaH2P04l NaHCO3, NaB04, CH3CO2H and NaOH. Depending on the reaction pH regulator, it may be necessary to first submit the solution of the hGH conjugate or its agonist and polymer variant, to pH regulator / ultrafiltration exchange to remove any unreacted polymer. For example, the conjugate solution of PEG-hGH or its agonist variant can be ultrafiltered through a low molecular weight exclusion membrane (10,000 to 30,000 Daltons) to remove most of the unwanted materials such as polymer. unreacted, surfactants, if present, or the like. The fractionation of the conjugates in a mixture containing the desired species is preferably carried out using an ion exchange chromatography medium. Said means can selectively bind conjugates of PEG-hGH or its agonist variant by differences in charge, which varies in a somewhat predictable manner. For example, the surface loading of hGH or its agonist variant is determined by the number of charged groups available on the surface of the protein. These charged groups typically serve as a potential point of attachment of poly (alkylene oxide) polymers. Therefore, the conjugates of hGH or its agonist variant will have a different charge compared to the other species, to allow selective isolation. For the method of the present invention, highly polar anionic or cationic exchange resins, such as quaternary amine or sulfopropyl resins, respectively, are used. Especially preferred are ion exchange resins. A non-limiting list of commercially available cation exchange resins suitable for use with the present invention are SP-hitrap®, SP Sepharose HP® and SP Sepharose® Fast Flow. Other suitable cation exchange resins can also be used, for example S and CM resins. A non-limiting list of anion exchange resins, including commercially available anion exchange resins, suitable for use with the present invention are Q-hitrap®, Q Sepharose HP®, and Q sepharose®, fast flow. Other suitable anion exchange resins, for example, DEAE resins, can also be used. For example, the anionic or cationic exchange resin is preferably packaged in a column and equilibrated by conventional means. A pH regulator having the same pH and osmolality as the solution of polymer conjugate and hGH or its agonist variant is used. The elution pH regulator preferably contains one or more selected salts of KCl, NaCl, K2HP04, KH2P04 >; Na2HP04, NaH2P04, NaHCO3, NaB04, and (NH4) 2C03. Then, the solution containing conjugate is adsorbed on the column, where the unreacted polymer and some high molecular weight species are not retained. When the charge is complete, a gradient flow of an elution pH regulator is applied to the column with increasing salt concentrations to elute the desired fraction of poly (alkylene oxide) conjugate and hGH or its agonist variant. The eluted combined fractions are preferably limited to uniform polymer conjugates, after the cationic or anionic exchange separation step. Then any hGH species or its unconjugated agonist variant can be rewashed from the column by conventional techniques. If desired, species of hGH or its mono- and multipeglylated agonist variant can be further separated from each other by means of an additional ion exchange chromatography or size exclusion chromatography. Techniques using multiple Socratic stages of increasing salt concentration or pH can also be used. Multiple steps of increasing concentration Socratic elution will result in the sequential elution of di- and then mono-hGH conjugates or their agonist-polymer variant. The temperature range for the elution is between about 4 ° C and about 25 ° C. Preferably, the elution is carried out at a temperature of about 4 ° C to about 22 ° C. For example, elution of the PEG-hGH fraction or its agonist variant is detected by UV absorbance at 280 nm. The collection of fractions can be achieved by simple elution profiles. A surfactant can be used in the process of conjugating the polyethylene glycol polymer with the portion of the hGH or its agonist variant. Suitable surfactants include ionic agents such as sodium dodecylsulfate. Other ionic surfactants such as lithium docecyl sulfate, quaternary ammonium compounds, taurocholic acid, caprylic acid, decano sulfonic acid, etc. may also be used. Nonionic surfactants can also be used. For example, materials such as polyoxyethylene sorbitans (Tweens), polyoxyethylene ethers (Tritons). See also Neugebauer, A Guide to the Properties and Uses of Detergents in Biology and Biochemistry (1992) Calbiochem Corp. The only limitations of the surfactants used in the methods of the invention are that they are used under conditions and concentrations that do not produce a substantial irreversible denaturation of hGH or its agonist variant, and do not completely inhibit conjugation of the polymer. The surfactants are present in the reaction mixtures in amounts of about 0.01-0.5%; preferably 0.05-0.5%; and more preferably from about 0.075-0.25%. Mixtures of surfactants are also contemplated. It is believed that the surfactants provide a temporary and reversible protection system during the conjugation process of the polymer. It has been shown that surfactants are effective for selectively rejecting conjugation of the polymer, but allowing lysine-based or amino-terminal-based conjugation to occur. HGH modified with polyethylene glycol or its modified agonist variant has a more lasting pharmacological effect, which can possibly be attributed to its longer in vivo half-life. Another embodiment of the invention relates to methods for preventing and / or treating a disease or disorder in which the use of GH, preferably hGH is beneficial, which comprises administering to a patient in need thereof a therapeutically effective amount of a modified hGH with polyethylene glycol of the invention or its agonist variant, alone or in combination with another therapeutic agent. The invention also relates to the use of a hGH modified with polyethylene glycol of the invention or its agonist variant to make a medicament for preventing and / or treating a disease or disorder in which the use of GH, preferably hGH, is beneficial. In addition, the invention also relates to a pharmaceutical composition comprising a hGH modified with polyethylene glycol of the invention or its agonist variant to prevent and / or treat a disease or disorder in which the use of GH, preferably hGH, is beneficial . Diseases and disorders in which GH is beneficial include, but are not limited to, growth hormone deficiency (GHD), growth hormone deficiency in adults (DHCa), Turner syndrome, growth retardation. in children born small for gestational age (PEG), Prader-Willi syndrome (PWS), chronic renal failure (CRF), AIDS wasting, aging, end-stage renal failure, cystic fibrosis, erectile dysfunction, fibromyalgia HIV lipodystrophy, osteoporosis, memory disorders, depression, Crohn's disease, skeletal dysplasias, traumatic brain injury, subarachnoid hemorrhage, Noonan syndrome, Down's syndrome, idiopathic low stature (EBI), end-stage renal disease (ESRD), very low birth weight (MBPN), rescue of bone marrow stem cells, metabolic syndrome, glucocorticoid myopathy, short stature due to the treatment of children with glucocorticoids, and growth retardation in low preterm children. In a more specific embodiment of the invention, the hGH modified with polyethylene glycol of the invention or its agonist variant, are used to prevent and / or treat disorders or diseases selected from the group consisting of GHD, Aug, PEG, SPW, Syndrome Turner and IRC. In another more specific embodiment of the invention, the hGH modified with polyethylene glycol of the invention or its agonist variant are used to prevent and / or treat disorders or diseases selected from the group consisting of idiopathic short stature, very low birth weight, brain injury traumatic, metabolic syndrome, and Noonan syndrome. Another embodiment of the invention relates to pharmaceutical compositions comprising a hGH modified with polyethylene glycol of the invention or its agonist variant, alone or in combination with another therapeutic agent, and at least one pharmaceutically acceptable excipient or carrier. Then, the hGH modified with polyethylene glycol of the invention or its agonist variant present, can be formulated into pharmaceutical products also containing a pharmaceutically acceptable diluent, an agent for preparing an isotonic solution, a pH conditioner and the like, in order to administer it to a patient. The above pharmaceutical products can be administered subcutaneously, intramuscularly, intravenously, pulmonarily, intradermally, or orally, depending on the purpose of the treatment. A dose may also be based on the class and condition of the disorder of a patient to be treated, typically between 0.1 mg and 5 mg per injection, and between 0.1 mg and 50 mg in an oral administration for an adult. As used herein, the modified polyethylene glycol-hGH or its agonist variant of the present invention can be used in combination with another therapeutic agent. As used herein, the terms "co-administration", "co-administered" and "combined with" in relation to compounds A and one or more other therapeutic agents are meant to mean, refer to and include the following: - simultaneous administration of said combination of A and the therapeutic agent (s) to a patient in need of treatment, when said components are formulated together in a single dosage form which releases said components substantially at the same time in said patient, - simultaneous administration of said combination of A and the therapeutic agent (s) to a patient in need of treatment, when said components are formulated separately in separate dosage forms, which are taken substantially at the same time by said patient, so that said components are released substantially at the same time in said patient, - sequential administration of said combination of A and the therapeutic agent (s) to a patient in need of treatment, when said components are formulated by separated into separate dosage forms, which are taken in consecutive times by said patient with a significant time interval between each administration, so that said components are released substantially at different times in said patient; and - sequential administration of said combination of A and the therapeutic agent (s) to a patient in need of treatment, when said compounds are formulated together in a single dosage form that releases said components in a controlled manner. so that they are administered at the same time, consecutively and / or overlapping at the same time and / or different times in said patient. Suitable examples of other therapeutic agents that can be used in combination with A, its pharmaceutically acceptable salts and / or its derivative forms include, but are not limited to: aromatase inhibitors such as exemestane, formestane, atamestane, fadrozole, letrozole, vorozole and anastrozole; regulators of free fatty acids including fibric acid derivatives (such as fenofibrate, clofibrate, gemfibrozil, bezafibrate and ciprofíbrato) and nicotinic acid derivatives such as acipimox; insulin sensitizing agents including, but not limited to, biguanides such as metformin, PPAR insulin sensitizing agents and thiazolodeniones such as troglitazone and rosiglitazone. Troglitazone, 5 - [[4- [3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H -] - benzopyran-2-yl) methoxy] phenyl] methyl- 3-2.4 -thiazolidinedione V411 (DIABII, Glaucanin) Pioglitazone (ACTOS, AD 4833, U 72107, U 72107A, U 72107E, ZACTOS) Chemical Name: 2,4-Thiazolidinedione, 5 - [[4- [2- (5-ethyl) monohydrochloride -2-pyridinyl) ethoxy] phenyl] methyl], (al-); Rosiglitazone (Avandia, BRL 49653, BRL 49653C) Chemical Name: 2,4-Thiazolidinedione, 5 - [[4- [2- (methyl-2-pyridinylamino) ethoxy] phenyl] methyl]; 25 Bexarotene-oral (LGD 1069 oral, Targretin oral, Targretin, Targretin oral, Targrexin oral) Chemical name: 4- [1- (3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthyl) ethenyl] benzoic acid; ZD 2079, (ICI D 2079) (Chemical name: (R) -N- [2-4- (carboxymethyl) phenoxy] ethyl) -N- (2-hydroxy-2-phenethyl) ammonium chloride: Netoglitazone, ( Isaglitazone, MCC 555, RWJ 241947) (Chemical Name: 5- [6 (2-fluorobenzyloxy) naphthalen-2-ylmethyl] thiazolidin-2,4-dione); INS (D-chiro-inositol) (Chemical Name: D-1, 2,3,4,5,6-hexahydroxycyclohexane), ON 2344 (DRF 2593); Dexlipotam, Chemical Name: 5 (R) - (1, 2-dithiolan-3-yl) pentanoic acid 35; HQL 975, Chemical Name: 3- [4- [2- (5-methyl-2-phenyloxazol-4-yl) ethoxy] phenyl] -2 (S) - (propylamino) propionic acid; IM 268, Chemical Name: 5,5'-methylene-bis (1,4-phenylene) bismethyleneb (thiazolidin-2,4-dione). Among the PPAR I agonists that are being developed include: Reglitazar (JTT 501, PNU 182716, PNU 716) (Chemical name: Isoxazolidien-3,5-dione, and 4 - [[4- (2-phenyl-5- methyl) -1, 3-oxazolyl] ethoxyphenyl-4] methyl, (4RS)); I (RP 297, Chemical Name: 10 5- (2,4-dioxothiazolidin-5-ylmethyl) -2-methoxy-N- [4- (trifluoromethyl) benzylbenzamide; R 119702 (Cl 1037, CS 011) Chemical Name: hydrochloride of (/ -) - 5- [4- (5-methoxy-1H-benzimidazol-2-ylmethoxy) benzyl] thiazolin-2,4-dione; DRF 2189, Chemical Name: 5 - [[4- [2- (1- indolyl) ethoxy] phenyl] methyl] thiazolidin-2,4-dione; inhibitors of cortisol synthesis such as ketoconazole, econazole or miconazole; growth hormones such as somatropin or somatonorm and their derivatives such as fusion protein growth hormone such as ALBUTROPIN; growth hormones with polyethylene glycol, such as pegylated growth hormone in cysteine, BT 005 (Bolder BioTechnology Inc.); growth hormone secretagogues, such as, for example, SM 130686 (Sumitomo) capromorelin (Pfizer), mecasermin (Fujisawa), sermorelin (Salk Institute, Bio-Technology General), somatrem, somatomedin (C Llanterente, Pharmacia Corporation) examorelina, ta bimorelin; CP 464709 (Pfizer), Ll 426410 and Ll 444711 (Lilly); 8- (aminoalkoxyimino) -8H-dibenzo [a, e] triazolo [4,5-cjcicioheptenos as described in WO2002057241, dibenzo [a, e] 1, 2,3-triazolo [4,5-c] [ 7] 2-substituted anulen-8-ones, as described in WO2002056873, growth hormone releasing peptides GHRP-6 and GHRP-1 as described in the US patent No. 4,411,890, and publications WO 89/07110, WO 89/07111, B-HT920, hexarelin and GHRP-2 as described in WO 93/04081, or growth hormone releasing hormone (GHRH, also referred to as GRF) and its analogs, somatomedins including IGF-1 and IGF-2 and its derivatives such as SomatoKine, a recombinant fusion of an insulin-like growth factor and its binding protein, BP-3, alpha-adrenergic agonists such as clonidine, xylazine, detomidine and medetomidine or 5HTID serotonin agonists such as sumitriptan or agents that inhibit somatostatin or its release such as physostigmine and pyridostigmine, TGRF 1-44 (Teratechnologies); L 165166 (Merck &Company); dipeptide derivatives as described in W09858947, inhibitors of dipeptidyl peptidase IV such as aminoacyl pyrrolidine-nitrile as described in US6521644, WO95 / 15309 and W098 / 19998; beta-aminoheterocyclic dipeptidyl peptidase inhibitors such as those described in US20030100563 and WO2003082817; and growth hormone releasing compounds, such as those described in US20030055261, US20030040483, EP 18072, EP 83864, WO 89/07110, WO 89/01711, WO 89/10933, WO 88/9780, WO 83 / 02272, WO 91/18016, WO 92/01711, WO 93/04081, WO 9514666, EP0923539, US patents Nos. 5,206,235, 5,283,241, 5,284,841, 5,310,737, 5,317,017, 5,374,721, 5,430,144, 5,434,261, 5,438,136, 5,494,919, 5,494,920, 5,492,916, 5,536,716 and 5,578,593, WO 94/13696, WO 94/19367, WO 95/03289, WO 95/03290, WO 95/09633, WO 95/11029, WO 95/12598, WO 95/13069, WO 95 / 14666, WO 95/16675, WO 95/16692, WO 95/17422, WO 95/17423, WO 95/34311, and WO 96/02530, piperidines, pyrrolidines and hexahydro-1 H-azepines as described in the documents US5804578, US5783582, WO2004007468, AMIDO-ESPIROPIPERIDINAS such as those described in WO0104119, 2-amino-5-pyrimidine-acetic acid compounds including 2 - [(5,6-dimethyl-2-benzoimidazolyl) amino] -4 -hydroxy-6-methyl-5-pyrimidine-acetic acid (2) and 2 - [(5,6-dimethyl-2-benzoimidadazolyl) amlno] -4-hydroxy-6-methyl-5-pyrimidine-acetic acid ethyl ester , as described in US6329383, benzimidazoles as described in EP1155014, peptidyl analogs related to GRF and the peptides of the patent of E.U.A. 4,411,890, antagonists of gonadotropin-releasing hormone, such as those described in WO0170228, WO0170227, WO0170228, WO0069433, WO0004013, W0995156, W09951595, W09951231-4, W09941251-2, W09921557, W09921553 and 6-AZAINDOL compounds as described in WO0053602, WO0053185, WO0053181, WO0053180, WO0053179, WO0053178, US6288078; IGT-1 secretagogues; insulin-like growth factor 2 (IGF-2 or somatomedin A) and IGF-2 secretagogues; myostatin antagonists that inhibit the receptor 3 of fibroblast growth factor (FGFR-3) tyrosine kinase. The polymeric substances included are also preferably soluble in water at room temperature. A non-limiting list of such polymers includes poly (alkylene oxide) homopolymers such as polyethylene glycol or polypropylene glycol, poly (oxyethylenated polyols), their copolymers and block copolymers, with the proviso that in block copolymers the solubility. As an alternative to PEG-based polymers, effectively non-antigenic materials such as dextran, polyvinylpyrrolidones, polyacrylamides, polyvinyl alcohols, carbohydrate-based polymers and the like can be used. Actually, activation of the a and terminal groups of these polymeric substances can be carried out in manners similar to those used to convert the polyalkylene oxides, and will therefore be apparent to those skilled in the art. Those skilled in the art will understand that the above list is merely illustrative, and that all polymeric materials described herein are contemplated. For the purposes of the present invention, "effectively non-antigens" means all materials that are understood in the art to be non-toxic and do not elicit an appreciable immunogenic response in mammals.

Definitions The following is a list of abbreviations and the corresponding meanings that are used interchangeably herein: g gram (s) mg milligram (s) ml milliliter (s) T.a. ambient temperature PEG polyethylene glycol The complete contents of all publications, patents, and patent applications cited in this description are incorporated herein by reference, as if each publication, patent or individual patent application was specifically and individually indicated to be Incorporates as a reference. Although the above invention has been described in some detail by way of illustration and example for the purpose of being understood clearly, it will be apparent to the person skilled in the art, in light of the teachings of this invention, that changes can be made and modifications without departing from the spirit and scope of the present invention. The following examples are provided for exemplary purposes only, and are not intended to limit the scope of the invention, which has been described above in general terms. In the following examples, hGH is that of SEQ ID NO: 1. It is understood that other members of the polypeptide family of hGH or its agonist variant can also be pegilated in a similar manner as exemplified in the following examples.

EXAMPLES EXAMPLE 1 PEGfranked from PM 40,000) -butyrylaldehyde-hGH This example demonstrates a method for generating substantially homogeneous preparations of mono-glycated hGH at the N-terminus by reductive alkylation. The branched methoxy-PEG-butyrylaldehyde reagent of about 40,000 MW (Shearwater Corp.) was selectively coupled by reductive amination to the N-terminus of hGH, taking advantage of the difference in the relative pKa value of the primary N-terminal amine versus the N-terminus. pKa values of the primary amines at the e-amino position of the lysine portions. The dissolved hGH protein was reacted at a concentration of 10 mg / ml in 25 mM Hepes (Sigma Chemical, St. Louis, MO) pH 7.0, (optionally 25 mM MES (Sigma Chemical, St. Louis, MO) pH 6.0, 10 mM sodium acetate (Sigma Chemical, St. Louis, MO) pH 4.5), with methoxy-PEG-butyrylaldehyde, M-PEG-ALD, (Shearwater Corp., Huntsville, AL) by the addition of M-PEG-ALD , to give a molar ratio of PEG: relative hGH of 2: 1. The reactions were catalyzed by addition of stock solution of 1 M NaCNBH4 (Sigma Chemical, St. Louis, MO), dissolved in H20, to a final concentration of 10-50 mM. The reactions were carried out in the dark at T.a. for 18-24 hours. The reactions were stopped by the addition of 1 M Tris (Sigma Chemical, St. Louis, MO), pH 7.6, to a final concentration of 50 mM Tris, or diluted in the appropriate pH buffer for immediate purification. Table 1 shows the percentage, determined by size exclusion chromatography, of the rriulti-PEGylated species, mono-PEGylated conjugate, unreacted PEG, and final purification performance of the PEG (branched 40K) -aldehyde and PEG (branched 40K ) -butyrylaldehyde. PEG-butyrylaldehyde results in more mono-PEGylated conjugate, lower levels of unreacted PEG, and higher final yield compared to PEG-aldehyde.

TABLE 1 EXAMPLE 2 PEG linear chain PM 30,000) -butyrylaldehyde-Hqh The methoxy-PEG reagent (linear MW 30,000) -butyrylaldehyde is coupled to the N-terminus of the hGH using the procedure described in Example 1.

EXAMPLE 3 PEG (linear chain MW 20,000) -butyrylaldehyde-hGH The methoxy-PEG reagent (line I MW 20,000) -butyrylaldehyde is coupled to the N-terminus of hGH using the procedure described in Example 1.

EXAMPLE 4 Purification of the pegylated hGH The pegylated hGH species were purified from the reaction mixture at > 95% (SEC analysis) using a single step of ion exchange chromatography.

Anion exchange chromatography The pegylated hGH species were purified from the reaction mixture at >95% (SEC analysis) using a single step of anion exchange chromatography. Monpegilated hGH was purified from the unmodified hGH species and multipegilated hGH using anion exchange chromatography. A typical PEG (20K) -butyrylaldehyde-hGH reaction mixture (5-100 mg protein) was purified, as described above on a Q-Sepharose Hitrap column (1 or 5 ml) (Amersham Pharmacia Biotech, Piscataway , NJ) or rapid flow Q-Sepharose column (26/20, 70 ml bed volume) (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in 25 mM HEPES, pH 7.3 (pH Regulator A). The reaction mixture was diluted 5-10X with buffer pH A and loaded onto the column with a flow rate of 2.5 ml / min. The column was washed with 8 column volumes of the pH regulator A. Subsequently, the different hGH species of the column were eluted in 80-100 column volumes of pH A regulator and a linear NaCl gradient of 0-100 mM. . The eluent was monitored by absorbance at 280 nm (A280) and 5 ml fractions were collected. The fractions were mixed according to the degree of pegylation, e.g. eg, mono, di, tri, etc. (evaluated as in example 15). The mixture was then concentrated to 0.5-5 mg / ml in a Centriprep YM10 concentrator (Amicon, Technology Corporation, Northborough, MA). The protein concentration of the mixture was determined by the A28o using an extinction coefficient of 0.78.

Cation exchange chromatography Cation exchange chromatography is carried out on a high performance SP Sepharose column (Pharmacia XK 26/20, bed volume 70 ml) equilibrated in 10 mM sodium acetate pH 4.0 (pH B regulator). The reaction mixture is diluted 10X with pH B regulator and loaded into the column at a flow rate of 5 ml / min. The column is then washed with 5 column volumes of the pH B regulator, followed by 5 column volumes of 12% pH C regulator (10 mM acetate pH 4.5, 1 M NaCl). Subsequently, the PEG-hGH species are eluted from the column with a linear gradient of pH C regulator at 12 to 27% in 20 column volumes. The eluent is monitored at 280 nm and 10 ml fractions are collected. The fractions are combined according to the degree of pegylation (mono, di, tri, etc.), exchanged in pH buffer of 10 mM acetate pH 4.5, and concentrated to 1-5 mg / ml in a stirred cell provided of an Amicon YM10 membrane. The protein concentration of the mixture is determined by A280 nm using an extinction coefficient of 0.78.

EXAMPLE 5 Biochemical characterization Mixtures of purified pegylated hGH were characterized by non-reducing SDS-PAGE, non-denaturing size exclusion chromatography, and peptide mapping.

High resolution liquid chromatography of size exclusion (SEC-HPLC) Non-denaturing SEC-HPLC The methoxy-PEG reaction was evaluated with different chemical bonding methods, sizes, connectors and geometries with the hGH, the anion exchange purification mixtures and the final purification products, using non-denaturing SEC-HPLC. The non-denaturing analytical SEC-HPLC was carried out using a Superdex 200, 7.8 mm X 30 cm column (Amersham Pharmacia Biotech, Piscataway, NJ) in 20 mM phosphate pH 7.2, 150 mM NaCl, with a flow rate of 0.5 ml / minute (optionally Tosohaas G4000PWXL Amersham Pharmacia Biotech, Piscataway, NJ). PEGylation greatly increases the hydrodynamic volume of the protein resulting in a shift to a shorter retention time. Moving species were observed in the reaction mixtures of PEG-aldehyde-hGH together with hGH. These PEGylated and non-PEGylated species were separated in chromatography with Q-Sepharose, and the resulting purified mono-PEG-aldehyde-hGH species, were subsequently shown to elute as a single peak in the non-denaturing SEC (> 95% purity). ). The Q-Sepharose chromatography step effectively separated the free PEG, hGH, and the multi- PEGylated hGH species from the mono-Pegylated hGH.

Denaturing SEC-HPLC The reaction of the polyethylene glycols-butyrylaldehyde with hGH, the anion exchange purification, and the purified final products were evaluated using the denaturing SEC-HPLC. Analytical denaturing SEC-HPLC is carried out using a 7.8 mm X 30 cm Tosohaas 3000SWXL column (Tosohaas Pharmacia Biotech, Piscataway, NJ) in 100 mM phosphate pH 6.8, 0.1% SDS at a flow rate of 0.8 ml / minute. PEGylation greatly increases the hydrodynamic volume of the protein resulting in a shift of a shorter retention time. The PEGylated and non-PEGylated species are separated by chromatography on Q-Sepharose.

SDS PAGE / PVDF transfer SDS-PAGE was used to evaluate the reaction of PEG-butyrylaldehyde with hGH, and the purified final products. SDS-PAGE was carried out in 10-20% Tris-tricine gels 1 mm thick (Invitrogen, Carlsbad, CA) under reducing and nonreducing conditions, and stained using the Novex Colloidal Coomassie® staining equipment. G-250 (Invitrogen, Carlsbad, CA). The bands are transferred to a PVDF membrane for subsequent identification of the N-terminal sequence.

Analytical anion exchange HPLC The reaction mixture of PEG-butyrylaldehyde / hGH, the purification fractions by anion exchange, and the final purified products were evaluated using analytical anion exchange HPLC. Anionic analytical exchange HPLC was carried out using an anion exchange column Tosohaas Q5PW or DEAE-PW, 7.5 mm X 75 mm (Tosohaas Pharmacia Biotech, Piscataway, NJ) in 50 mM Tris, pH 8.6 with a flow rate of 1 ml / min. The samples were eluted with a linear gradient of 5-200 mM NaCl.

N-terminal sequence and peptide mapping Automated Edman degradation chemistry was used to determine the sequence of the N-terminal protein. An Applied Biosystems Model 494 Procise sequencer (Perkin Elmer, Wellesley, MA) was used for degradation. The respective PTH-AA derivatives were identified by RP-HPLC analysis in a serial fashion using an Applied Biosystems Model 140C PTH analyzer equipped with a Perkin Elmer / Brownlee 2.1 mm d.i. column, PTH-C18. Digestions with trypsin were carried out at a concentration of 1 mg / ml and typically 50 μg of material was used per digestion. Trypsin was added so that the ratio of trypsin to PEG-hGH was 1: 30 (w / w). The tris pH regulator was present at a concentration of 30 mM, pH 7.5. The samples were incubated at room temperature for 16 ± 0.5 hours. The reactions were quenched by the addition of 50 μl of 1 N HCl per ml of digestion solution. The samples were diluted, before placing the samples in the sample autoinjector, with a final concentration of 0.25 mg / ml in 6.25% acetonitrile. First acetonitrile (up to 19.8% acetonitrile) was added, mixed well, and then water was added to the final volume (four times the initial volume). The additional digestion solution can be separated and stored for up to 1 week at -20 ° C. A Waters Alliance 2695 HPLC system was used for the analysis, but other systems would produce similar results. The column used was a 25 cm x 4.6 mm Astee C-4 polymer column with 5 μm particles. The experiments were carried out at room temperature with a typical load of 50 μg of protein per sample. The pH regulator A is trifluoroacetic acid in 0.1% water; the pH regulator B is trifluoroacetic acid in 0.085% acetonitrile. The gradient was as follows: Time% A% B% C% D Flow Rate Curve 0. 00 0.0 0.0 100.00 0.0 1,000 1 90.00 0.0 0.0 55.00 45.00 1,000 6 90.10 0.0 0.0 0.0 100.00 1,000 6 91.00 0.0 0.0 0.0 100.00 1,000 6 91.10 0.0 0.0 100.00 0.0 1,000 6 95.00 0.0 0.0 100.00 0.0 1,000 6 The column is heated to 40 ° C using a heating jacket The maximums were detected using a Waters 996 PDA detector that collects data between 210 and 300 nm. The chromatogram obtained at 214 nm was used for the analysis of the sample.

Triptych maps were made for hGH, PEG (branched 40K) -aldehyde, and PEG (branched 40K) -butyrylaldehyde (Figures 1A, 1B, 1C). The N-terminal tryptic fragment was designated T-1. Table 2 shows the percentage of T-1 present compared to hGH without PEGylar. These data suggest that 90% of the PEG modification is at the N-terminus, with the portion apparently bound to one of the different possible lysine portions, when PEG-aldehyde is used compared to more than 98% at the N-terminus when PEG-butyrylaldehyde is used.

TABLE 2 EXAMPLE 6 Pharmacodynamic studies Weight gain in the rat Female Sprague Dawley rats, hypophysectomized in Taconic Labs, were previously classified according to the growth rate during a period of 7 to 11 days. The rats were divided into groups of eight. Group 1 consisted of rats given daily or day 0 and day 6 subcutaneous dose of vehicle. In group 2, a daily subcutaneous dose of GH (30 μg / rat / dose) was administered. In group 3, subcutaneous doses of GH were administered on day 0 and day 6 (180 μg / rat / dose). In group 4, subcutaneous doses of PEG (branched 40k) -butyrylaldehyde-hGH were administered on day 0 and 6 (180 μg / rat / dose). Weight gain, weighing at least on alternate days during the study. Figures 3 and 4.

Length of the rat tibia The animals of the 11-day weight gain studies were sacrificed on day 11, the left tibias were removed and analyzed by X-rays, and the lengths of the bones were measured using a caliper. Fig. 5 IGF-1 studies Animals from the six-day weight gain studies were used. Blood samples were taken at different times during the study, and serum IGF-1 levels were determined by ELISA. Figure 6 Serum biochemical studies Animals from the weight gain studies of 11 days to evaluate the serum biochemical values on day 7 after the cumulative administration of 1.8 mg / kg on day 0 and 6, as shown in table 3.

TABLE 3 hGH PEG-hGH Vehicle Test (11 days with 300μg / kg / day) (1.8 mg / kg on day 0 and 6) ALB 4.07 + 0.04 4.06 ± 0.05 4.18 ± 0.03 * t (g / dL) FAL 311 ± 15 309 ± 16 280 ± 14 (U / L) ALT 53.6 + 2.4 51.1 + 2.2 51.3 ± 2.6 (U / L) AST 149 ± 7 131 ± 4 137 ± 11 (U / L) BUN 37.4 ± 1.6 26.7 ± 1.5 * 27.9 + 0.6 * (mg / dL) Ca2 + 10.9 + 0.1 11.5 + 0.1 * 11.0 + 0.1 1 (mg / dL) Col 85.1 + 2.8 76.9 + 3.6 * 115.3 + 2.8 * t (mg / dL) CRE 0.62 ± 0.02 0.60 + 0.01 0.59 + 0.01 (mg / dL) Glucose 73.4 ± 4.7 79.6 ± 4.7 67.1 ± 8.9 (mg / dL) Fos 8.26 ± 0.28 9.59 ± 0.16 * 8.42 + 0.23t (mg / dL) PT 6.36 ± 0.10 6.48 ± 0.10 6.39 ± 0.06 (mg / dL) ABT 10.0 ± 0.5 7.6 ± 0.4 * 11.0 ± 0.4t (μmol / L) SDH 11.9 ± 1.0 9.7 ± 1.4 10.6 ± 0.7 (U / L) Triglycerides 57.4 ± 4.0 47.8 ± 5.7 45.7 ± 3.9 * (mg / dL) LDH 786 ± 72 762 ± 94 914 ± 189 (U / L) Na + 147 ± 0.5 146 ± 0.8 145 ± 0.6 * (mmol / L) K + 5.94 ± 0.09 6.31 + 0.16 * 6.71 + 0.17 * t (mmol / L) CI "103 ± 0.5 102 ± 0.5 102 ± 0.6 (mmol / L) * p <0.10 vs. vehicle, 'p < 0.05 vs Hgh ALB, albumin; FAL, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; Ca2 +, calcium; Cabbage, cholesterol; CRE, creatinine Fos, phosphate; PT, toral protein; ABT, total bile acid; SDH, sorbitol dehydrogenase; LDH lactic dehydrogenase; Na +, sodium:; ? +, potassium; CI ", chloride.

The nitrogen urea concentration in blood on day 11 decreased significantly (p <0.10) to the same extent with respect to the vehicle group in the animals treated with hGH and PHA-794428. This indicates the increased use of nitrogen as a result of the new protein synthesis during enhanced growth.

Pharmacokinetic studies Pharmacokinetic studies were carried out on male rats Normal Sprague-Dawley cannulated. The injections were administered as a single subcutaneous bolus of 100 μg / kg / rat of GH p PEG-GH using six rats per group. Blood samples were taken for one to five days as appropriate for evaluation of the relevant FC parameters. Blood levels of GH and PEG-GH were monitored in each sample using immunoassay.

Immunoensavo of hGH The concentration levels of protein hGH and pegylated hGH in the plasma of mouse and monkey cynomologus were determined, using the fluorescence immunoassay with the AutoDELFIA hGH (PerkinElmer). The levels of IGF-1 in rat and human were controlled by the immunoassay equipment (Diagnostic System Laboratories).

Non-compartmental pharmacokinetic properties for the hGH-PEG conjugate of Example 1 in non-human primates The hGH-PEG conjugate of Example 1 was administered to cynamologus monkeys as intravenous (v) or subcutaneous (s) bolus injections of 0.18 mg / kg (Table 4). The FC parameters were determined using average data for n = 3 animals. Concentrations in the plasma were measured using the fluorescence immunoassay autoDELFIA kit (PerkinElmer) and a predetermined standard curve for the PEG-GH conjugate.

TABLE 4

Claims (11)

NOVELTY OF THE INVENTION REVIVAL DICTION EN

1. - The use of a hGH modified with polyethylene glycol having the structure of formula I or II, Formula I or mPEG-0 (CH2CH20) n (CH2) lt, CHrNH- Formula II wherein n is an integer between 1 and 10; m is an integer between 1 and 10; R is the human growth hormone or methionyl-growth hormone, as a pharmaceutical product.

2. The use of a hGH modified with polyethylene glycol having the structure of formula I or II, Formula I or mPEG-0. { CH2CH2O) n (CH2) mCH2-NH-R Formula II wherein n is an integer between 1 and 10; m is an integer between 1 and 10; R is the human growth hormone or methionyl-growth hormone, alone or combined with another therapeutic agent, in the manufacture of a medicament for the prevention and / or treatment of a disease or disorder in which the use of growth hormone is beneficial, wherein said disease or disorder is selected from the group consisting of growth hormone deficiency (DHC) ), hormone deficiency of growth in adults (DHCa), Turner syndrome, delay in Growth in children who were born small for gestational age (PEG), Prader-Willi syndrome (PWS), chronic renal failure (CRF), injury traumatic brain injury, subarachnoid hemorrhage, Noonan syndrome, idiopathic low stature (EBI), very low birth weight (VLBW), short stature due to the treatment of children with glucocorticoids, and growth retardation in low preterm infants.

3. The use as claimed in claim 2, wherein said disease or disorder in which the use of GH is beneficial, is selected from the group consisting of DHC, DHCa, Turner syndrome, PEG, SPW, IRC and EBI.

4. The use as claimed in any of claims 1 to 3, wherein n is equal to 4 and m is equal to 3.

5. The use as claimed in claim 4, wherein said hGH modified with polyethylene glycol has the structure of formula I, with n equal to 4 and m equal to 3.

6. The use as claimed in claim 1 or 2, wherein said human growth hormone comprises the amino acid sequence of SEQ ID NO:

7. The use as claimed in claim 6, wherein more than 90% of said polyethylene glycol is conjugated to an amino terminal phenylalanine of the amino acid sequence of SEQ ID NO: 1. 8.- Use as claims in claim 7, wherein more than 95% of said polyethylene glycol is conjugated to an amino terminal phenylalanine of the amino acid sequence of SEQ ID NO: 1. 9. The use as claimed in claim 1 or 2, in where each mPEG has a molecular weight of approximately 20 kDa. 10. A composition comprising a conjugate of human growth hormone-PEG of formula I or II combined with another therapeutic agent, and at least one pharmaceutically acceptable carrier Formula I or mPEG-0 (CH2CH2O) n (CH2) II, CHrNH-R Formula II wherein n is an integer between 1 and 10; m is an integer between 1 and 10; R is the human growth hormone or methionyl-growth hormone. 11. The composition according to claim 10, further characterized in that said hGH modified with polyethylene glycol has the structure of formula I with n equal to 4 and m equal to 3.