HK1098360A - N-terminally monopegylated human growth hormone conjugates, process for their preparation, and methods of use thereof - Google Patents

Description

N-terminal mono-pegylated human growth hormone conjugates, methods for making them, and methods of use thereof

The present invention claims priority from U.S. application N ° 10/771895, filed 2, month 4, 2004, which is hereby incorporated by reference in its entirety as if written herein.

Technical Field

[001] The present invention relates to chemical modifications, including pegylation of human growth hormone (hGH) and agonist variants thereof, by which chemical and/or physiological properties of hGH can be altered. The pegylated hGH may have increased plasma persistence duration, reduced clearance, increased stability, reduced antigenicity, reduced polyethylene glycol heterogeneity, or a combination thereof. The invention also relates to methods for the modification of hGH. Furthermore, the present invention relates to pharmaceutical compositions comprising the modified hGH. A further embodiment is the use of the modified hGH for the treatment of growth and development disorders.

Background

[002] Human growth hormone (hGH) is a protein comprising a single chain of 191 amino acids cross-linked by two disulfide bridges and has a molecular weight of 22kDa in the monomeric form. Human GH is secreted by the pituitary gland and can also be produced by recombinant genetic engineering. hGH will cause growth in all body tissues capable of growth. hGH plays an important role not only in promoting growth during the growth phase in humans but also in maintaining normal body composition, anabolism and lipid metabolism (k.barneis. and u.keller, Baillieres clin.endocrino.meta.10: 337 (1996)).

[003]Recombinant hGH has been commercially available for many years. There are two therapeutically useful types of recombinant hGH formulations on the market: authentic recombinant hGH, e.g., Genotropin^TMOr Nutropin^TMAnd analogs having an additional methionine residue at the N-terminus such as somatomerm^TM. hGH is useful for stimulating linear growth in patients with hypopituitary dwarfism, also known as Growth Hormone Deficiency (GHD), or with turner's syndrome, but is also proposed for other indications,including long term treatment of growth deficiency in children born at Small Gestational Age (SGA), for treating patients with Parnewidi syndrome (PWS), Chronic Renal Insufficiency (CRI), AIDS wasting and aging. Adult growth hormone deficient (aGHD) patients have a variety of problems, such as characteristic changes in body composition including increased fat mass, lean body mass and decreased extracellular fluid, as well as decreased bone mineral density, abnormal lipid metabolism and cardiovascular dysfunction. Many of these problems can be ameliorated by hGH supplementation therapy (j.verhelst J and r.abs.drugs.; 62: 2399 (2002)).

[004] One of the major biological effects of Growth Hormone (GH) is the promotion of growth in young mammals and the maintenance of tissue in older mammals. The organ systems affected include bone, connective tissue, muscle, and internal organs such as the liver, intestine, and kidney. Growth hormones exert their effects by interacting with specific receptors on the membrane of the target cell. hGH is a member of a family of homologous hormones, including placental lactogen, prolactin, and other gene and species variants or growth hormones (Nicoll, c.s. et al (1986) endocrinereviewss 7: 169). Among these hormones, hGH differs in that it exhibits broad species specificity and binds to a cloned somatotropin (Leung, D.W. et al, [1987] Nature 330; 537) or a receptor for prolactin (Boutin, J.M. et al, [1988] Cell; 53: 69). The cloned Gene of hGH has been expressed in a secretory form in Escherichia coli (Escherichia coli) (Chang, C.N. et al, [1987] Gene 55: 189), and its DNA and amino acid sequences have been reported (Goeddel, et al, [1979) Nature 281: 544; gray et al [1985] Gene 39: 247).

[005] Human growth hormone (hGH) is involved in a number of normal human growth and development regulations. This pituitary hormone shows a number of biological effects including linear growth (somatization), lactation, activation of macrophages, and also insulin-like and diabetogenic effects (Chawla, R, K. (1983) Ann. Rev. Med.34, 519; Edwards, C.K., et al, (1988) Science239, 769; Thomer, M.O., et al, (1988) J.Clin. invest.81: 745). Growth hormone deficiency in children results in dwarfism, a disease that has been successfully treated by exogenous administration of hGH for over twenty years.

[006] In adults as well as children, hGH maintains normal body composition by increasing nitrogen accumulation and stimulating skeletal muscle growth and through mobilization of body fat. Visceral adipose tissue is particularly sensitive to hGH. In addition to enhancing lipolysis, hGH decreases the uptake of triglycerides into body fat stores. Serum concentrations of IGF-I (insulin-like growth hormone-I) and IGFBP3 (insulin-like growth hormone binding protein 3) were increased by hGH.

[007] Human growth hormone (hGH) is a single chain polypeptide (molecular weight 21,500) consisting of 191 amino acids. Disulfide linkages are located at 53 and 165 and at 182 and 189. Niall, Nature, New Biology, 230: 90(1971). hGH is a potent anabolic agent, particularly because of the accumulation of nitrogen, phosphorus, potassium, and calcium. Treatment of pituitary-excised rats with GH restores the growth rate of at least a portion of the rats. Moore et al, Endocrinology 122: 2920-2926(1988). Among them, its most significant effect in hypopituitary (GH-deficient) subjects is the acceleration of bone-growth plate-cartilage linear growth, leading to increased height. Kaplan, Growth Disorders in Children and Adolescents (Springfield, IL: Charles C.Thomas, 1964).

[008] hGH causes a variety of physiological and metabolic effects in a variety of animal models, including linear bone growth, lactation, macrophage activation, insulin-like and diabetogenic effects, etc. (R.K. Chawla et al, Annu. Rev. Med.34: 519 (1983); O.G.P.Isaksson et al, Annu. Rev. physiol.47, 483 (1985); C.K. Edwards et al, Science239, 769 (1988); M.O.Thomer and M.L.Vance, J.Clin.invest.82: 745 (1988); J.P.Hughes and H.G.Friesen, Ann.Rev.physiol.47: 469 (1985)). GH secretion has been reported to decline with age, particularly in postmenopausal women. Millard et al, neurobiol. aging, 11: 229-; takahashi et al, Neuroendocrinology M, L6-137-142 (1987). See also Rudman et al, j.clin.invest, 67: 1361-1369(1981) and Blackman, endocrinologiund Aging, 16: 981(1987). Furthermore, it has been reported that some of the signs of aging, including reduced lean body mass, expansion of adipose tissue mass, and thinning of the skin, can be reduced by treatment with GH three times a week. See, e.g., Rudman et al, n.eng.j.med., 323: 1-6(1990) and together with the article by Dr Vance in the same journal phase (pages 52-54). These biological effects result from the interaction of hGH and specific cellular receptors. Two different human receptors, the hGH liver receptor (D.W. Leung et al, Natuire 330: 537(1987)), and the human prolactin receptor (J.M. B.B.U.M., mol. Endocrinology.3: 1455(1989)) have been cloned. However, other receptors are possible, including the human placental lactogen receptor (M.Freeman, M.Comer, G.Komer and S.Handwerger, Endocrinol.120: 1865 (1987)). These cognate receptors contain a glycosylated extracellular hormone binding domain, a single transmembrane domain, and a cytoplasmic domain that vary considerably in sequence and size. One or more receptors are thought to play a crucial role in the physiological response to hGH.

[009] It is generally observed that physiologically active proteins administered into the body can show their pharmacological activity only for a short period of time due to their high clearance in vivo. Moreover, the relative hydrophobicity of these proteins may limit their stability and/or solubility.

[0010] In order to reduce the clearance rate, improve the stability or eliminate the antigenicity of therapeutic proteins, methods have been proposed in which the protein is chemically modified with a water-soluble polymer. This type of chemical modification can effectively block the proteolytic enzyme from physical contact with the protein backbone itself, thus preventing degradation. Chemical attachment of certain water-soluble polymers can effectively reduce renal clearance by increasing the hydrodynamic volume of the molecule. Additional advantages include, in some cases, increased stability and circulation time of the therapeutic protein, increased solubility and decreased immunogenicity. Poly (alkylene oxide), particularly poly (ethylene glycol) (PEG), is one such chemical moiety that has been used in the preparation of therapeutic protein products (the verb "pegylated" means the attachment of at least one PEG molecule). The attachment of poly (ethylene glycol) has been shown to prevent proteolysis, Sada, et al, j.fermentative bioengineering 71: 137, 139(1991), and methods for attaching certain poly (ethylene glycol) moieties can be provided. See U.S. Pat. No.4,179,337, 1979, 12/18, Davis et al, "Non-Immunogenic Polypeptides"; and U.S. Pat. No.4,002,531, Royer, "ModifyingEnzymes with Polyethylene Glycol and Product Produced Thereby", published 11.1.1977. For a review see Abuchowski et al, Enzymes as Drugs (edited by J.S. Holdererg and J.Roberts, pp.367-383 (1981)).

[0011] Other water soluble polymers such as ethylene/propylene glycol, carboxymethylcellulose, dextran, poly (vinyl alcohol), poly (vinyl pyrrolidone), poly (-1, 3-dioxolane), copolymers of poly (-1, 3, 6-trioxane), ethylene/maleic anhydride copolymers, poly-amino acids (homopolymers or random copolymers) are also used.

[0012]Many examples of pegylated therapeutic proteins have been described. ADAGEN^®A pegylated formulation of adenosine deaminase, has been approved for the treatment of severe combined immunodeficiency disorders. ONCASPAR^®I.e. a pegylated L-asparaginase, has been approved for the treatment of allergic allergy patients. Pegylated superoxide dismutase has been in clinical trials for the treatment of craniocerebral injuries. Pegylated interferon-alfa (U.S.5,738,846, 5,382,657) has been approved for the treatment of hepatitis; pegylated glucocerebrosidase and pegylated hemoglobin have been reported to have been in preclinical testing. Another example is PEGylated IL-6, EF 0442724, entitled "Modified hIL-6", which discloses poly (ethylene glycol) added to IL-6.

[0013] Another specific therapeutic protein that has been chemically modified is granulocyte colony stimulating factor (G-CSF). G-CSF induces rapid proliferation and release of neutrophils into the bloodstream and thus provides anti-infective efficacy. European patent publication EP 0401384 entitled "chemical Modified granular Stimulating Factor" published on 12.1990 describes materials and methods for preparing G-CSF to which poly (ethylene glycol) is attached. Modified G-CSF and analogs thereof are also reported in EP 0473268 entitled "Continuous Release pharmaceutical compositions Comprising a polymeric conjugate ToA Water Soluble Polymer", published on 3.4.1992, which describes the use of various G-CSF and derivatives Covalently attached to a Water Soluble particulate Polymer such as poly (ethylene glycol). Modified polypeptides having human granulocyte colony stimulating factor activity are reported in EP 0335423 published at 10/4 in 1989. U.S. Pat. No.5,824,784 provides methods for N-terminally modifying proteins or analogs thereof and compositions resulting therefrom, including novel N-terminally chemically modified G-CSF compositions. U.S. Pat. No.5,824,778 discloses chemically modified G-CSF.

[0014] For poly (ethylene glycol), a number of methods have been used to attach poly (ethylene glycol) molecules to proteins. Typically, poly (ethylene glycol) molecules are attached to proteins through reactive groups on the proteins.

[0015] Amino groups such as those at the lysine residue or at the N-terminus are suitable for such attachment. For example, Royer (U.S. patent No.4,002,531, supra) claims that a reductive alkylation reaction be used to attach poly (ethylene glycol) to the enzyme. Chamow et al, Biocon jungetechem.5: 133-140(1994) reported the modification of CD4 immunoadhesins with monomethoxy poly (ethylene glycol) acetaldehyde by reductive alkylation. The authors reported that 50% of CD4-Ig was modified with MePEG under conditions that allowed control of the degree of pegylation. As above, at page 137. The authors also reported that the in vitro binding capacity of the modified CD4-Ig (binding to protein gp 120) decreased at a rate related to the degree of pegylation (MePEGylation) as above. U.S. Pat. No.4,904,584, Shaw, published 2/27 of 1990, relates to the modification of the number of lysine residues in proteins used to link poly (ethylene glycol) molecules via reactive amino groups.

[0016] WO 93/00109 relates to a method for stimulating GH sensitive tissue in mammals or birds comprising maintaining a continuous, effective plasma GH concentration for 3 or more days. One method of achieving such plasma concentrations is said to be through the use of GH conjugated to a macromolecular substance such as PEG (polyethylene glycol). Binding to macromolecular species is said to result in an increase in half-life. WO 93/00109 has reported pegylated human growth hormone using mPEG acetaldehyde-5000 and mPEG N-hydroxysuccinimide ester (mPEG-NHS-5000). Using mPEG-NHS resulted in a heterogeneous mixture of pegylated forms of various hGH. WO 93/00109 also discloses the use of mPEG-maleimide to PEGylate cysteine hGH variants.

[0017] WO 99/03887 discloses pegylated cysteine variants of human growth hormone. This conjugate, designated BT-005, is said to be more effective than hGH in stimulating weight gain and to have a longer half-life in growth hormone deficient rats.

[0018]Clark et al (Journal of Biological Chemistry 271: 21969-21977, 1996) also reported pegylated human growth hormone using succinimidyl esters of carboxymethylated PEG. Clark et al describe size-enhanced hGH derivatives using mPEG-NHS-5000, which selectively bind to primary amines. Increased levels of PEG modification decreased affinity for its receptor and increased EC in cell-based assays₅₀Up to 1500 times. Olson et al, Polymer Preprints 38: 568-569, 1997 disclose the use of N-hydroxysuccinimide (NHS) PEG and Succinimidyl Propionate (SPA) PEG to obtain various PEGylated hGH.

[0019] WO 94/20069 discloses previously pegylated hGH as part of a formulation for pulmonary delivery.

[0020] US 4,179,337 discloses a process for pegylating enzymes and hormones to obtain physiologically active non-immunogenic, water-soluble polypeptide conjugates. GH is mentioned as an example of a hormone to be PEGylated.

[0021] EP458064 a2 discloses pegylation of an introduced or naturally occurring cysteine residue in growth hormone. EP 458064A 2 further mentions the integration of two cysteines into a loop known as the Ω -loop at the wild-type bovine growth hormone residue 102-112, more specifically EP 458064A 2 discloses the replacement of residues 102 and 112 of bovine growth hormone with Ser for Cys and Tyr for Cys, respectively.

[0022] WO 95/11987 proposes attaching PEG to the thio group of a cysteine either present in the parent molecule or introduced by directed mutagenesis. WO 95/11987 relates to PEGylation of protease linker-1, however PEGylation of hGH and other proteins in general has also been proposed.

[0023] WO 99/03887 discloses, for example, the modification of growth hormone by the insertion of additional cys 25 serine residues and the attachment of PEG to the introduced residues.

[0024] WO 00/42175 relates to a method for producing proteins containing free cysteine residues for PEG attachment. WO 00/42175 discloses the following mutants of hGH: T3C, S144C and T148C and their cysteines were pegylated.

[0025] WO 97/11178 (and also US 5849535, US 6004931 and US 6022711) relates to the use of GH variants as agonists or antagonists of hGH. WO 97/11178 also discloses pegylation of hGH, including lysine pegylation and introduction or substitution of lysine (e.g. K168A and K172R). WO 9711178 also discloses a G120K substitution.

[0026] WO 03/044056 discloses various pegylated hghs, including branched 40K PEG acetaldehyde hGH conjugates.

[0027] Previous reports of PEGylated hGH required multiple PEG linkages to achieve hydrodynamic volumes greater than the 70K molecular weight split point of renal filtration as described (Knauf, M.J., et al, J.biol.chem.263: 15064-15070, 1988), but multiple PEG linkages resulted in undesirable product heterogeneity.

[0028] Administration of rhGH is currently performed daily for long periods, and thus less frequent administration would be highly desirable. hGH molecules with longer circulating half-lives will reduce the number of required uses and potentially provide superior therapeutic hGH levels, while enhancing therapeutic efficacy.

[0029] Despite many attempts to pegylate hGH, there is still a need for pegylated hGH molecules with suitable properties to be promising commercial products. The present invention provides PEG-hGH conjugates with a single PEG preferentially attached at the N-terminal phenylalanine of hGH, which conjugates have advantages over other PEG-hGH conjugates. The use of mPEG acetaldehyde-5000 or mPEG N-hydroxysuccinimide ester (mPEG-NHS-5000) to attach various low molecular weight (5kd) PEGs at the alpha-or epsilon-amino sites (N-terminal and 9 lysines in hGH) has been described in WO 93/00109, Clark et al (Journal of Biological Chemistry 271: 21969-. This leads to a heterogeneous population. For example, an hGH with 9 lysines may have some molecules with 10 PEG linkages, some with 9 linkages, some with 8 linkages, some with 7 linkages, some with 6 linkages, some with 5 linkages, some with 4 linkages, some with 3 linkages, some with 2 linkages, some with 1 linkage, and some without PEG linkages. Also, in a molecule with several PEGs, the PEGs may not be attached at the same position on different molecules. This resulting heterogeneity is disadvantageous in the manufacture of conjugates, purification, and identification difficulties, high costs, and high unrepeatability when developing therapeutic products. Another approach (WO 00/42175) employs hGH variants containing a free cysteine residue for PEG attachment. However, this approach can result in incorrectly folded proteins with incorrectly paired disulfide bonds and in heterogeneous pegylation products where PEG is attached to some or all of the cysteines. Attachment of multiple PEGs to multiple sites can result in less stable associations between the PEG and the different sites, which can dissociate at different rates. This makes it difficult to predict the pharmacokinetics of the product accurately, resulting in inaccurate dosing. Heterogeneous products also suffer from adverse problems in the regulatory approval (regulatory approval) for the acquisition of therapeutic products.

[0030] Thus, it would be desirable to obtain a pegylated hGH molecule having a single PEG attached at a single site. The present invention addresses this need in a number of ways.

Summary of The Invention

[0031] The present invention relates to chemically modified hGH and agonist variants thereof having at least one enhanced chemical or physiological property selected from, but not limited to, reduced clearance, increased plasma persistence duration, increased stability, increased solubility, and reduced antigenicity. Thus, as described in more detail below, the present invention has a number of aspects relating to chemical modification of polypeptides, including but not limited to hGH and agonist variants thereof, as well as to specific modification using poly (ethylene glycol) butyraldehyde moieties.

[0032] The invention also relates to methods of producing chemically modified hGH and agonist variants thereof. In particular, the present invention relates to methods of using butyraldehyde to produce chemically modified hGH, which results in greater N-terminal ligation selectivity.

[0033] The present invention also relates to compositions comprising chemically modified hGH and agonist variants thereof alone or in combination with other therapeutic agents.

[0034] The present invention also relates to the use of the chemically modified hGH and agonist variants thereof of the present invention alone or in combination with other therapeutic agents for the prevention and/or treatment of conditions and/or diseases in which GH therapy is useful.

Brief description of the drawings

[0035] FIG. 1 is an HPLC trace of trypsin mapping analysis of the reaction of hGH with 40K branched butyraldehyde hGH or with 40K branched acetaldehyde hGH. The top panel is the trypsin pattern of 40K branched butyraldehyde hGH. The middle panel is the trypsin map of 40K branched acetaldehyde hGH. The bottom panel is the trypsin map of non-pegylated hGH. T1 is an N-terminal tryptic fragment.

[0036] FIG. 2 shows the amino acid sequence of human growth hormone (SEQ ID NO: 1).

[0037] FIG. 3 shows the efficacy of 40K branched butyraldehyde hGH in the rat body weight gain assay. 4-5 week old (100-. When rats entered the animal facility, animals were maintained at a constant room temperature of 80 ° f and weighed daily for 4-10 days to estimate basal growth rate. Starting on day 0, rats (. about.100 g) in the control group then received subcutaneous injections of 0.3mg/kg hGH (filled circles) or PBS (open circles) daily for 11 consecutive days. The 40K branched butyraldehyde hGH test group (filled squares) received a single dose of 1.8mg/kg PHA-794428 on days 0 and 6. Each group had 8-10 animals. Mean growth +/-SEM is plotted.

[0038] FIG. 4 shows the dose-responsive growth promoting effect of 40K branched butyraldehyde hGH in rats. This efficacy study was performed in a similar manner as described in fig. 3, except that multiple single doses of 40K branched butyraldehyde hGH were administered (day 0 only), and the study was performed for 6 days. The control group received injections of 0.3mg/kg hGH (filled circles) or PBS vehicle (open circles) once daily for 6 consecutive days. The 40K branched butyraldehyde hGH was administered at a dose of 1.8mg/kg (filled squares), 0.6mg/kg (open squares), 0.2mg/kg (filled triangles), or 0.067mg/kg (open triangles). Each group had 8 rats.

[0039] Fig. 5 shows tibial growth in response to 40K branched butyraldehyde hGH. Hypophysectomized rats were treated as described in fig. 3. Animals were sacrificed on day 11, the left tibia was removed and examined by X-ray, and the bone length was measured with a caliper. The mean length is plotted +/-SEM. Asterisks indicate significant differences from the control (p < 0.05).

[0040] FIG. 6 shows plasma IGF-1 levels for a six day efficacy study. Animals were treated as described in figure 4. Blood samples were taken at different times and serum IGF-1 levels were determined by ELISA. Mean values +/-SEM are plotted.

Detailed Description

[0041] hGH and its agonist variants, which are members of the recombinant protein family, are described in US 4,658,021 (methionyl human growth hormone-Met-1-191 hGH) and US5,633,352. Methods for their recombinant production and use are described in detail in US 4,342,832, 4,601,980; US 4,898,830; US5,424,199 and US5,795,745.

[0042] Any purified and isolated hGH and agonist variants thereof produced by host cells such as e.coli and animal cells transformed or transfected by using recombinant gene technology may be used in the present invention. Additional hGH variants are described in US6,143,523 and WO 92/09690 published 6/11 1992. Among them, hGH produced from transformed E.coli or an agonist variant thereof is particularly preferred. Such hGH or agonist variants thereof may be obtained in large quantities in high purity and homogeneity. For example, the above hGH or agonist variants thereof may be used according to US 4,342,832, 4,601,980; US 4,898,830; the processes disclosed in US5,424,199 and US5,795,745. The term "having substantially the following amino acid sequence" means that the above amino acid sequence may include one or more amino acid changes (deletions, additions, insertions, or substitutions) as long as such changes do not cause any adverse function dissimilar to hGH or agonist variants thereof. More preferably, hGH or agonist variants thereof having substantially the amino acid sequence including therein at least one lysine, aspartic acid, glutamic acid, unpaired cysteine residue, free N-terminal alpha-amino group or free C-terminal carboxyl group are used.

[0043] The term "hGH polypeptide or hGH protein" as used herein includes all hGH polypeptides, preferably polypeptides from mammals, more preferably from humans and rodents, as well as variants, analogs, orthologs, homologs and derivatives thereof and fragments thereof, which are characterized by promoting growth during the growth phase and maintaining normal body composition, anabolism and lipid metabolism. Preferably, the term "hGH polypeptide or protein" refers to SEQ ID NO: 1 and variants, homologs and derivatives thereof that exhibit essentially the same biological activity (promoting growth during the growth phase and maintaining normal body composition, anabolism and lipid metabolism). More preferably, the term "hGH polypeptide or protein" refers to the polypeptide of SEQ ID NO 1.

[0044] The term "hGH polypeptide variant" as used herein refers to a polypeptide from the same species but different from the reference hGH polypeptide. Typically, the differences are limited such that the amino acid sequences of the reference polypeptide and the variant are similar as a whole and identical in many regions. Preferably, the hGH polypeptide is at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference hGH polypeptide, preferably SEQ ID NO: 1. For polypeptides having an amino acid sequence that is at least, e.g., 95% identical to a query amino acid, it means that the amino acid sequence of the target polypeptide is identical to the query sequence, except that the target polypeptide sequence may contain up to 5 amino acid changes per 100 amino acid sequences of the query. These changes to the reference sequence may occur at any position between the amino-or carboxy-terminal or those terminal positions of the reference amino acid sequence, interspersed either individually between residues in the reference sequence or in one or more contiguous groups within the reference sequence. The query sequence may be the entire amino acid sequence of the reference sequence or any fragment thereof as determined as described herein.

[0045] Such hGH polypeptide variants may be naturally occurring variants, such as naturally occurring allelic variants encoded by one of a plurality of alternative forms of hGH occupying a given locus on the chromosome of an organism, or isoforms encoded by naturally occurring splice variants derived from a single original transcript. Alternatively, hGH polypeptide variants may be variants that are not known to occur naturally and may be generated using mutagenesis techniques known in the art.

[0046] It is known in the art that one or more amino acids can be deleted from the N-terminus or C-terminus of a biologically active peptide or protein without substantial loss of biological function (see, e.g., Ron et al, (1993), Biol chem., 2682984-2988; incorporated herein by reference in its entirety).

[0047] One of ordinary skill in the art will also recognize that some amino acid sequences of hGH polypeptides may be altered without significant effect on the structure or function of the protein. Such mutants include deletions, insertions, inversions, repeats and substitutions, which are selected to have little effect on activity according to the general rules known in the art. For example, guidance on how to generate phenotypically silent amino acid substitutions is provided by Bowie et al, (1990), Science 247: 1306-1310, which is incorporated by reference in its entirety, indicates that there are two main methods for studying the tolerance of amino acid sequences to changes.

[0048] The first method relies on an evolutionary process in which mutations are either accepted or excluded by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific sites in cloned hGH and selects or screens to determine sequences that remain functional. These studies have revealed that proteins are unexpectedly tolerant to amino acid substitutions. The authors further indicate which amino acid changes may be allowed at certain positions of the protein. For example, most buried amino acid residues require nonpolar side chains, while few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al, (1990) supra and in the references listed therein.

[0049] What is generally considered to be conservative substitutions are the interchange between the aliphatic amino acids Ala, Val, Leu and Phe; the interchange of the hydroxyl residues Ser and Thr, the interchange of the acidic residues Asp and Glu, the substitution between the amide residues Asn and Gln, the interchange of the basic residues Lys and Arg, and the interchange in the aromatic residues Phe, Tyr. Moreover, the following amino acid groups generally represent equivalent changes: (1) ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr; (2) cys, Ser, Tyr, Thr; (3) val, Ile, Leu, Met, Ala, Phe; (4) lys, Arg, His; (5) phe, Tyr, Trp, His.

[0050] The term hGH polypeptide also encompasses all hGH polypeptides encoded by hGH analogues, orthologs and/or species homologues. As used herein, the term "hGH analog" refers to hGH of different and unrelated organisms, which perform the same function in each organism but which are not derived from an ancestral structure common to ancestors of the organisms. In contrast, similar hghs are produced independently and subsequently evolved to perform the same function (or similar functions). In other words, a similar hGH polypeptide is a polypeptide having a rather different amino acid sequence but performing the same biological activity, i.e. promoting growth and maintaining normal body composition, anabolism and lipid metabolism during the growth phase. As used herein, the term "hGH ortholog" refers to hGH within two different species, the sequences of which are related to each other by a common homologous hGH in the ancestral species, but which have become different from each other by evolution. As used herein, the term "hGH homolog" refers to hGH of different organisms, which hGH performs the same function in each organism and which hGH originates from an ancestral structure common to ancestors of the organism. In other words, a homologous hGH polypeptide is a polypeptide that has a rather similar amino acid sequence and exerts the same biological activity, i.e. promotes growth and maintains normal body composition, anabolism and lipid metabolism during the growth phase. Preferably, hGH polypeptide homologs may be defined as polypeptides exhibiting at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to a reference hGH polypeptide, preferably SEQ ID NO: 1.

[0051] Thus, the hGH polypeptide according to the invention may be, for example: (i) wherein one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be an hGH polypeptide encoded by the genetic code; or (ii) an hGH polypeptide in which one or more amino acid residues comprise a substituent group; or (iii) hGH in which the hGH polypeptide is fused to another compound, such as a compound to increase the half-life of the polypeptide (e.g., polyethylene glycol); or (iv) an hGH polypeptide in which additional amino acids are fused to the polypeptide of the above form, such as an IgG Fc fusion domain polypeptide or a leader or secretory sequence or a sequence used for purification of the polypeptide of the above form or a preprotein sequence;

[0052]hGH polypeptides may be monomeric or multimeric. The multimer may be a dimer, trimer, tetramer, or a polypeptide comprising at least 5 monomersA multimer of the unit. Multimers may also be homodimers or heterodimers. The multimers of the invention may be the result of hydrophobic, hydrophilic, ionic, and/or covalent bonding and/or may be indirectly linked through, for example, a liposome structure. In one example, covalent attachment is between heterologous sequences contained in a fusion protein comprising an hGH polypeptide or fragment thereof (see, e.g., U.S. patent No.5,478,925, the disclosure of which is incorporated herein by reference in its entirety). In another example, the hGH polypeptide or fragment thereof is linked to one or more polypeptides, which may be hGH polypeptides or heterologous polypeptides, via a peptide linker such as those described in U.S. patent No.5,073,627 (incorporated herein by reference). Another method for preparing multimeric hGH polypeptides involves the use of hGH polypeptides fused to a leucine or isoleucine zipper polypeptide sequence known to promote multimerization of the proteins in which they are contained, wherein said fusion is using techniques known to those skilled in the art, including the techniques of WO 94/10308. In another example, the hGH polypeptide may be encoded by inclusion in a composition comprising Flag^®Fusion of polypeptide sequences to Flag of hGH polypeptide^®Interactive linkage between polypeptide sequences. hGH multimers may also be produced using chemical techniques known in the art, such as cross-linking using linker molecules, wherein the linker molecule length optimization techniques are known in the art (see, e.g., US5,478,925), or using techniques known in the art to form one or more intermolecular cross-links between cysteine residues within a polypeptide sequence desired to be contained in a multimer (see, e.g., US5,478,925), the addition of cysteine or biotin to the C-terminus or N-terminus of hGH, as well as techniques to produce multimers containing one or more such modified polypeptides (see, e.g., US5,478,925) or any of the 30 techniques for producing liposomes containing hGH multimers (see, e.g., US patent No.5,478,925), the disclosure of which is incorporated by reference in its entirety.

[0053] As used herein, the term "hGH polypeptide fragment" refers to a polypeptide comprising an hGH polypeptide, preferably SEQ ID NO: 1, or a contiguous stretch of the amino acid sequence of a portion of the polypeptide of 1.

[0054] More specifically, the hGH polypeptide fragment comprises at least 6, preferably at least 8-10, more preferably 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 125, 150, 175, 191 consecutive amino acids of an hGH polypeptide according to the invention. hGH polypeptide fragments may additionally be described as a subclass of hGH polypeptides comprising at least 6 amino acids, wherein "at least 6" is defined as defined at 6 and represents a subset of hGH polypeptides comprising SEQ ID NO: 1 of the polypeptide of 1 is any integer between integers of the C-terminal amino acid. Further included are classes of hGH polypeptide fragments of at least 6 amino acids in length as described above, further defined by their N-terminal and C-terminal positions. Also encompassed by the term "hGH polypeptide fragments" as a separate class are all polypeptide fragments which may be specifically defined by N-terminal and C-terminal positions and which are about at least 6 amino acids long as described above. That is, any combination of N-terminal and C-terminal positions that may be occupied by fragments of at least 6 contiguous amino acid residues in the sequence listing or any given amino acid sequence of the invention is encompassed by the invention.

[0055] Obviously, the above classes of polypeptide fragments of the invention may alternatively be described by the formulae "a to b"; wherein "a" equals the N-most terminal amino acid position and "b" equals the C-most terminal amino acid position of the polynucleotide; and further wherein "a" equals 1 and the number of amino acids of the hGH polypeptide sequence minus 6, and wherein "b" equals an integer between 7 and the number of amino acids of the hGH polypeptide sequence; and wherein "a" is an integer at least 6 less than "b".

[0056] The above hGH polypeptide fragments can be immediately imagined using the above description and are thus not listed separately in order to avoid unnecessarily extending the present specification. Furthermore, the above fragments do not necessarily need to have hGH biological activity, although polypeptides having such activity are preferred embodiments of the invention, as they may be useful, for example, in immunoassays, epitope mapping, epitope appending, as vaccines and as molecular weight markers. The above fragments may also be used to generate antibodies to specific portions of the polypeptide.

[0057] Also encompassed by the term "hGH polypeptide fragment" is the domain of the hGH polypeptide. Such domains ultimately include linear or structural motifs and signals, including, but not limited to, leucine zippers, helix-turn-helix motifs, post-translational modification sites such as glycosylation sites, ubiquitination sites, alpha-helix and beta-sheet, signal sequences encoding signal peptides that direct secretion of the encoded protein, sequences involved in transcriptional regulation such as homeobox, acid sequence (acicstretch), enzymatic activity sites, substrate binding sites, and cleavage sites. Such domains may exhibit specific biological activities such as DNA or RNA-binding activity, protein secretion, transcriptional regulation, enzymatic activity, substrate binding activity, and the like.

[0058] Domains have a size that typically comprises between 3 and 191 amino acids. In a preferred embodiment, the domain comprises an integer number of amino acids between 6 and 191. The domains may be synthesized using methods known to those skilled in the art, including those disclosed herein for preparing hGH polypeptides to produce anti-hGH antibodies. Methods for determining the amino acids that make up a domain with a particular biological activity include mutagenesis studies and assays to determine the biological activity being tested.

[0059] Particularly preferred fragments in the context of the present invention are hGH polypeptides that retain substantial biological activity, i.e. promote growth during the growth phase and maintain normal body composition, anabolism and lipid metabolism.

[0060] Alternatively, polypeptides of the invention may be searched for motifs, domains and/or signals in a database using computer methods known to those skilled in the art. Searchable databases include Proteins (Hofmann et al, (1999) Nucleic Acids Res.27: 215-219; Bucher and Bairoch (1994) procedures 2 and International Conference on Intelligent Systems for Molecular biology. Altman et al, pp53-61, AAAIPress, 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) Proteins Acids 21 (9): 1700-6), Print (Atwoood et al, (182) Nucleic Acids Res.28 (1): 263-6), Blocks (Henikoff et al, (2000) Proteins Res.21 (9): 1700-6), and Sbame 3 (2000) Nucleic Acids 2 (1996) 2000) Acpex.3 (2000) and Sbx.7-20 (2000) Nucleic Acids 3 (2000) 3. Sbc) (2000) Nucleic Acids 3 (2000-7-2, 2000) (Sbc et al, (2000) Nucleic Acids 2, 7, 2000, 2-7, 2-7, 3,2, 3,2, 3,2, 3,2, Smart (Schultz et al, (1998) Proc. Natl. Acad. Sci. U S A95, 5857-5864), Dali/FSSP (Holm and Sander (1996) Nucleic Acids Res.24 (1): 206-9; Holm and Sander (1997) Nucleic Acids Res.25 (1): 231-4; Holm and Sander (1999) Nucleic Acids Res.27 (1): 244-7), HSSP (Sander and Schneider (1991) Proteins9 (1): 56-68), CATH (Orenzin et al, (1997) Structure 5 (8): 1093-108; Pearl et al, (2000) biom. Soc. Trans.28 (2): 269-75), SCOP (Murzin et al, (1995) J. mol. C. 631. 10): 5; Pear.70-2000; Natl. J. 19865; Nature A. 75; Nature A. J. Act.631. 75; Natl. C. A. 2000; Nature A. 19871; Nature A. 2000; Nature et al; Nature A. 25; 231-33; Nature A. 25; 231-4; 1997; Nature et al; Nature C. acide. acide.75; Nature et al; Nature G et al; Nature et al. (1997; Nature W.),278; Nature et al, (1999) proteins37 (3): 360-78; attwood et al, (2000) Nucleic Acids Res.28 (1): 225-7) the disclosure of each of which is incorporated by reference in its entirety. For a review of the available databases, see volume 28, phase 1 of Nucleic Acid Research (2000), the disclosure of which is incorporated herein by reference in its entirety.

[0061] The term "hGH polypeptide fragment" also encompasses epitope-bearing fragments. These epitopes may be antigenic epitopes or both antigenic and immunogenic epitopes. An immunogenic epitope is defined as a portion of a protein that elicits an antibody response in vivo when the polypeptide is an immunogen. On the other hand, the region of the polypeptide to which the antibody binds is defined as an epitope. An antigen may comprise a minimum of 3 amino acids in a spatial conformation that is unique to the epitope. Typically an epitope comprises at least 6 such amino acids, and more typically comprises at least 8-10 such amino acids.

[0062] The hGH epitope-bearing fragment according to the invention may be any fragment between 6 amino acids in length and the full-length sequence of the hGH polypeptide, preferably a fragment between 6 and 50 amino acids. An epitope-bearing fragment can be defined by the number of contiguous amino acid residues (as a subclass) as described above or by the particular N-terminal and C-terminal positions (as a class).

[0063] Fragments for use as epitopes may be generated by any conventional method (see, e.g., Houghten (1985), Proc. Natl. Acad. Sci. USA 82: 5131. SP. 5135 and U.S.4,631,21, the disclosures of which are incorporated herein by reference in their entirety). Methods for determining the amino acids that make up an epitope include X-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping, such as those described by Geysen et al, (1984), proc.natl.acad.sci.u.s.a.81: 3998-4002; PCT publications WO 84/03564 and WO 84/03506, the disclosures of which are incorporated herein by reference in their entirety, describe the Pepscan method. Another example is Jameson and Wolf, (1988), comp.appl.biosci.4: 181-186 (which reference is incorporated by reference in its entirety). The Jameson-Wolf antigen analysis can be performed, for example, with the computer program PROTEAN, using default parameters (Windows version 4.0, DNASTAR, Inc.).

[0064] The present invention also provides for the exclusion of any hGH fragment class defined by N-terminal and C-terminal positions or any fragment subclass defined by the size of amino acid residues as described above. Any number of fragments as described above, determined by N-terminal and C-terminal positions or by amino acid size, may be excluded as individual classes. The present invention also provides in the same way the exclusion of any hGH domain or epitope-bearing fragment.

[0065] The hGH polypeptides of the invention may be prepared in a suitable manner. Such hGH polypeptides and fragments thereof may be produced from natural sources, purified by recombinant techniques including in vitro translation techniques or recombinant cells expressing hGH cDNA, or a combination of these Methods using techniques known to those skilled in the art (see, e.g., "Methods in enzymology, Academic Press, 1993", see, among others, Methods for purifying Proteins; Creighton, (1983) Proteins: Structures and molecular principles, W.H.Freeman & Co.2nd Ed., T.E., New York; and Hunkapiller et al, (1984) Nature.310 (5973): 105-11, see, chemical synthesis of Proteins, and Davis et al, (1986) Basic Methods in molecular biology, ed., Elsevier Press, NY, for recombinant techniques, the disclosure of which is incorporated herein by reference in its entirety). The polypeptides of the invention are preferably provided in isolated form and may be partially purified or preferably substantially purified.

[0066] The terms "polynucleotide having at least x% identity to a reference polynucleotide" and "polypeptide having at least x% identity to a reference polypeptide" encompass polynucleotides or polypeptides having a sequence of residues (nucleotides or amino acids, respectively) that exhibits a percent identity, as defined below, equal to or greater than x, as compared to the sequence of said reference polynucleotide or polypeptide, respectively.

[0067] The percent identity is determined after the two polynucleotide or polypeptide sequences are optimally aligned over a comparison window, where the portion of the polynucleotide or polypeptide sequences in the comparison window may contain additions or deletions of one or more residues in order to optimize the sequence alignment. The comparison window contains a certain number of positions (corresponding to residues or gaps of residue insertions/deletions) corresponding to the window size. Each window position may represent one of the following:

1 °/having a residue (nucleotide or amino acid) at that position in the first aligned sequence and a different residue at the same position in the second aligned sequence, in other words, the second sequence has a substituted residue at that position as compared to the first sequence.

2 °/with one residue (nucleotide or amino acid) at that position in the first aligned sequence and the same residue at the same position in the second aligned sequence.

3 °/having a residue (nucleotide or amino acid) at that position in the first aligned sequence and no amino acid at the same position in the second aligned sequence, in other words, the second sequence shows a deletion at that position compared to the first sequence.

The number of positions within the comparison window belonging to the first above-defined class is referred to as R1.

The number of positions within the comparison window belonging to the second above-defined class is referred to as R2.

The number of positions within the comparison window belonging to the third above-defined class is referred to as R3.

[0068] Percent identity (% id) can be calculated by any of the following formulas:

% id ═ R2/(R1+ R2+ R3). times.100, or

％id＝(R2+R3)/(R1+R2+R3)×100

[0069] Alignment of the sequences for comparison can be performed using any of a variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, FASTDB, WU-BLAST, Gapped-BLAST, PSI-BLAST (Pearson and Lipman, (1988), Proc. Natl. 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, Thpsomon et al, (1994), Nuc. acids Res. 22: 4673-4680, Higgins et al, (1996), Meth. enzymol.266: 402; Brlag et al, (1990) Comp. Swi. 6: 4673-27; Johnd. 1999), and Biochem et al, (1999) incorporated by reference, Symand-12, and Biochem et al, (1990).

[0070] In a particular embodiment, the Smith-Waterman method is used with scoring matrices such as PAM, PAM250, preferably with BLOSUM matrices such as BLOSUM60 or BLOSUM62, and with default parameters (gap opening penalty ═ 10 and gap extension penalty ═ 1) or preferably with user-determined parameters that are better than the default parameters.

[0071] In another particular embodiment, the protein and nucleic acid sequences are aligned using the basic Alignment Search Tool ("BLAST") program using default parameters or using user-provided modified parameters. 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, the disclosures of which are incorporated herein by reference in their entirety). Less preferably, PAM or PAM250 Matrices may also be used (see, e.g., Schwartz and Dayhoff, (1978), eds., substrates for detecting distance Relationships: Atlas of Protein Sequence and Structure, Washington: National biological research Foundation, the disclosure of which is incorporated herein by reference in its entirety).

[0072] In another specific embodiment, the polynucleotide or polypeptide sequences are aligned using the FASTDB computer program based on Brutlag et al, (1990), supra. Preferred parameters for FASTDB alignment of DNA sequences are: matrix equals to unity, k-tuple equals to 4, Mismatch Penalty equals to 1, Joining Penalty equals to 30, Randomization group length equals to 0, Cutoff Score equals to 1, Gap Penalty equals to 5, Gap Size Penalty equals to 0.05, Window Size equals to 500, or the length of the target nucleotide sequence, shorter one is selected. Preferred parameters for the FASTDB amino acid sequence alignment are: matrix equals PAM 0, k-tuple equals 2, MismatchPenalty equals 1, Joining Penalty equals 20, Randomzation Group25Length equals 0, Cutoff Score equals 1, Window Size equals sequence Length, Gap Penalty equals 5, Gap SizePenalty equals 0.05, Window Size equals 500, or the Length of the target amino acid sequence, shorter ones are selected.

[0073] According to the present invention, poly (ethylene glycol) is covalently bound through the amino acid residue of hGH or agonist variants thereof. A variety of activated poly (ethylene glycols) having many different functional groups, linkers, configurations and molecular weights are known to those skilled in the art and can be used to generate PEG-hGH conjugates or PEG-hGH agonist variant conjugates (for review see Roberts M.J. et al, adv. Drug Del. Rev.54: 459. 476, 2002; Harris J.M. et al, Drug Delivery Sytems 40: 538. 551, 2001). The present invention relates to methods of directing selective conjugation of PEG moieties to the N-terminus with aldehyde chemistry using butyraldehyde linking moieties. Butyraldehyde linker resulted in increased N-terminal specificity compared to acetaldehyde linker (table 1 and figure 1).

[0074] One embodiment of the present invention is a human growth hormone-PEG conjugate having the structure of formula I or formula II:

or

mPEG-O(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 human growth hormone, methionyl growth hormone or a human growth hormone variant.

[0075] In a particular embodiment, n is between 1 and 5 and m is between 1 and 5.

[0076] 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 is 7 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.

[0077] A particular embodiment is a human growth hormone-PEG conjugate having the structure:

or

mPEG-O(CH₂CH₂O)₄CH₂CH₂CH₂CH₂-NH-R

Wherein R is human growth hormone, methionyl human growth hormone or a human growth hormone variant.

[0078] Another specific embodiment of the present invention is a human growth hormone-PEG conjugate, wherein the human growth hormone comprises or consists of SEQ ID NO: 1.

[0079] A particular embodiment of the 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%, more preferably 98% of the polyethylene glycol is bound to SEQ ID NO: 1 on the amino-terminal phenylalanine of the amino acid sequence of 1.

[0080] Another particular embodiment of the invention is a polypeptide wherein more than 90% of the polyethylene glycol is bound to SEQ ID NO: 1 on the amino-terminal phenylalanine of the amino acid sequence of 1.

[0081] Another particular embodiment of the invention is a polypeptide wherein more than 95% of the polyethylene glycol is bound to SEQ ID NO: 1 on the amino-terminal phenylalanine of the amino acid sequence of 1.

[0082] Another particular embodiment of the invention is a polypeptide wherein more than 98% of the polyethylene glycol is bound to SEQ ID NO: 1 on the amino-terminal phenylalanine of the amino acid sequence of 1.

[0083] The poly (ethylene glycol) used in the present invention is not limited to any particular form or molecular weight range. The poly (ethylene glycol) may have a molecular weight between about 500 and about 100,000 daltons. The term "about" means that some molecules will weigh less and some less than the stated molecular weight, which refers to the average molecular weight, in the preparation of polyethylene glycol. It will be appreciated that there is some degree of polydispersity associated with polymers such as poly (ethylene glycol). PEG with low dispersibility is preferably used. Typically, PEG having a molecular weight of about 500 to about 60,000 is used. Particular PEGs of the invention have a molecular weight in the range of about 1,000 to about 40,000. In another specific embodiment, the PEG has a molecular weight greater than about 5,000 to about 40,000. In another specific embodiment, the PEG has a molecular weight of about 20,000 to about 40,000. Other sizes of PEG may be used depending on the desired therapeutic properties (e.g., duration of desired sustained release, effect on biological activity, if any, degree of antigenicity or lack of antigenicity and other known effects of polyethylene on therapeutic proteins). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 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 daltons.

[0084] In another embodiment, the poly (ethylene glycol) is a branched PEG having more than one PEG moiety attached (U.S.5,932,462; U.S.5,342,940; U.S.5,643,575; U.S.5,919,455; U.S.6,113,906; U.S.5,183,660; Kodera Y., Bioconjugate Chemistry 5: 283-. In a preferred embodiment, the molecular weight of each poly (ethylene glycol) of the branched PEG is about 5,000-20,000. In a particular embodiment, each poly (ethylene glycol) of the branched PEG has a molecular weight of about 20,000.

[0085] Poly (alkylene oxide), particularly poly (ethylene glycol), is conjugated to hGH or agonist variants thereof through a terminal active group, which conjugation may or may not leave a linking moiety (spacer arm) between PEG and protein. To form hGH conjugates of the invention or agonist variants thereof, polymers such as poly (ethylene glycol) are converted to the activated form, as is known to those skilled in the art. Reactive groups are, for example, terminal reactive groups that mediate a bond between a chemical moiety on a protein and poly (ethylene glycol). Typically, one or both of the terminal polymer hydroxyl end groups (i.e., the alpha and omega terminal hydroxyl groups) are converted to reactive functional groups that enable covalent bonding. This process is often referred to as "activation" and the poly (ethylene glycol) product having reactive groups is hereinafter referred to as "activated poly (ethylene glycol)". In a particular embodiment, the polymer hydroxyl end groups at one end are converted to or protected with a non-reactive group. In a particular embodiment, the polymer hydroxyl end group at one end is converted to or protected with a methyl group. As used herein, the term "mPEG" refers to PEG protected at one end with a methyl group. mPEG can be structurally represented as:

CH₃O-(CH₂CH₂O)_n-H

[0086] polymers containing alpha and epsilon linkages are referred to as "di-activated poly (vinyl oxide)" and are referred to as "difunctional". Polymers containing the same reactive groups at the alpha and epsilon terminal hydroxyl groups are sometimes referred to as "homobifunctional" or "homobifunctional". Polymers containing different reactive groups on the alpha and epsilon terminal hydroxyl groups are sometimes referred to as "heterobifunctional" (see, e.g., WO01/26692) or "heterobi-activated". Polymers containing a single reactive group are referred to as "mono-activated" polyalkenyl oxides or "mono-functional". Other substantially non-antigenic polymers are similarly "activated" or "functionalized".

[0087] The activated polymer is thus suitable for mediating the bond between a chemical moiety on a protein, such as an alpha-or epsilon-amino group, a carboxyl group or a thiol group, and poly (ethylene glycol). The di-activated polymer may react in this manner with two protein molecules or in another embodiment with one protein molecule and one reactive small molecule to effectively form a protein polymer or protein-small molecule conjugate by cross-linking.

[0088]In a preferred embodiment of the invention, the alpha-amino group or epsilon-amino group of the N-terminal lysine of hGH or agonist variants thereof is used to form a secondary amine or amide bond with the activated PEG. In the present inventionAnother preferred aspect of the invention, e.g., Chamow et al, Bioconjugate chem.5: 133-140(1994), U.S. Pat. No.4,002,531, WO 90/05534 and U.S. Pat. No.5,824,784 by using a suitable reducing agent such as NaCNBH₃、NaBH₃Reductive alkylation of pyridine borane, etc., forms a secondary amine bond between the alpha-amino or epsilon-amino group of the N-terminal lysine of hGH or agonist variants thereof and the single or branched PEG acetaldehyde.

[0089] 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 most preferably at least 98% of the poly (ethylene glycol) is on the alpha-amino group at the amino terminus.

[0090] The binding reaction, known as pegylation, has in the past been performed in solution with a molar excess of polymer and does not take into account where the polymer will bind to the protein. However, this general technique has generally proven inadequate for binding biologically active proteins to non-antigenic polymers while maintaining sufficient biological activity. One way to maintain the biological activity of hGH or agonist variants thereof is to substantially avoid binding of reactive groups of those hGH or agonist variants thereof associated with the receptor binding site during the polymer binding process. It is another aspect of the present invention to provide methods for conjugating poly (ethylene glycol) to hGH or agonist variants thereof while maintaining high levels of persistence activity.

[0091] Chemical modification by covalent bonds can be performed under any suitable conditions typically employed in the reaction of a biologically active substance with activated poly (ethylene glycol). The binding reaction is performed under relatively mild conditions to avoid inactivation of hGH or agonist variants thereof. Mild conditions include maintaining the reaction solution at a pH of 3-10 and a reaction temperature within about 0 ℃ to 37 ℃. In case the active amino acid residue of hGH or agonist variants thereof has a free amino group, the above modification is preferably performed in a series of suitable buffers (pH 3-10) including, but not limited to, phosphate buffer, MES, citrate buffer, acetate buffer, succinate buffer or HEPES at 4 ℃ -37 ℃ for 1-48 hours. The pH is preferably maintained between 4 and 7 when the N-terminal amino group is attacked with an agent such as PEG acetaldehyde. The activated poly (ethylene glycol) may be used in a molar amount of about 0.01 to 100 times, preferably about 0.01 to 2.5 times the number of free amino groups of hGH or variants thereof. On the other hand, if the reactive amino acid residue of hGH or agonist variants thereof has a free carboxyl group, the above modification is preferably carried out at 4 ℃ -37 ℃ for 1-24 hours at a pH of about 3.5 to about 5.5, for example, the modification with poly (oxyethyldiamine) is carried out in the presence of carbodiimide (pH 4-5). The activated poly (ethylene glycol) may be used in a molar amount of 0.01-300 times the number of free carboxylic groups of hGH or agonist variants thereof.

[0092] In a separate embodiment, the upper limit of the amount of polymer included in the binding reaction exceeds an amount of about 1: 1, which is an amount that makes it possible to react the activated polymer and hGH or agonist variant thereof without forming a substantial amount of high molecular weight species, i.e. more than about 20% of the conjugate contains more than about-chain polymer per molecule of hGH or agonist variant thereof. 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 conjugate, which can then be separated from any high molecular weight species.

[0093] In another aspect of the invention, bifunctional activated PEG derivatives may be used to generate polymeric hGH or agonist variant thereof-PEG molecules, wherein a plurality of hGH or agonist variant molecules thereof are cross-linked by PEG. Although the reaction conditions described herein may result in significant amounts of unmodified hGH or agonist variants thereof, these unmodified hGH or agonist variants thereof may conveniently be recycled to future reactions for additional binding reactions. The methods of the present invention surprisingly result in very few, i.e. less than about 30% and more preferably, less than about 10%, high molecular weight species and species containing more than one polymer chain per hGH or agonist variant thereof. These reaction conditions will be compared to those typically used in polymer binding reactions where the activated polymer is in a multiple molar excess to the target. In a further aspect of the invention, the polymer is present in an amount of about 0.1-50 equivalents per equivalent of hGH or agonist variant thereof. In a further aspect of the invention, the polymer is present in an amount of about 1-10 equivalents per equivalent of hGH or agonist variant thereof.

[0094] The conjugation reactions of the present invention initially provide a reaction mixture or combined library containing mono-and di-PEG-hGH conjugates, 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 polymerized PEG-hGH or agonist variant species thereof. After unreacted species and high molecular weight species have been removed, a composition comprising predominantly mono-and di-polymer-hGH or agonist variant conjugates thereof is recovered. Assuming that the conjugate comprises mostly a single polymer chain, the conjugate is substantially homogeneous. These modified hGH or agonist variants thereof have at least about 0.1% of the in vitro Biological activity associated with native or unmodified hGH or agonist variants thereof, as measured using standard FDC-P1 cell proliferation assays (Clark et al, Journal of Biological Chemistry 271: 21969-21977, 1996), receptor binding assays (US 5,057,417) or growth of pituitary-excised rats (Clark et al, Journal of Biological Chemistry 271: 21969-21977, 1996). However, in a preferred aspect of the invention, the modified hGH or agonist variant thereof has about 25% in vitro biological activity, more preferably the modified hGH or agonist variant thereof has about 50% in vitro biological activity, more preferably the modified hGH or agonist variant thereof has about 75% in vitro biological activity, and most preferably the modified hGH or agonist variant thereof has equivalent in vitro biological activity.

[0095] The methods of the invention preferably comprise a rather limited ratio of polymer to hGH or agonist variant thereof. Thus, it has been found that hGH or agonist variant conjugates thereof are primarily limited to species containing only one polymer chain. Furthermore, the attachment of the polymer to the active group of hGH or agonist variants thereof is considerably less random than when a higher molar excess of polymer linker is used. After the binding reaction has been quenched, the unmodified hGH or agonist variant thereof present in the reaction mixture may be recycled into future reactions using ion exchange or size exclusion chromatography or similar separation techniques.

[0096]The poly (ethylene glycol) -modified hGH or agonist variants thereof, i.e. the chemically modified protein according to the invention, can be isolated from the reaction mixture by conventional methods for purifying proteins, such as dialysis, salting out, ultrafiltration, ion exchange chromatography, Hydrophobic Interaction Chromatography (HIC), gel chromatography and electrophoresis. Ion exchange chromatography is particularly effective in removing unreacted poly (ethylene glycol) and hGH or agonist variants thereof. In a further embodiment of the invention, mono-and di-polymer-hGH or agonist variant species thereof are separated from the reaction mixture to remove high molecular weight species and unmodified hGH or agonist variant thereof. Separation is effected by placing the mixed species in a buffer solution containing about 0.5-10mg/mL hGH or agonist variant-polymer conjugate thereof. Suitable solutions have a pH of about 4 to about 10. The solution preferably contains one or more of KCl, NaCl, K₂HPO₄、KH₂PO₄、Na₂HPO₄、NaH₂PO₄、NaHCO₃、NaBO₄、CH₃CO₂H and NaOH.

[0097] Depending on the reaction buffer, the hGH or agonist variant polymer conjugate solution thereof may first have to be buffer exchanged/ultrafiltered to remove any unreacted polymer. For example, a solution of PEG-hGH or agonist variant conjugate thereof may be ultrafiltered through a low molecular weight cut-off (10,000-30,000 daltons) membrane to remove most of the undesired species such as unreacted polymer, surfactant, if any, and the like.

[0098] The conjugates are preferably fractionated into libraries containing the desired species using ion exchange chromatography media. Such a medium is capable of selectively binding PEG-hHG or an agonist variant conjugate thereof through differences in charge that vary in a somewhat predictable manner. For example, the surface charge of hGH or agonist variants thereof is determined by the number of available charged groups on the protein surface. These charged groups often serve as potential attachment points for poly (alkylene oxide) polymers. Thus, hGH or agonist variant conjugates thereof will have a different charge than the other species to enable selective separation.

[0099]Strongly polar anion or cation exchange resins such as quaternary ammonium or sulfopropyl resins, respectively, are used in the process of the present invention. Ion exchange resins are particularly preferred. A series of commercial cation exchange resins suitable for use in the present invention are SP-hitrap^®、SP Sepharose HP^®And SP Sepharose^®fast flow, but is not limited thereto. Other suitable cation exchange resins such as S and CM resins may also be used. A series of anion exchange resins suitable for use in the present invention, including commercially available anion exchange resins, are Q-hitrap^®、Q Sepharose HP^®And Q sepharose^®fast flow, but is not limited thereto. Other suitable cation exchange resins such as DEAE resins may also be used.

[00100]For example, anion or cation exchange resins are preferably packed into a column and equilibrated by conventional methods. A buffer having the same pH and osmolality as the conjugated polymer hGH or agonist variant solution thereof is used. The elution buffer preferably contains one or more of KCl, NaCl, K₂HPO₄、KH₂PO₄、Na₂HPO₄、NaH₂PO₄、NaHCO₃、NaBO₄And (NH)₄)₂CO₃A salt buffer. The conjugate-containing solution is then adsorbed onto the column without unreacted polymer and some macromolecular species remaining therein. After loading is complete, a gradient elution buffer with increasing salt concentration is applied to the column to elute the desired poly (alkylene oxide) -conjugationhGH or agonist variant fractions thereof. After the cation or anion exchange separation step, the eluted pooled fractions are preferably limited to the same polymer conjugate. Any unconjugated hGH or agonist variant species thereof may then be recovered by elution from the column by conventional techniques. The mono-and poly-pegylated hGH or agonist variant species thereof may be further separated from each other by additional ion exchange chromatography or size exclusion chromatography, if desired.

[00101] Techniques utilizing multiple, increasing concentrations of salt or a constant solvent elution step of pH may also be used. Multiple increasing concentrations of the constant solvent elution step will result in sequential elution of the di-and mono-polymer conjugate-hGH or agonist variants thereof.

[00102] The temperature range for elution is between about 4 ℃ and about 25 ℃. Preferably, the elution is carried out at about 4 ℃ to about 22 ℃. For example, elution of the PEG-hGH or agonist variant fraction thereof is detected by UV absorbance at 280 nm. Fraction collection was accomplished by a simple time elution profile.

[00103] Surfactants may be used in the process of conjugating the poly (ethylene glycol) polymer to hGH or an agonist variant portion thereof. Suitable surfactants include ionic agents such as Sodium Dodecyl Sulfate (SDS). Other ionic surfactants such as lithium dodecylsulfonate, quaternary ammonium compounds, taurocholic acid, caprylic acid, decanesulfonic acid, and the like, may also be used. Nonionic surfactants may also be used. For example, materials such as poly (oxyethylene) sorbitan species (tweens), poly (oxyethylene) esters (tritons) species may be used. See also Neugebauer, A Guide to the Properties and Uses of Detergents in Biology and Biochemistry (1992) Calbiochem Inc. The only limitation of the surfactants used in the methods of the present invention is that they are used under conditions and concentrations that do not cause substantial irreversible denaturation of hGH or agonist variants thereof and do not completely inhibit polymer conjugation. The surfactant is present in the reaction mixture in an amount of about 0.01 to 0.5%, preferably about 0.05 to 0.5%, most preferably about 0.075 to 0.25%. Mixtures of surfactants are also contemplated.

[00104] It is believed that the surfactant provides a temporary, reversible protection system during the polymer conjugation process. Surfactants have been shown to be effective in selectively hindering polymer conjugation to allow lysine-based or amino-terminal based conjugation to proceed.

[00105] The poly (ethylene glycol) modified hGH or agonist variants thereof of the present invention have a more durable pharmacological effect, which may be attributed to their prolonged half-life in vivo.

[00106] Another embodiment of the present invention relates to a method for the prevention and/or treatment of diseases or conditions in which the use of GH, preferably hGH, is beneficial, said method comprising administering a therapeutically effective amount of a poly (ethylene glycol) modified hGH or agonist variant thereof of the present invention alone or in combination with other therapeutic agents to a patient in need thereof. The present invention also relates to the use of a poly (ethylene glycol) modified hGH or agonist variant thereof of the present invention in the manufacture of a medicament for the prevention and/or treatment of a disease or condition in which the use of GH, preferably hGH, is beneficial. Furthermore, the present invention also relates to a pharmaceutical composition comprising a poly (ethylene glycol) -modified hGH or agonist variant thereof according to the present invention for use in the prevention and/or treatment of a disease or condition wherein the use of GH, preferably hGH, is beneficial.

[00107] Diseases or conditions in which use of GH is beneficial include, but are not limited to, Growth Hormone Deficiency (GHD), adult growth hormone deficiency (aGHD), Teller's syndrome, growth deficiencies in children born Short of Gestational Age (SGA), Par-Weierz syndrome (PWS), Chronic Renal Insufficiency (CRI), AIDS wasting and aging, late stage renal failure, cystic fibrosis, erectile dysfunction, HIV lipodystrophy, fibromyalgia, osteoporosis, memory disorders, depression, crohn's disease, skeletal dysplasia, traumatic brain injury, subarachnoid hemorrhage, noonan syndrome, down's syndrome, congenital short stature (ISS), advanced kidney disease (ESRD), Very Low Birth Weight (VLBW), bone marrow stem cell rescue, metabolic syndrome, glucocorticoid myopathy, short stature in children due to glucocorticoid therapy, and growth deficiencies associated with short, early-maturing children.

[00108] In a more specific embodiment of the invention, the poly (ethylene glycol) modified hGH and agonist variants thereof of the present invention are used for the prevention and/or treatment of a disease selected from the group consisting of GHD, ahgd, SGA, PWS, turner's syndrome and CRI.

[00109] In another more specific embodiment of the invention, the poly (ethylene glycol) modified hGH or agonist variant thereof of the invention is for use in the prevention and/or treatment of a disorder or disease selected from the group consisting of congenital short stature, very low birth weight, traumatic brain injury, metabolic syndrome and noonan syndrome.

[00110] Another embodiment of the present invention relates to a pharmaceutical composition comprising a poly (ethylene glycol) modified hGH or agonist variant thereof of the present invention alone or in combination with other therapeutic agents, and at least one pharmaceutically acceptable excipient or carrier. The poly (ethylene glycol) modified hGH or agonist variants thereof of the present invention may then be formulated into pharmaceutical formulations also containing pharmaceutically acceptable diluents, agents for preparing isotonic solutions, pH adjusting agents and the like for their administration to patients.

[00111] The above pharmaceutical preparation may be administered subcutaneously, intramuscularly, intravenously, pulmonarily, intradermally or orally depending on the purpose of treatment. The dosage may also be based on the kind and condition of the disease to be treated in the patient, and is usually 0.1mg to 5mg by injection to an adult and 0.1mg to 50mg by oral administration.

[00112] As used herein, a poly (ethylene glycol) modified hGH or agonist variant thereof of the present invention may be used in combination with an additional therapeutic agent. As used herein, the terms "co-administration," "co-administered," and "in combination with" when referring to compound a and one or more other therapeutic agents are intended to mean, and at dosages and including the following:

-when the components are formulated together into a single dosage form, administering simultaneously the combination of a and therapeutic agent to a patient in need of treatment, said dosage form releasing said components to said patient at substantially the same time.

-administering the combination of a and therapeutic agent to a patient in need of treatment substantially simultaneously when the components are formulated separately from each other into separate dosage forms, said separate dosage forms being taken by the patient substantially simultaneously, whereby said components are released to said patient substantially simultaneously.

-when the components are formulated separately from each other into separate dosage forms, sequentially administering such a combination of a and a therapeutic agent to a patient in need of treatment with a significant time interval between each administration, wherein said separate dosage forms are ingested by the patient in consecutive times, whereby said components are released to said patient in substantially different times; and

-when these components are formulated together in a single dosage form releasing the components in a controlled manner, this combination of a and therapeutic agent is administered sequentially to a patient in need of treatment, whereby the components are administered simultaneously, consecutively and/or overlapping by the patient at the same and/or different times.

[00113] Suitable examples of other therapeutic agents that may be used in combination with a, their pharmaceutically acceptable salts, and/or their derived forms include, but are not limited to: aromatase inhibitors such as exemestane, formestane, atamestane, fadrozole, letrozole, vorozole and anastrozole; free fatty acid modulators including fibric acid derivatives (e.g., fenofibrate, clofibrate, gemfibrozil, bezafibrate and ciprofibrate) and nicotinic acid derivatives such as acipimox; insulin sensitizers, including, but not limited to, biguanides such as metformin, PPAR γ insulin sensitizers and thiazolodeneiones such as troglitazone and rosiglitazone, troglitazone being 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, U72107, U72107 a, U72107E, ZACTOS), having the chemical name: 2, 4-thiazolidinedione, 5- [ [4- [2- (5-ethyl-2-pyridyl) ethoxy ] phenyl ] methyl ] -, monohydrochloride, (a/-); rosiglitazone (Avandia, BRL 49653C) has the chemical name: 2, 4-thiazolidinedione, 5- [ [4- [2- (methyl-2-pyridylannino) ethoxy ] phenyl ] methyl ]; oral administration of 25 bexarotene (oral LGD 1069, oral Targretin, oral Targretyn, oral Targrexin) with the chemical name: 4- [1- (3, 5,5, 8, 8-pentamethyl-5, 6,7, 8-tetrahydro-2-naphthyl) ethenyl ] benzoic acid; ZD 2079, (ICI D2079) (chemical name: R) -N- [2-4- (carboxymethyl) 30 phenoxy ] ethyl) -N- (2-hydroxy-2-phenylethyl) ammonium chloride: netoglitazone, (Isagllitazone, MCC 555, RWJ 241947) (chemical name: 5- [6 (2-fluorobenzyloxy) naphthalen-2-ylmethyl ] thiazolidine-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) pentaloic 35 acid; HQL 975, chemical name: 3- [4- [2- (5-methyl-2-phenylazol-4-yl) ethoxy ] phenyl ] -2(S) - (propylamino) propionic acid; YM 268, chemical name: 5, 5' -methylene-bis (1, 4-phenylene) bismethylenebis (thiazolidine-2, 4-dione). I PPAR agonists under development include: reglitazar (JTT 501, PNU 182716, PNU 716) (chemical name: isoxazolidine-3, 5-dione, i 4- [ [4- (2-phenyl-5-methyl) -1, 3-oxazolyl ] ethoxyphenyl-4 ] methyl-, (4 RS)); i (RP 297, chemical name: 105- (2, 4-dioxothiazolidin-5-ylmethyl) -2-methoxy-N- [4- (trifluoromethyl) benzylbenzamide, R119702 (CI 1037, CS 011), chemical name (/ -) -5- [4- (5-methoxy-1H-benzimidazol-2-ylmethoxy) benzyl ] thiazoline-2, 4-dione, hydrochloride salt, 15 DRF 2189, chemical name: 5- [ [4- [2- (1-indolyl) ethoxy ] phenyl ] methyl ] thiazolidine-2, 4-dione, cortisol synthesis inhibitors such as ketoconazole, econazole or miconazole, growth hormones such as growth hormone (somatropin) or recombinant growth hormone (somatomerm) and derivatives thereof such as human growth hormone fusion protein such as ALBUTROPIN, polyethylene glycol somatotropin Growth hormones such as cysteine-ethylene glycol growth hormone, BT005(Bolder BioTechnology); growth hormone secretagogues such as SM 130686(Sumitomo) capromorin (Pfizer), mecamylamine (Fujisawa), Sermorelin (Salk institute, Bio-Technology General), recombinant growth hormone (somatrem), somatropin (C Llorente; Pharmacia company) Isarelin, Tamorelin; CP 464709(Pfizer), LY 426410, and LY 444711 (Lilly); 8- (aminoalkoxyimino) -8H-dibenzo [ a, e ] triazolo [4, 5-c ] cycloheptenes as disclosed in WO2002057241, 2-substituted dibenzo [ a, e ]1, 2, 3-triazolo [4, 5-c ] [7] annulen-8-ones as described in WO2002056873, growth hormone releasing peptides GHRP-6 and GHRP-1 as described in U.S. Pat. No.4,411,890 and publications WO 89/07110, WO 89/07111, B-HT920, hexarelin and GHRP-2 or growth hormone releasing hormone (GHRH, also named GRF) and its analogs as described in WO 93/04081, growth hormones including IGF-1 and IGF-2 and derivatives thereof such as somatoKine-insulin-like growth hormone-1 and its binding protein, BP-3, a recombinant fusion protein of 8- (aminoalkoxyimino) -8H-dibenzo [ a, e ] cycloheptenes as described in WO 2002057273, growth hormone-2 and its analogs, growth hormone-like proteins, and its derivatives, Alpha-2-adrenergic agonists such as clonidine, xylazine, dexmeditomidine and medetomidine or 5-hydroxytryptamine 5HTID agonists such as sunnitiptan or agents which inhibit somatostatin or its release such as physostigmine and pyridostigmine, ThGRF 1-44 (Theatetechnologies); l165166 (Merck & Company); dipeptide derivatives as described in WO9858947, dipeptidyl peptidase IV inhibitors as described in US6521644, WO95/15309 and WO98/19998 such as amino-acylpyrrolidinecarbonitrile; beta-amino heterocyclic dipeptidyl peptidase inhibitors such as those described in US20030100563 and WO 2003082817; growth hormone releasing compounds as 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 patent numbers 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 as described in US5804578, US5783582, WO2004007468, Pyrrolidines and hexahydro-1H-aza * types, AMIDOPASPIROPIPERIDINES, such as those described in WO0104119, 2-amino-5-pyrimidineacetic acid compounds, including ethyl 2- [ (5, 6-dimethyl-2-benzimidazolyl) amino ] -4-hydroxy-6-methyl-5-pyrimidineacetate (2) and 2- [ (5, 6-dimethyl-2-benzimidazolyl) amino ] -4-hydroxy-6-methyl-5-pyrimidineacetate as described in US6329383, benzimidazoles as described in EP1155014, similar peptidyl compounds related to GRF and peptides of US patent 4,411,890, antagonists of gonadotropin releasing hormone such as those described in WO0170228, WO0170227, WO0170228, WO 1230069433, WO0004013, W0995156, WO9951595, WO 9951-4, WO 4199251-2, Those described in WO9921557, WO9921553 and 6-azaindole compounds as described in WO0053602, WO0053185, WO0053181, WO0053180, WO0053179, WO0053178, US 6288078; an IGF-1 secretagogue; insulin-like growth hormone-2 (IGF-2 or somatotropin A) and IGF-2 secretagogues; myostatin antagonists and compounds that inhibit fibroblast growth hormone receptor-3 (FGFR-3) tyrosine kinase.

[00114] The included polymers are also preferably water soluble at room temperature. A series of such polymers include poly (alkylene oxide) homopolymers such as poly (ethylene glycol) or poly (propylene glycol), poly (oxyethylenated polyols), copolymers thereof, and block copolymers thereof, but are not limited thereto, so long as the water solubility of the block copolymer is maintained.

[00115] As an alternative to PEG-based polymers, effectively non-antigenic materials such as dextran, poly (vinyl pyrrolidone), poly (polyacrylamide), poly (vinyl alcohol), carbohydrate-based polymers, and the like, may also be used. Indeed, the activation of the α -and ε -end groups of these polymers can be effectively performed in a manner similar to that used to convert poly (alkylene oxides) and thus will be apparent to the skilled artisan. Those of ordinary skill in the art will recognize that the foregoing list is merely illustrative and that all polymers having the properties described herein are contemplated. For purposes of the present invention, "effectively non-antigenic" refers to all materials known in the art to be non-toxic and not elicit a significant immunogenic response in mammals.

Definition of

[00116] The following is a list of abbreviations and corresponding meanings that may be used interchangeably herein:

g

mg of

mL or mL

RT Room temperature

PEG poly (ethylene glycol)

[00117] As each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference herein, the entire contents of all publications, patents, and patent applications cited in this disclosure are hereby incorporated by reference herein.

[00118] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that changes and modifications may be made thereto without departing from the spirit and scope of the invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention, which has been described above in broad terms.

[00119] In the examples below, hGH is SEQ ID NO: 1 hGH. It will be appreciated that other members of the hGH or agonist variant family of polypeptides may also be pegylated in a manner similar to that exemplified in the examples hereinafter.

Examples

Example 1

Branched 40,000MW PEG-butyraldehyde hGH

[00120]This example illustrates the production of a substantially homogeneous, N-terminally monopegylated hGH formulation by reductive alkylation. Using N-terminal amino acidsAmine relative pK_aValue versus pK for the primary amine at the epsilon-amino site of the lysine residue_aIn contrast, a methoxy-branched PEG butyraldehyde reagent of about 40,000MW (Shearwater Corp.) was selectively conjugated to the N-terminus of hGH by reductive amination. hGH protein dissolved at 10mg/mL in 25mM Hepes (Sigma Chemical St. Louis, Mo.) pH 7.0 (optionally 25mM MES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 10mM sodium acetate (Sigma Chemical, St. Louis, Mo.) pH 4.5) was reacted with methoxy-PEG-butyraldehyde, M-PEG-ALD (Shearwater, Huntsville, AL) by adding M-PEG-ALD to produce a relative PEG: hGH molar ratio of 2: 1. The reaction was catalyzed by adding 1M NaCNBH4 stock solution (Sigma Chemical, St. Louis, Mo.) dissolved in water to a final concentration of 10-50 mM. The reaction was carried out at RT for 18-24 hours in the dark. Immediate purification was achieved by stopping the reaction by adding 1M Tris (Sigma Chemical, St. Louis, Mo.) to a final concentration of Tris of pH 7.6 to 50mM or by diluting the reaction into an appropriate buffer.

[00121] Table 1 shows the percent of poly-pegylated species, mono-pegylated conjugates, unreacted PEG, and the final purification yields of 40K branched PEG-acetaldehyde and 40K branched PEG-butyraldehyde as determined by size exclusion chromatography. PEG-butyraldehyde results in increased mono-pegylated conjugates, decreased unreacted PEG levels, and increased final yields compared to PEG-acetaldehyde.

TABLE 1

Comparison of 40K branched PEG-ALD-hGH and 40K branched PEG-butyraldehyde-hGH
			Species in the reaction mixture
	40K PEG-acetaldehyde hGH	40K PEG-butyraldehyde-hGH
Poly-PEG products	4.02％	5.03％
			mono-PEG products	48.70％	61.02％
Unreacted hGH	41.80％	29.20％
Final purification yield	30.80％	44.70％

Example 2

Straight chain 30,000MW PEG-butyraldehyde hGH

[00122] The methoxy-linear 30,000 MWPEG-butyraldehyde reagent was conjugated to the N-terminus of hGH using the method described in example 1.

Example 3

Straight chain 20,000MW PEG-butyraldehyde hGH

[00123] The methoxy-linear 20,000 MWPEG-butyraldehyde reagent was conjugated to the N-terminus of hGH using the method described in example 1.

Example 4

Purification of Pegylated hGH

[00124] The pegylated hGH species was purified from the reaction mixture to > 95% using a single ion exchange chromatography step (SEC analysis).

Anion exchange chromatography

[00125]The PEG hGH species was purified from the reaction mixture to > 95% using a single anion exchange chromatography step (SEC analysis). Single-pegylated hGH was purified from unmodified hGH and multi-pegylated hGH using anion exchange chromatography. A typical 20K butyraldehyde hGH reaction mixture (5-100mg protein) was purified as described above on a Q-Sepharose Hitrap column (1 or 5mL) (Amersham Pharmacia Biotech, Piscataway, N.J.) or a Q-Sepharose flow column (26/20, 70mL bed volume) equilibrated in 25mM HEPES, pH 7.3 (buffer A). The reaction mixture was diluted 5-10 fold with buffer A and loaded onto the column at a flow rate of 2.5 mL/min. The column was washed with 8 column volumes of buffer a. Subsequently, the various hGH species were eluted from the column with 80-100 column volumes of buffer A and a linear NaCl gradient of 0-100 mM. At 280nm (A)₂₈₀) The eluate was monitored for absorbance, and 5mL fractions were collected. Fractions were pooled according to degree of pegylation, e.g., mono-, di-, tri-pegylated, etc. (as assessed in example 15). The combined fractions were then concentrated to 0.5-5mg/mL in a Centriprep YM10 concentrator (Amicon, Technology, Inc., Northborough, Mass.). An extinction coefficient of 0.78 was used, passing A₂₈₀The protein concentration of the pooled fractions was determined.

Cation exchange chromatography

[00126] Cation exchange chromatography was performed on a SP Sepharose high performance column (Pharmacia XK 26/20, 70ml bed volume) equilibrated in 10mM sodium acetate pH 4.0 (buffer B). The reaction mixture was diluted 10-fold with buffer B and loaded onto the column at a flow rate of 5 mL/min. The column was then washed with 5 column volumes of buffer B followed by 5 column volumes of 12% buffer C (10mM acetate pH 4.5, 1M NaCl). The PEG-hGH species was then eluted from the column with a linear gradient of 12-27% buffer C over 20 column volumes. The eluate was monitored at 280nm and 10mL fractions were collected. Fractions were pooled according to degree of pegylation (mono-, di-, tri-, etc.), exchanged into 10mM acetate pH 4.5 buffer and concentrated to 1-5mg/mL in stimulated cells with Amicon YM10 membrane. The protein concentration of the pooled fractions was determined by a280nm using an extinction coefficient of 0.78.

Example 5

Biochemical identification

[00127] The pooled fractions of purified pegylated hGH were identified by non-reducing SDS-PAGE, non-denaturing size exclusion chromatography and peptide chromatography.

Size exclusion high performance liquid chromatography (SEC-HPLC)

Undenatured SEC-HPLC

[00128] Ligation chemistry, size, linker and geometric diversity of methoxy-PEG reaction with hGH, anion exchange purification of pooled fractions and final purified product were evaluated using undenatured SEC-HPLC. Analytical non-denaturing SEC-HPLC was performed at a flow rate of 0.5 mL/min in 20mM phosphate pH 7.2, 150mM NaCl using a column Superdex 2007.8 mM × 30cm (Amersham Bioscience, Piscataway, N.J.) (optionally TosohaasG4000PWXL Amersham Bioscience, Piscataway, N.J.). Pegylation greatly increases the hydrodynamic volume of the protein, resulting in a shift to an earlier retention time. New species and unmodified hGH can be observed in PEG-acetaldehyde hGH reaction mixtures. These pegylated and non-pegylated species were separated on Q-Sepharose chromatography and the resulting purified mono-PEG-acetaldehyde hGH species was subsequently shown to elute as a single peak (> 95% purity) on non-denaturing SEC. The Q-Sepharose chromatography step effectively removed free PEG, hGH and polyethylenglycolated hGH species from the mono-pegylated hGH.

Denaturing SEC-HPLC

[00129] Reaction of butyraldehyde polyethylene glycol with hGH, anion exchange purification, and final purified product were evaluated using denaturing SEC-HPLC. Analytical denaturing SEC-HPLC was performed at a flow rate of 0.8 mL/min using a Tosohaas 3000SWXL column of 7.8mM × 30cm (Tosohaas Pharmacia Biotech, Piscataway, NJ) in 100mM phosphate pH 6.8, 0.1% SDS. Pegylation greatly increases the hydrodynamic volume of the protein, resulting in a shift to an earlier retention time. The pegylated and unpegylated species were separated on Q-Sepharose chromatography.

SDS PAGE/PVDF transition

[00130]SDS-PAGE was used to evaluate the reaction of PEG butyraldehyde with hGH and the purified final product. SDS-PAGE was performed on 1mm thick 10-20% Tristricine gels (Invitrogen, Calsbarda) under reduced and unreduced conditions and with Novex Colloidal Coomassie^TMStaining with G-250 staining kit (Invitrogen, Calsbad, Calif.). Bands were imprinted onto PVDF membranes for subsequent N-terminal sequence identification.

Analytical anion exchange HPLC

[00131] Analytical anion exchange HPLC was used to evaluate the PEG butyraldehyde/hGH reaction mixture, the anion exchange purification fractions, and the final purified product. Analytical anion exchange HPLC was performed at a flow rate of 1 mL/min using a Tosohaas Q5PW or DEAE-PW anion exchange column, 7.5mm × 75mm (Tosohaas Pharmacia Biotech, Piscatavir, N.J.) at 50mM Tris ph 8.6. The sample was eluted with a linear gradient of 5-200mM NaCl.

N-terminal sequences and peptide spectroscopy

[00132]Automated Edman degradation chemistry for NH determination₂-terminal protein sequence. An Applied Biosystems Model 494 Procise sequencer (Perkin Elmer, Welsli, Mass.) was used for degradation. Individual PTH-AA derivatives were identified by RP-HPLC analysis in an on-line fashion using an Applied Biosystems Model140C PTH analyzer equipped with a PerkinElmer/Brownlee 2.1mm i.d. PTH-C18 column.

[00133] Trypsin digestion was performed at a concentration of 1mg/mL, and typically 50. mu.g of material was used per digestion. Trypsin was added so that the ratio of trypsin to PEG-hGH was 1: 30 (w/w). Tris buffer was 30mM, pH 7.5. The samples were incubated at room temperature for 16. + -. 0.5 hours. The reaction was quenched by the addition of 50. mu.L of 1N HCl per mL of digestion solution. The samples were diluted in 6.25% acetonitrile to a final concentration of 0.25mg/ml before being placed in an autosampler. Acetonitrile (19.8% acetonitrile) was added first, stirred gently, and then water was added to the final volume (4 times the initial volume). Additional digestion solution may be removed and stored at-20 ℃ for one week.

[00134] A Waters Alliance 2695HPLC system was used for the analysis, but other methods should produce similar results. The column used was a 25cm x 4.6mm column based on AstecC-4 polymer with 5 μm particles. The experiment is usually carried out at ambient temperature with 50. mu.g protein per sample. Buffer a was 0.1% trifluoroacetic acid in water; buffer B was 0.085% trifluoroacetic acid in acetonitrile. The gradient is as follows:

time A% B% C% D% flow Curve

0.00 0.0 0.0 100.0 0.0 1.000 1

90.00 0.0 0.0 55.0 45.0 1.000 6

90.10 0.0 0.0 0.0 100.0 1.000 6

91.00 0.0 0.0 0.0 100.0 1.000 6

91.10 0.0 0.0 100.0 0.0 1.000 6

95.00 0.0 0.0 100.0 0.0 1.000 6

[00135] The column was heated to 40 ℃ using a heating mantle. Data between 210 and 300nm were collected with a Waters 996 PDA detector to detect peaks. Chromatography at 214nm extraction was used for sample analysis.

[00136] Trypsin mapping analysis was performed on hGH, 40K branched PEG-aldehyde and 40K branched butyraldehyde (FIG. 1). The N-terminal trypsin fragment is designated T-1. The percentage of T-1 present compared to non-PEGylated hGH is shown in Table 2. The data show that 90% of the PEG modifications using PEG-aldehyde are at the N-terminus while the others are apparently attached to one of several possible lysine residues, whereas with PEG-butyraldehyde more than 98% of the modifications are at the N-terminus.

TABLE 2

	% of T-1 present	T-1 present in% compared to non-PEGylated hGH
			hGH	28.0％
PEG-acetaldehyde/hGH	2.6％	9.2％
			PEG-butyraldehyde/hGH	0.3％	1.2％

Example 6

Study of drug efficacy

Weight gain in rats

[00137] Female Sprague Dawley rats hypophysectomized at Taconic Lab were pre-screened for growth rates for 7-11 days. Rats were divided into 8 groups. The rats of group 1 were administered vehicle daily or daily on day 0 and day 6 subcutaneous doses. Rats of group2 were administered GH (30 μ g/rat/dose) at daily subcutaneous doses. Group 3 rats were administered GH (180. mu.g/rat/amount) subcutaneously on day 0 and day six. Group 4 rats were dosed subcutaneously with 40k of branched PEG-butyraldehyde-hGH (180 μ g/rat/amount) on day 0, day six. Weight gain in hypophysectomized rats was monitored by weighing at least every other day during the study. Fig. 3& 4.

Tibial length of rat

[00138] Animals in the 11 day weight gain study were sacrificed on day 11, the left tibia was removed and examined with X-ray and bone length was measured using calipers. FIG. 5

Study of IGF-1

[00139] Animals from a six-day weight gain study were used. Blood samples were taken at various times during the study and serum IGF-1 levels were determined by ELISA. FIG. 6

Serum biochemical study

[00140] As shown in Table 3, animals in the 11 day weight gain study were used to assess biochemical values of serum at day 7 after cumulative administration of 1.8mg/kg at day 0 and day six.

TABLE 3

Assay substance	Carrier	hGH (11 days 300. mu.g/kg/day)	PEG-hGH (1.8 mg/kg on day 0 and sixth day)
				ALB(g/dL)ALP(U/L)ALT(U/L)AST(U/L)BUN(mg/dL)Ca2+(mg/dL) Cholesterol (mg/dL) CRE (mg/dL) glucose (mg/dL) phosphate (mg/dL) TP (g/dL)	4.07±0.04311±1553.6±2.4149±737.4±1.610.9±0.185.1±2.80.62±0.0273.4±4.78.26±0.286.36±0.10	4.06±0.05309±1651.1±2.2131±426.7±1.5*11.5±0.1*76.9±3.6*0.60±0.0179.6±4.79.59±0.16*6.48±0.10	4.18±0.03**280±1451.3±2.6137±1127.9±0.6*11.0±0.1*115.3±2.8**0.59±0.0167.1±8.98.42±0.23*6.39±0.06

Assay substance	Carrier	hGH (11 days 300. mu.g/kg/day)	PEG-hGH (1.8 mg/kg on day 0 and sixth day)
				TBA (. mu.mol/L) SDH (U/L) triglyceride (mg/dL) LDH (U/L) Na+(mmol/L)K+(mmol/L)Cl-(mmol/L)	10.0±0.511.9±1.057.4±4.0786±72147±0.55.94±0.09103±0.5	7.6±0.4*9.7±1.447.8±5.7762±94146±0.86.31±0.16*102±0.5	11.0±0.4*10.6±0.745.7±3.9*914±189145±0.6*6.71±0.17**102±0.6

P < 0.10 ratio vector, * p < 0.05 ratio hGH

ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; ca²⁺Calcium; chol, cholesterol; CRE, creatinine; phos, phosphate; TP, total protein amount; TBA, total bile acid; SDH, sorbitol dehydrogenase; LDH, lactic acid depletionA catalase; na (Na)⁺Sodium; k⁺Potassium; cl^-And chlorine.

[00141] On day 11, the blood urea nitrogen concentration in the animals treated with hGH and PHA-794428 was significantly reduced to the same extent (p < 0.10) relative to the vehicle-treated group. This indicates enhanced nitrogen utilization due to enhanced synthesis of new proteins during growth.

Pharmacokinetic Studies

[00142] Pharmacokinetic studies were performed in normal, intubated Sprague-Dawley male rats. 6 rats were used per group and 100. mu.g/kg/rat of GH or PEG-GH were injected as a single subcutaneous bolus. Blood samples were taken for testing of the corresponding PK parameters, suitably on days 1-5. The blood concentrations of GH and PEG-GH were monitored for each sample using an immunoassay.

hGH immunoassay

[00143] Protein concentration levels of hGH and pegylated hGH in mouse and cynomolgus plasma were determined using the hGH AutoDELFIA kit fluorescence immunoassay (PerkinElmer). Rat and human IGF-1 levels were monitored by immunoassay kits (Diagnostic System Laboratories).

Atrioventricular pharmacokinetic properties of the hGH-PEG conjugate of example 1 in a non-human primate.

[00144] The hGH-PEG conjugate of example 1 was administered to cynomolgus monkeys (table 4) at 0.18mg/kg intravenous (iv) or subcutaneous (sc) bolus injection. PK parameters were determined from the mean data of 3 animals. Plasma concentrations were measured using the AutoDELFIA kit fluorescence immunoassay (Perkinelmer) and a pre-determined standard curve for the PEG-GH conjugate.

TABLE 4

Amount (mg/kg)	Intravenous 0.18/subcutaneous 0.18
		CL, intravenous (ml/hr/kg)	0.8
Vss(ml/kg)	28.0
		T1/2, intravenous (hours)	25.0
T1/2, subcutaneous (hours)	61.2
		SC AUC (μ g/ml hour)	195
SC bioavailability (%)	84
		Tmax, subcutaneous (hour)	32

Sequence listing

Sequence listing

<110>Pharmacia Corporation

Finn，Rory

<120> N-terminal mono-PEGylated human growth hormone conjugates, methods for making them, and methods of use thereof

<130>32152A

<150>10/771,895

<151>2004-02-04

<160>1

<170>PatentIn version 3.2

<210>1

<211>191

<212>PRT

<213> Intelligent people

<400>1

Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg

1 5 10 15

Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu

20 25 30

Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro

35 40 45

Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg

50 55 60

Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu

65 70 75 80

Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Ser Leu Arg Ser Val

85 90 95

Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp

100 105 110

Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu

115 120 125

Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser

130 135 140

Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr

145 150 155 160

Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe

165 170 175

Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe

180 185 190

Claims (10)

1. A method for the prevention and/or treatment of a disease or condition in which the use of growth hormone is beneficial, said method comprising separately administering to a patient in need thereof a therapeutically effective amount of a poly (ethylene glycol) -modified hGH having the structure of formula I or II,

formula I

Or

mPEG-O(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 human growth hormone or methionyl growth hormone,

or in combination with another therapeutic agent, wherein said disease or condition for which the use of growth hormone is beneficial is selected from the group consisting of erectile dysfunction, HIV lipodystrophy, fibromyalgia, osteoporosis, memory disorders, depression, crohn's disease, skeletal dysplasia, traumatic brain injury, subarachnoid hemorrhage, noonan's syndrome, down's syndrome, congenital short stature (ISS), advanced renal disease (ESRD), Very Low Birth Weight (VLBW), bone marrow stem cell rescue, metabolic syndrome, glucocorticoid myopathy, short stature in children due to glucocorticoid therapy, and short, premature children with concomitant growth deficit.

2. The method of claim 1, wherein the disease or condition in which use of GH is beneficial is selected from the group consisting of congenital short stature, very low birth weight, traumatic brain injury, metabolic syndrome, and noonan syndrome.

3. The method of claim 1 or 2, wherein n equals 4 and m equals 3.

4. The method of claim 3, wherein said poly (ethylene glycol) modified hGH has a structure of formula I with n equal to 4 and m equal to 3.

The method of claim 1, wherein said human growth hormone comprises the amino acid sequence of SEQ ID NO: 1.

6. The method of claim 5, wherein greater than 90% of the polyethylene glycol binds to the amino acid sequence of SEQ ID NO: 1 on the amino terminal phenylalanine of the amino acid sequence of 1.

7. The method of claim 6, wherein greater than 95% of the polyethylene glycol binds to the amino acid sequence of SEQ ID NO: 1 on the amino terminal phenylalanine of the amino acid sequence of 1.

8. The method of claim 1, wherein each mPEG has a molecular weight of about 20 kDa.

9. A composition comprising a conjugate of human growth hormone-PEG of formula I or II in combination with another therapeutic agent and at least one pharmaceutically acceptable carrier,

formula I

Or

mPEG-O(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 human growth hormone or methionyl growth hormone.

10. The composition of claim 9, wherein said poly (ethylene glycol) modified hGH has a structure of formula I wherein n is equal to 4 and m is equal to 3.