KR20070042567A - Glycerol branched polyethylene glycol human growth hormone conjugates, process for their preparation, and methods of use thereof - Google Patents

Abstract

The present invention relates to PEGylation of human growth hormone (hGH) using glycerol branched chain PEG. The present invention also relates to a method for PEGylating hGH. The present invention also relates to pharmaceutical compositions comprising PEGylated hGH. A further embodiment is the use of PEGylated hGH for the treatment of growth or developmental disorders.

Description

Glycerol BRANCHED POLYETHYLENE GLYCOL HUMAN GROWTH HORMONE CONJUGATES, PROCESS FOR THEIR PREPARATION, AND METHODS OF USE THEREOF}

The present invention relates to PEGylation of human growth hormone (hGH), by which PEGylation can change the chemical and / or physiological properties of hGH. PEGylated hGH conjugates can have increased plasma residence time, reduced clearance rate, improved stability, reduced antigenicity, reduced PEGylation heterogeneity, or a combination thereof. The invention also relates to a method of modifying hGH. In addition, the present invention relates to pharmaceutical compositions comprising modified hGH. A further embodiment is the use of modified hGH for the treatment of growth and developmental disorders.

This application claims priority under U.S. Provisional Application No. 60 / 605,945, filed Aug. 31, 2004, under Section 119, which is incorporated herein by reference in its entirety, as described herein.

Natural hGH is a protein comprising a single chain of 191 amino acids cross-linked by two disulfide bridges and the monomeric form has a molecular weight of 22 kDa. Human GH is secreted by the pituitary gland and can also be produced by recombinant genetic engineering techniques. hGH will cause growth in all body tissues that can grow. hGH not only plays an important role in promoting growth during human growth in humans, but also plays an important role in maintaining normal body composition, assimilation and lipid metabolism (K. Barneis. And U. Keller, Baillieres Clin. Endocrinlo. Metab. 10: 337 (1996)).

Recombinant hGH has been on the market for many years. Two types of recombinant hGH preparations useful as a therapeutic that is, regular, for example, in the notes pin (Genotropin ^TM) or neutroavidin pin (Nutropin ^TM), and analogs, for example with an additional methionine residue at the N- terminus, Soma Somatonorm ^™ is commercially available. hGH is used to stimulate linear growth in patients with hypopituitary dwarfism, also known as growth hormone deficiency (GHD) or Turner's syndrome, but in young children born during pregnancy Other uses have also been proposed, including long-term treatment of failure and treatment of patients with Prader-Willi syndrome (PWS), chronic renal failure (CRI), AIDS depletion or aging. Adult growth hormone deficiency (aGHD) patients have a variety of problems, including increased fat mass, reduced lean body mass and extracellular fluids, reduced bone mineral density, metabolic abnormalities in lipids, and cardiovascular function. Have characteristic changes in body composition, including disability. Many of these problems are ameliorated by hGH replacement therapy (J. Verhelst J and R. Abs. Drugs .; 62: 2399 (2002)).

The main biological effect of growth hormone (GH) is to promote growth in young mammals and to maintain tissue in older mammals. Affected organ systems include skeletal, connective tissue, muscle, and intestines such as the liver, intestines, and kidneys. GH exerts its effect through interaction with specific receptors on target cell membranes. hGH is a member of the cognate hormone class, including placental lactogen, prolactin, and other genetic and species variants of GH (Nicoll, CS, et al. (1986) Endocrine Reviews 7: 169). hGH exhibits a wide range of species specificities and is expressed in cloned somatogenic receptors (Leung, DW, et al. [1987] Nature 330; 537) or prolactin receptors (Boutin, JM, et al. [1988] Cell; 53: 69). It binds from other hormones in these hormone classes. The cloned gene for hGH is Escherichia coli ) in secreted form (Chang, CN, 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).

hGH participates in most of the normal human growth and developmental regulation. This pituitary hormone has a number of biological effects, including linear growth (somatogenesis), lactation, activation of macrophages, in particular insulin-like and diabetic effects (Chawla, R, K. (1983) Ann. Rev. Med. 34, 519; Edwards, CK et al. (1988) Science 239, 769; Thomer, M. 0., et al. (1988) J. CHn. Invest. 81: 745). GH deficiency in children results in small certification, which has been successfully treated for more than 10 years by external administration of hGH.

In children as well as adults, hGH maintains normal body composition through increased nitrogen reserves, stimulation of skeletal muscle growth, and mobilization of body fat. Visceral adipose tissue responds specifically to hGH. In addition to enhanced lipolysis, hGH reduces the entry of triglycerides into body fat deposits. Serum concentrations of IGF-1 (insulin-like growth factor-1) and IGFBP3 (insulin-like growth factor binding protein 3) are increased by hGH.

hGH is a powerful anabolic agent, especially because it contains nitrogen, phosphorus, potassium and calcium. Treatment of pituitary rats with GH can restore at least a portion of the growth rate of these rats (Moore et al., Endocrinology 122: 2920-2926 (1988)). The most striking of the effects of GH in patients with pituitary insufficiency (GH-deficiency) is accelerated linear growth of bone-growth-plate-cartilage that increases kidneys (Kaplan, Growth Disorders in Children and Adolescents (Springfield, IL: Charles C. Thomas, 1964).

hGH exhibits a variety of physiological and metabolic effects, including linear bone growth, lactation, macrophage activation, insulin-like and diabetic effects, etc. in various animal models (RK Chawla et al., Annu. Rev. Med. 34: 519 (1983); 0. GP lsaksson et al., Annu. Rev. Physiol. 47, 483 (1985); CK Edwards et al., Science 239, 769 (1988); M. 0. Thomer and ML Vance , J. Clin. Invest. 82: 745 (1988); JP Hughes and HG Friesen, Ann. Rev. Physiol. 47: 469 (1985)). It has been reported that GH secretion decreases with age, especially in postmenopausal women (Millard et al., Neurobiol. Aging, 11: 229-235 (1990); Takahashi et al., Neuroendocrinology M, L6-137-142 (1987)). ). See also Rudman et al., J. Clin. Invest., 67: 1361-1369 (1981) and Blackman, Endocrinology and Aging, 16: 981 (1987). Moreover, there are reports that several of the signs of aging, including decreased defatting weight, increased fat tissue weight, and thinning of the skin, can be alleviated by three GH treatments per week. See, eg, Rudman et al., N. Eng. J. Med, 323: 1-6 (1990) and articles published in the same journal publication by Dr. Vance (pp. 52-54). See. This biological effect is derived from the interaction between hGH and specific cellular receptors. Two different human receptors, hGH liver receptors (DW Leung et al., Nature 330: 537 (1987)) and human prolactin receptors (JM Boutin et al., Mol. Endocrinology. 3: 1455 (1989)) were cloned have. However, other receptors, including human placental lactogen receptors, are also possible (M. Freemark, M. Comer, G. Komer, and S. Handwerger, Endocrinol. 120: 1865 (1987)). These cognate receptors have glycosylated extracellular hormone binding domains, single transmembrane domains and cytoplasmic domains that differ significantly in sequence and size. It is assumed that one or more receptors play a critical role in the physiological response to hGH.

In general, it is observed that physiologically active proteins administered into the body may exhibit their pharmacological activity only for a short time due to their high removal rate in the body. In addition, the relative hydrophobicity of these proteins can limit their stability and / or solubility.

Several methods of chemically modifying proteins into water-soluble polymers have been proposed to reduce the removal rate of therapeutic proteins, improve stability, or eliminate antigenicity. This type of chemical modification can prevent degradation by effectively blocking proteolytic enzymes from physical contact with the protein backbone itself. Chemical bonding of some water soluble polymers can effectively reduce elongation removal due to increased hydrodynamic volume of the molecule. Additional advantages in certain cases include increased stability and circulation time, increased solubility, and reduced immunogenicity of the therapeutic protein. Poly (alkylene oxide) In particular, poly (ethylene glycol) (PEG) is a chemical moiety used in the preparation of therapeutic protein products (the verb “pegylating” means binding one or more PEG molecules). The binding of poly (ethylene glycol) has been shown to protect against proteolysis (Sada, et al., J. Fermentation Bioengineering 71: 137-139 (1991)), and methods of binding specific poly (ethylene glycol) residues are used. It is possible. US Pat. No. 4,179,337, issued December 18, 1979 to Davis et al., Non-lmmunogenic Polypeptides; And US Patent No. 4,002,531 issued January 11, 1977 to Royer, Modifying Enzymes with Polyethylene Glycol and Product Produced Thereby. See Abuchowski et al., In Enzymes as Drugs. (J. S. Holcerberg and J. Roberts, eds. Pp. 367-383 (1981)) for review.

Other water-soluble polymers such as copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, poly (vinyl alcohol), poly (vinyl pyrrolidone), poly (-1,3-dioxolane), poly (-1 , 3,6-trioxane), ethylene / maleic anhydride copolymer, poly-amino acid (homopolymer or random copolymer) are used.

In the case of poly (ethylene glycol), various means are used to bind the poly (ethylene glycol) molecules to the protein. In general, poly (ethylene glycol) molecules are bound to a protein through reactive groups found on the protein. Amino groups such as amino groups on lysine residues or amino groups at the N-terminus are convenient for this linkage. For example, Royer (US Pat. No. 4,002,531, supra) states that reductive alkylation was used to bind poly (ethylene glycol) molecules to enzymes. Chamow et al., Bioconjugate Chem. 5: 133-140 (1994) report the modification of monomethoxypoly (ethylene glycol) aldehydes to CD4 immunoconjugates via reductive alkylation. US Pat. No. 5,824,784 demonstrates PEGylation of G-CSF, including PEGylation at the N-terminus under reducing alkylation conditions.

WO 93/00109 relates to a method of stimulating a GH reactive tissue of a mammal or a bird comprising maintaining a continuous and effective plasma GH concentration for a period of at least 3 days. One way to achieve such plasma concentrations is described using GH coupled to macromolecular materials such as PEG (polyethylene glycol). Coupling with macromolecular materials is described to improve half-life. As known (Knauf, MJ et al, J. Biol. Chem. 263: 15064-15070, 1988) mPEG aldehyde-5000 and mPEG N to achieve hydrodynamic volumes exceeding the 70K molecular weight cut-off of kidney filtration. PEGylated hGH using hydroxysuccinimidyl ester (mPEG-NHS-5000) is reported in WO 93/00109. The use of mPEG-NHS results in a heterogeneous mixture consisting of multiple PEGylated forms of hGH. In addition, WO 93/00109 discloses the use of mPEG-maleimide for PEGylating cysteine hGH variants.

WO 99/03887 discloses cysteine variant GH that is PEGylated. This conjugate, referred to as BT-005, is reported to be more effective at stimulating weight gain in GH deficient rats and to have a longer lifespan than hGH.

HGH PEGylated with succinimidyl esters of carboxymethylated PEG is also reported in Clark et al., Journal of Biological Chemistry 271: 21969-21977, 1996. This document describes hGH derivatives of increased size using mPEG-NHS-5000, which is selectively conjugated to primary amines. Increasing the PEG modification level reduced its affinity for the receptor and increased the EC ₅₀ by 1500-fold in cell-based assays. Olson et al., Polymer Preprints 38: 568-569, 1997, disclose N-hydroxysuccinimide (NHS) PEG and succinimidyl propionate (SPA) PEG to achieve multiple PEGylated hGH species. Initiate the use of.

WO 94/20069 discloses the expectation of PEGylated hGH as part of an agent for pulmonary delivery.

US Pat. No. 4,179,337 discloses a method of PEGylating enzymes and hormones to obtain water soluble polypeptide conjugates having physiological activity and non-immunogenicity. GH is mentioned as an example of the hormone being PEGylated.

EP 458064 A2 discloses PEGylation of cysteine residues introduced into or naturally present in somatotropin. European Patent 458064 A2 further mentions the introduction of two cysteine residues into a loop called an omega loop which is described as being located at residues 102-112 of wild type bovine somatotropin, more specifically European Patent No. 458064 A2 discloses replacing residues 102 and 112 of bovine somatotropin with Ser to Cys and Tyr to Cys, respectively.

WO 95/11987 proposes binding PEG to thiol groups of cysteine residues present in the parent molecule or introduced by site directed mutagenesis. WO 95/11987 relates to the PEGylation of protease Nexin-1, but general PEGylation of hGH and other proteins is also proposed.

WO 99/03887 discloses GH modified by, for example, replacing the serine at position 25 with a cysteine residue and binding PEG to the introduced cysteine residue.

WO 00/42175 relates to a method of preparing a protein having free cysteine residues for the binding of PEG. WO 00/42175 discloses muteins T3C, S144C and T148C, and their cysteine PEGylation of hGH.

WO 97/11178 (and US Pat. Nos. 5,584,535, 6004931, and 602711) relate to the use of GH variants as agonists or antagonists of hGH. WO 97/11178 also discloses PEGylation of hGH, including lysine PEGylation and the introduction or substitution of lysine (eg, K168A and K172R). International Patent Application Publication No. WO 9711178 also discloses substitution G120K.

WO 03/044056 discloses various PEGylated hGH species, including lysine branched chain 40K PEG aldehyde hGH conjugates.

US patent application 2004/0127417 discloses a lysine branched PEG butyraldehyde hGH conjugate.

International Patent Application Publication No. WO 04/46222, US Patent Application No. 2005/0058620, Japanese Patent Application No. 08-059818, Japanese Patent Application No. 11-228685 and Japanese Patent Application No. 2000-001541 Disclosed are polyalkylene glycol derivatives having a reactive group at the primary carbon at position 1 and having a polyalkylene glycol chain at positions 2 and 3.

Since rhGH is currently administered daily for long periods of time, it would be highly desirable to lower the frequency of administration. HGH molecules with longer circulating half-lives can provide more optimal therapeutic hGH levels while reducing the number of administrations required and accompanying enhanced therapeutic effects.

Despite many attempts to develop long-lasting forms of hGH, including PEGylation of hGH, there is still a need for PEGylated hGH molecules with properties suitable for practical use. The present invention provides PEG-hGH conjugates with a single PEG bound primarily to the N-terminal phenylalanine of hGH, which provides an advantage over other PEG-hGH conjugates. Multiple low molecular weights at the α-amino site or ε-amino site (N-terminus and 9 lysines in hGH) using mPEG aldehyde-5000 or mPEG N-hydroxysuccinimidyl ester (mPEG-NHS-5000) (5 Kd) PEG binding methods are described in WO 93/00109, Clark et al., Journal of Biological Chemistry 271: 21969-21977, 1996, and Olson et al., Polymer. Preprints 38: 568-569, 1997). This method results in a heterogeneous population. For example, hGH with 9 lysines may have several molecules bound with 10 PEGs, some molecules bound with 9 PEGs, some molecules bound with 8 PEGs, some molecules bound with 7 PEGs, 6 PEGs Several molecules bound to one molecule, some molecules bound to five PEGs, some molecules bound to four PEGs, several molecules bound to three PEGs, several molecules bound to two PEGs, several molecules bound to one PEG And several molecules to which PEG is not bound. Among the molecules with several PEGs, PEGs may not be bound at the same position on different molecules. The method resulting in inhomogeneity is a disadvantage that makes conjugation, purification and characterization difficult, expensive and highly reproducible when developing therapeutics. Another method (WO 00/42175) is to use hGH variants that carry free cysteine residues for the binding of PEG. However, this method produces incorrectly folded proteins with inaccurate paired disulfide bonds, resulting in non-natural hGH variants and resulting in heterogeneous PEGylation products with PEG bound in some cysteines or all cysteines. You can also Having multiple PEGs bound at multiple sites can result in molecules with more labile bonds between the PEG and the various sites, which can dissociate at different rates. This makes it difficult to accurately predict the pharmacokinetics of the product resulting in incorrect dosing. Heterogeneous products also add unwanted problems in obtaining regulatory approval for therapeutic agents.

Thus, it would be desirable to have a PEGylated hGH molecule with a single PEG bound at a single site. The present invention addresses this need in a number of ways.

1 is a size exclusion HPLC report showing the elution profile of the purified single-PEGylated glycerol branched 43K PEG aldehyde hGH reaction product on TSK G4000PWXL column.

FIG. 2 is an HPLC record of trypsin map analysis of hGH and glycerol branched 43K PEG aldehyde hGH. The top panel is the trypsinization map of hGH. Bottom panel is trypsinization map of glycerol branched chain 43K PEG aldehyde hGH. T1 is an N-terminal trypsin fragment.

3 shows the amino acid sequence of hGH (SEQ ID NO: 1).

4 shows the efficacy of glycerol branched chain 43K PEG aldehyde hGH in an 11-day rat weight gain assay. Pituitary female Sprague-Dawley rats, 4 to 5 weeks old, were purchased from Harlan Labs. When housed in an animal facility, the animals were maintained at a constant room temperature of 80 ° F. After 3 days of acclimation, the rats were weighed daily for 4-10 days to establish basal growth rate. Then, starting from 0, the rats (about 100 g) in the control group for the continuous about 0.3 mg / kg hGH (▲) or PBS vehicle (●) were subcutaneously injected daily for 11 days once. Glycerol branched 43K PEG aldehyde hGH test group (■) who were administered with 1.8 mg / kg of glycerol branched 43K PEG aldehyde hGH chain single dose on days 0 and 6 days. There were six animals per army. Constructed values represent mean weight gain ± SEM.

Figure 5 shows tibial growth for 11 days in response to glycerol branched chain 43K PEG aldehyde hGH. The animal was the animal treated in FIG. At 11 days, the animals were sacrificed, the animals were sacrificed, X-rays were taken of the left tibia, and bone lengths were measured using a caliper. Average length +/- SEM was constructed. Asterisks indicate significant differences from control (P <0.05). There were six animals per group.

FIG. 6 shows 7 days of blood urea nitrogen levels in response to glycerol branched chain 43K PEG aldehyde hGH. Blood samples were taken from the animals treated in FIG. Serum was prepared and urea nitrogen levels were measured. Mean ± SEM was constructed (6 animals per group). Asterisks indicate significant differences from control (P <0.05).

FIG. 7 shows a six day dose escalation efficacy study for glycerol branched chain 43K PEG aldehyde hGH. This growth study was performed in a manner similar to that described in FIG. 4 except that various single doses of glycerol branched chain 43K PEG aldehyde hGH were administered on day 0 only and the study was conducted for 6 days. The control group was subcutaneously injected once daily with 0.3 mg / kg hGH (◆) or PBS vehicle (○) for 6 consecutive days. The glycerol branched chain 43K PEG aldehyde hGH test group received a single dose of glycerol branched chain 43K PEG aldehyde hGH on day 0. Glycerol branched 43K PEG aldehyde hGH doses were 1.8 mg / kg (■), 0.6 mg / kg (×), 0.2 mg / kg (+) and 0.067 mg / kg (▲). There were six animals per group.

8 shows serum IGF-1 levels for 6 days efficacy study. Animals were treated as described in FIG. 7. Blood samples were taken at various time points plotted and serum IGF-1 levels were measured by ELISA. Group (n = 6) means were used to calculate the IGF-1 response using a one-way analysis of the deviation from the measured values, a series of 1.8 mg / kg, 0.6 mg / kg, 0.2 mg AUCdO-6 (ng / ml * 24h) values of 37,839, 28,1292, 22,958 and 20,040 were measured for the / kg, and 0.067 mg / kg doses, respectively.

9 shows PK / PD evaluations after administration of a single dose of glycerol branched chain 43K PEG aldehyde hGH to pituitary rats. The effect of a single 1.8 mg / kg subcutaneous administration of glycerol branched 43K PEG aldehyde hGH on plasma drug levels (a) or plasma IGF-1 response (b) is shown.

The present invention provides a glycerol branched chain, which may have one or more improved chemical or physiological properties selected from, but not limited to, reduced clearance, increased plasma residence time, increased stability, improved solubility, and reduced antigenicity. Poly (ethylene glycol) moiety relates to PEGylated hGH. Thus, as will be described in more detail below, the present invention has a number of aspects relating to chemically modifying hGH using glycerol branched chain poly (ethylene glycol) residues.

In addition, the present invention provides a) increased stability of the glycerol backbone, b) increased receptor binding, c) reduced cost, d) increased N-terminal selectivity of binding, e) increased solubility, and f) reduced antigenicity. at least one improved property over lysine based branched PEG-hGH conjugates, including, but not limited to, g) increased stability of the conjugate, h) increased ease of manufacture, and i) reduced proteolysis. Can have

The present invention also relates to a method of preparing PEGylated hGH. In particular, the present invention relates to a method for preparing PEGylated hGH using glycerol branched chain PEG.

The present invention also relates to compositions comprising PEGylated hGH, alone or in combination with another therapeutic agent. The present invention also relates to the use of PEGylated hGH of the present invention, alone or in combination with another therapeutic agent, in the prevention and / or treatment of disorders and / or diseases in which GH treatment is useful.

The present invention relates to a glycerol branched chain PEG-hGH conjugate. In certain embodiments, the glycerol branched PEG derivative has an aldehyde reactive group and, optionally, a linker between PEG and a reactive functional group located at the primary carbon at position 1 of the glycerol backbone. It has a polyalkylene glycol chain at positions 2 and 3 to produce hGH conjugates as described in 04/46222 or US patent application 2005/0058620. The linker is not particularly limited as long as it is a covalent bond. Preferably, the linker includes an alkylene group; And alkylene groups having ester bonds, urethane bonds, amide bonds, ether bonds, carbonate bonds or quaternary amino groups. Preferred alkylene groups include methylene, ethylene, trimethylene, propylene, isopropylene, tetramethylene, butylene, isobutylene, pentamethylene and hexamethylene groups.

Certain embodiments of the invention are PEG-hGH conjugates represented by Formula I:

Where

n is an integer between 60 and 75;

m is an integer between 450 and 460;

R is a hGH polypeptide.

In certain embodiments n is an integer between about 64 and about 72.

In specific embodiments, the (CH ₂ CH ₂ O) _n residue has an average molecular weight of about 2.6 Kd to about 3.5 Kd, specifically the average molecular weight is about 3 Kd.

In specific embodiments, the (CH ₂ CH ₂ O) _m residues each have an average molecular weight of about 17.6 Kd to about 22 Kd, specifically the average molecular weight is about 20 Kd.

In certain embodiments, the (CH ₂ CH ₂ O) _n residues have an average molecular weight of about 3 Kd, and the (CH ₂ CH ₂ O) _m residues each have an average molecular weight of about 20 Kd.

The term "about" as used in connection with the molecular weight of a PEG moiety means that in the preparation of PEG some molecules will have a molecular weight that is more or less than the molecular weight described and the molecular weight described refers to the average molecular weight. It should be understood that there is some polydispersity associated with polymers such as poly (ethylene glycol). Preference is given to using PEG with low polydispersity. In certain embodiments, one of the terminal polymer hydroxyl end-groups is converted or capped with a methyl group. As used herein, the term “mPEG” refers to PEG capped by a methyl group at one end. mPEG may be represented as the formula CH ₃ O- (CH ₂ CH ₂ O) _n -H.

The term “human growth hormone polypeptide”, “hGH polypeptide” or “hGH protein” as used herein encompasses all hGH polypeptides characterized by promoting growth in the growth phase and maintaining normal body composition, assimilation and lipid metabolism. Preferably, the term “hGH polypeptide” refers to the hGH polypeptide of SEQ ID NO: 1.

The hGH polypeptides of the invention can be prepared in any suitable manner. Such hGH polypeptides and fragments thereof may be purified from natural sources using techniques known to those skilled in the art, prepared by recombinant techniques, including in vitro translation techniques or expression in recombinant cells capable of expressing hGH cDNA. Or by combining these methods (see, for example, Methods in Enzymology, Academic Press, 1993); For chemical synthesis of proteins, see Creighton, (1983) Proteins: Structures and Molecular Principles, WH Freeman & Co. 2nd Ed., TE, New York; and Hunkapiller et al., (1984) Nature. 310 (5973): 105-11); For recombination techniques, see Davids et al. (1986) Basic Methods in Molecular Biology, ed., Elsevier Press, NY, which are incorporated herein by reference in their entirety. The polypeptides of the present invention are preferably provided in isolated form and may be partially or preferably substantially purified.

Particular embodiments of the invention are PEG-hGH conjugates, wherein more than 80%, more preferably 81%, more preferably 82%, more preferably 83%, more preferably 84%, more preferably Is 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% PEG is SEQ ID NO: 1 Is conjugated to the amino-terminal phenylalanine of hGH.

Another embodiment of the invention is a substantially homogeneous preparation, optionally comprising a N-terminal PEGylated hGH with a pharmaceutically acceptable diluent, carrier or adjuvant, which is a PEG at a site other than the N-terminus. It does not contain essentially hGH. The term "substantially homogeneous agent" is greater than 80%, more preferably 81%, more preferably 82%, more preferably 83%, more preferably 84%, more preferably 85%, more preferred Preferably 86%, more preferably 87%, more preferably 88%, more preferably 89%, more preferably 90%, more preferably 91%, more preferably 92%, more preferably Means an agent wherein 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97%, more preferably 98% of hGH is mono-PEGylated do.

In one embodiment of the invention, Chamow et al., Bioconjugate Chem. 5: 133-140 (1994), US Pat. No. 4,002,531, WO 90/05534 and US Pat. No. 5,824,784. As described below, reductive alkylation with an appropriate reducing agent such as NaCNBH ₃ , NaBH ₃ , pyridine borane, etc., forms a secondary amine bond between the N-terminal primary α-amino group of the hGH polypeptide and the glycerol branched chain PEG aldehyde. do. Incubation of the glycerol branched PEG aldehyde with the hGH polypeptide causes the PEG residues to be added to the amino group via Schiff base formation. This bond is converted to a stable secondary amine by reduction with a reducing agent. Reductive alkylation processes are shown in the following schemes (Chamow et al. Sources).

Conjugation reactions called "PEGylation reactions" have been carried out in solutions containing a molar excess of polymer, regardless of where the polymer is attached to the protein. However, this general technique has proven inadequate for conjugated bioactive proteins to non-antigenic polymers while retaining sufficient bioactivity. One way to maintain hGH bioactivity is to substantially avoid conjugation of hGH reactive groups associated with receptor binding site (s) in the polymer coupling process. Another aspect of the invention is to provide a method of conjugated poly (ethylene glycol) to hGH while maintaining a high retained activity.

Chemical modification via covalent bonds can be carried out under any suitable conditions commonly used in the reaction of biologically active materials with activated poly (ethylene glycol). Conjugation reactions are carried out under relatively mild conditions to avoid inactivation of hGH. Mild conditions include maintaining the pH of the reaction solution at 3 to 10 and maintaining the reaction temperature within about 0 ° C to 37 ° C. If the reactive amino acid residue in hGH has a free amino group, the modification is preferably at 4 ° C. to 37 ° C. with a non- appropriate buffer (pH 4 to 10) including phosphate, MES, citrate, acetate, succinate or HEPES. It is performed for 1-48 hours in the limited list. In targeting the N-terminal amino group with a reagent such as PEG aldehyde, pH 4-8 is preferably maintained. Activated poly (ethylene glycol) can be used at about 0.01 to 100 times the molar amount of the free amino group number of hGH, preferably about 0.01 to 2.5 times.

Although the reaction conditions described herein can result in significant amounts of unmodified hGH, the unmodified hGH can be easily recycled in future batches for further conjugated reactions. The process of the present invention produces surprisingly very low, i.e., less than about 20%, more preferably less than about 10%, high molecular weight species and species having more than one polymer strand per hGH. This reaction condition is in contrast to the reaction conditions typically used for polymer conjugated reactions in which the activated polymer is present in several molar excesses to the target.

The conjugated reaction of the present invention initially provides a reaction mixture or pool containing mono-PEGylated hGH conjugate, unreacted hGH, unreacted polymer and less than about 20% high molecular weight species. High molecular weight species include conjugates having more than one polymer strand and / or polymerized PEG-hGH species. After unreacted species and high molecular weight species are removed, the composition containing predominantly mono-PEGylated hGH conjugate is recovered. Assuming that most conjugates comprise a single polymer strand, the conjugates are substantially homogeneous. This modified hGH can be analyzed by standard FDC-P1 cell proliferation assay (Clark et al. Journal of Biological Chemistry 271: 21969-21977, 1996), receptor binding assay (US Pat. No. 5,057,417) or pituitary excised rat growth (Clark et al. Have at least about 0.1% in vitro biological activity associated with natural or unmodified hGH as measured using the Journal of Biological Chemistry 271: 21969-21977, 1996). However, in a preferred embodiment of the invention, the modified hGH has about 25% in vitro biological activity, more preferably the modified hGH has about 50% in vitro biological activity, and more preferably the modified hGH has about 75%. % In vitro biological activity, most preferably the modified hGH has an equivalent or improved in vitro biological activity.

The process of the invention preferably comprises a somewhat limited ratio of polymer to hGH. Thus, it has been found that hGH conjugates are mainly limited to species containing only one strand of polymer. Moreover, the binding of polymer to hGH reactive groups is substantially less random than when higher molar excess of polymer linker is used. After the conjugation reaction is quenched, the unmodified hGH present in the reaction pool can be recycled in future reactions using ion exchange or size exclusion chromatography or similar separation techniques.

Poly (ethylene glycol) -modified hGH is a common method used for purification of proteins such as dialysis, salting-out, ultrafiltration, ion exchange chromatography, hydrophobic interaction chromatography (HIC), gel chromatography Or it can be purified from the reaction mixture by electrophoresis. Ion exchange chromatography is particularly effective at removing unreacted poly (ethylene glycol) and hGH. In a further embodiment of the invention, mono-PEGylated hGH species are isolated from the reaction mixture to remove high molecular weight species and unmodified hGH. Separation is performed by placing the mixed species in a buffer solution containing about 0.5-10 mg / mL of the polymer-hGH conjugate. Suitable buffer solutions have a pH of about 4 to about 10. The solution is preferably at least one buffer salt selected from KCl, NaCl, K ₂ HPO ₄ , KH ₂ PO ₄ , Na ₂ HPO ₄ , NaH ₂ PO ₄ , NaHCO ₃ , NaBO ₄ , CH ₃ CO ₂ H and NaOH. It contains.

Depending on the reaction buffer, the polymer-hGH conjugate solution may first need to be treated by buffer exchange / ultrafiltration to remove any unreacted polymer. For example, the PEG-hGH conjugate solution can be ultrafiltered through a low molecular weight cut-off (10,000 Daltons to 30,000 Daltons) membrane to remove most unwanted materials such as unreacted polymers, surfactants, etc., if present. have.

Fractionation of the conjugate into a pool containing the desired species is preferably carried out using an ion exchange chromatography medium. Such media can selectively bind to PEG-hGH conjugates through charge differences that change in a somewhat predictable manner. For example, the surface charge of hGH is determined by the number of charged groups available on the surface of the protein. This charged group typically acts as a potential point of attachment of the poly (alkylene oxide) polymer. Therefore, the hGH conjugate will have a different charge than other species to allow for selective separation.

Strongly polar anionic or cation exchange resins such as quaternary amine or sulfopropyl resins, respectively, can be used in the process of the invention. Anion exchange resins are particularly preferred. Non-limiting lists of commercially available cation exchange resins suitable for use in the present invention are SP-hitrap ^® , SP Sepharose HP ^® and SP Sepharose ^® fast flow. Other suitable cation exchange resins such as S and CM resins may also be used. Non-limiting lists of anion exchange resins including commercially available anion exchange resins suitable for use in the present invention are Q-Hraplab ^® , Q Sepharose HP ^® and Q Sepharose ^® Fast Flow. Other suitable anion exchange resins such as DEAE resin can also be used.

For example, anion or cation exchange resins are packed into columns and preferably equilibrated by conventional means. A buffer having the same pH and osmotic pressure as the hGH containing solution conjugated with the polymer is used. Elution buffers preferably comprise at least one salt selected from KCl, NaCl, K ₂ HPO ₄ , KH ₂ PO ₄ , Na ₂ HPO ₄ , NaH ₂ PO ₄ , NaHCO ₃ , NaBO ₄ and (NH ₄ ) ₂ CO ₃ . It contains. The conjugate-containing solution is then adsorbed onto a column that has no unreacted polymer and some high molecular weight species. After loading is complete, a gradient stream of elution buffer with increasing salt concentration is applied to the column to elute the desired fractions containing polyalkylene oxide and hGH conjugated. After the cation or anion exchange separation step, the collected eluted fractions are preferably defined as homogeneous polymer conjugates. Thereafter, any non-conjugated hGH species can be washed off the column again by conventional techniques. If necessary, further ion exchange chromatography or size exclusion chromatography can further separate the single-PEGylated hGH species and the multi-PEGylated hGH species from each other.

Techniques using multiple isocratic steps with increased salt concentration or pH may also be used. Multiple isocratic elution steps of increasing concentration will elute the di-hGH-polymer conjugate followed by the single-hGH-polymer conjugate.

The temperature range for elution is from about 4 ° C to about 25 ° C. Preferably, the elution is carried out at a temperature of about 4 ° C to about 22 ° C. For example, elution of the PEG-hGH fraction is detected by UV absorbance at 280 nm. Fraction harvesting can be achieved through a simple time dissolution profile.

Surfactants can be used in the process of conjugated poly (ethylene glycol) polymers to hGH moieties. Suitable surfactants include ionic-type materials such as sodium dodecyl sulfate (SDS). Other ionic surfactants may also be used, such as lithium dodecyl sulfate, quaternary ammonium compounds, taurocholic acid, caprylic acid, decane sulfonic acid, and the like. Non-ionic surfactants can also be used. For example, materials such as poly (oxyethylene) sorbitan (twin), poly (oxyethylene) ether (triton) can be used. See also Neugebauer, A Guide to the Properties and Uses of Detergents in Biology and Biochemistry (1992) Calbiochem Corp. The only limitation to the surfactants used in the process of the present invention is that they must be used under conditions and concentrations that do not result in substantial irreversible denaturation of hGH 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 0.05% to 0.5%, most preferably about 0.075% to 0.25%. Mixtures of surfactants are also contemplated.

It is believed that the surfactant provides a temporary and reversible protection system during the polymer conjugation process. Surfactants have been found to be effective at selectively interfering with polymer conjugation while allowing lysine-based or amino end-based conjugation to proceed.

The poly (ethylene glycol) -modified hGH of the present invention has a longer lasting pharmacological effect that can be attributed to its extended in vivo half life.

Another embodiment of the invention provides for the prevention of diseases or disorders in which the use of a GH, preferably hGH, in which a therapeutically effective amount of the poly (ethylene glycol) -modified hGH of the invention or an agent variant thereof, alone or in combination with another therapeutic agent is advantageous. And / or administering to a patient in need thereof, a method of preventing and / or treating said disease or disorder. The invention also relates to the use of the poly (ethylene glycol) -modified hGH or agonist variant thereof of the invention in the manufacture of a medicament for the prevention and / or treatment of a disease or disorder for which the use of GH, preferably hGH is advantageous. will be. Further, the present invention relates to a pharmaceutical composition comprising the poly (ethylene glycol) -modified hGH of the present invention or an agent variant thereof for the prevention and / or treatment of a disease or disorder in which the use of GH, preferably hGH is advantageous. It is also.

Diseases or disorders that favor the use of GH include growth hormone deficiency (GHD), adult growth hormone deficiency (aGHD), Turner syndrome, growth failure in small born children during pregnancy, Prader-Willi syndrome (PWS), and chronic renal failure ( CRI), AIDS depletion, aging, end stage renal failure, cystic fibrosis, erectile dysfunction, HIV dyslipidemia, fibromyalgia, osteoporosis, memory disorders, depression, Crohn's disease, skeletal dysfunction, traumatic brain injury, subarachnoid hemorrhage, Nunan syndrome (Noonan's) syndrome, Down syndrome, idiopathic short stature (ISS), late renal disease (ESRD), ultralow birth weight (VLBW), bone marrow stem cell rescue, metabolic syndrome, glucocorticoid myopathy, glucocorticoids in children Short stature due to treatment, and failure to keep up with growth for short premature children.

In a more specific embodiment of the invention, the poly (ethylene glycol) -modified hGH or agonist variant thereof of the invention is for the prevention and / or prevention of a disorder or disease selected from the group consisting of GHD, aGHD, SGA, PWS, Turner syndrome and CRI Used for treatment.

In another more specific embodiment of the invention, the poly (ethylene glycol) -modified hGH or agonist variant thereof of the invention is a disorder selected from the group consisting of idiopathic short stature, ultralow birth weight, traumatic brain injury, metabolic syndrome and nunan syndrome or Used for the prevention and / or treatment of diseases.

Another embodiment of the invention relates to a pharmaceutical composition comprising the poly (ethylene glycol) -modified hGH of the invention and one or more pharmaceutically acceptable excipients or carriers, alone or in combination with another therapeutic agent. The poly (ethylene glycol) -modified hGH of the present invention may then be formulated into a pharmaceutically acceptable diluent, a substance for the preparation of an isotonic solution, a pH-adjusting agent, and the like, and administered to a patient.

The medicament may be administered subcutaneously, intramuscularly, intravenously, pulmonary, intradermal or orally depending on the therapeutic purpose. The dosage may be based on the type and condition of the disorder of the patient to be treated, and is normally 0.1 mg to 5 mg for injection and 0.1 mg to 50 mg for oral administration to adults.

As used herein, the poly (ethylene glycol) -modified hGH or agent variant of the invention can be used in conjunction with another therapeutic agent. As used herein, the terms “co-administration”, “co-administration” and “together” as used to refer to Compound A and one or more other therapeutic agents, mean, refer to, and include administration as follows: :

When Compound A and the therapeutic agent (s) are formulated together in a single dosage form that is released substantially simultaneously to a patient in need of treatment, concurrent administration of the combination of Compound A and the therapeutic agent (s) to a patient in need of treatment box;

When Compound A and the therapeutic agent (s) are formulated separately from one another in separate dosage forms that are ingested substantially simultaneously by the patient in need of treatment, so that Compound A and the therapeutic agent are substantially simultaneously released to the patient in need of treatment. Administering the combination of (s) substantially simultaneously to a patient in need thereof;

Compound A and the therapeutic agent (s) are formulated separately from one another in separate dosage forms that are sequentially ingested at substantial time intervals between each administration by the patient in need of treatment, thereby substantially differenting the patient in need of treatment. When released in time, the combination of Compound A and therapeutic agent (s) is administered continuously to a patient in need thereof; And

Compound A and therapeutic agent (s) are formulated together in a single dosage form that is released in a controlled manner to be administered simultaneously, continuously and / or overlapping simultaneously and / or at different time points by the patient in need of treatment. If any, the combination of Compound A and the therapeutic agent (s) is administered sequentially to the patient in need thereof.

Suitable examples of other therapeutic agents that may be used with Compound A, its pharmaceutically acceptable salts, and / or derivatives thereof include aromatase inhibitors such as exemestane, formemtan, atamestane, padrosol, letrozole, Borosol and anastrozole; Free fatty acid modulators, including fibric acid derivatives (eg, fenofibrate, clofibrate, gemfibrozil, benzafibrate and ciprofibrate) and nicotinic acid derivatives such as acipimox; Biguanides such as metformin, PPAR gamma insulin sensitizers, and thiazolodenions such as troglitazone and rosiglitazone, troglitazone, 5-[[4- [3,4-dihydro-6-hydroxy-2,5,7, 8-tetramethyl-2H-]-benzopyran-2-yl) methoxy] phenyl] methyl3-2,4-thiazolidinedione V411 (DIABII, glowcanine) pioglitazone (ACTOS, AD 4833, U 72107, U 72107A, U 72107E, ZACTOS) Chemical names: 2,4-thiazolidinedione, 5-[[4- [2- (5-ethyl-2-pyridinyl) ethoxy] phenyl] methyl]-, monohydrochloride, insulin sensitizers, including but not limited to (a /-); Rosiglitazone (Avandia, BRL 49653, BRL 49653C) Chemical names: 2,4-thiazolidinedione, 5-[[4- [2- (methyl-2-pyridinylarino) ethoxy] phenyl] methyl]; 25 Bexarotene-oral (LGD 1069 oral, Targretine oral, Targretin, Targretyn oral, targrexine oral) Chemical name: 4- [1- (3,5 , 5,8,8-penta methyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] benzoic acid; ZD 2079, (ICI D 2079) (chemical name: R) -N- [2-4- (carboxymethyl) 30 phenoxy] ethyl) -N- (2-hydroxy-2-phenethyl) ammonium chloride: nettoggle Litazone, (isaglitazone, MCC 555, RWJ 241947) (chemical name: 5- [6- (2-fluorobenzyloxy) naphthalen-2-ylmethyl] thiazolidine-2,4-dione); INS (D-Kiro-inositol) (chemical name: D-1,2,3,4,5,6-hexahydroxycyclohexane), ON 2344 (DRF 2593); Dexsleypotam, chemical name: 5 (R)-(1,2-dithiolan-3-yl) pentalo 35 acid; HQL 975, chemical name: 3- [4- [2- (5-methyl-2-phenyloxazol-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), including but not limited to. I PPAR agonists in development include reglitaza (JTT 501, PNU 182716, PNU 716) (chemical names: isoxazolidiene-3,5-dione, 1,4-[[4- (2-phenyl-5-methyl-) 1,3-oxazolyl] ethoxyphenyl-4-] methyl-, (4RS)); I (RP 297, chemical name: 10,5- (2,4-dioxothiazolidin-5-ylmethyl)- 2-methoxy-N- [4- (trifluoromethyl) benzylbenzamide; R 119702 (Cl 1037, CS 011) Chemical name: (/-)-5- [4- (5-methoxy-1H-benz Imidazol-2-ylmethoxy) benzyl] thiazoline-2,4-dione hydrochloride; 15 DRF 2189, chemical name: 5-[[4- [2- (1-indolyl) ethoxy] phenyl] methyl] thia Zolidine-2,4-dione; cortisol synthesis inhibitors such as ketoconazole, econazol and myconazole; GH such as somatropin or somatonorm and derivatives thereof such as hGH fusion proteins such as ALBUTROPIN; PEG-GH such as Cysteine-PEGylated GH, BT 005 (Bolder BioTechnology Inc.); GH secretagogues such as SM 130686 (Sumitomo), Capromorelin (Pfizer), Mecassermine (Fuj) isawa), Sermorelin (Salk Institute, Bio-Technology General), Somatrem, Somatomedin (C Llorente; Pharmacia Corporation), Examorelin, Tabimorelin; CP 464709 (Pfizer), LY 426410 and LY 444711 ( Lilly), 8- (aminoalkoxyimino) -8H-dibenzo [a, e] triazolo [4.5-c] cycloheptene, disclosed in WO 2002/057241, WO 2002/057241; 2-substituted dibenzo [a, e] 1,2,3-triazolo [4,5-c] [7] anulen-8-ones disclosed in 056873; US Pat. No. 4,411,890, and International Patent Application Publications GH-releasing peptides GHRP-6 and GHRP-1 disclosed in WO 89/07110 and WO 89/07111; B-HT920, Hexarelin and GHRP-2 or GH disclosed in WO 93/04081. Releasing hormone (GHRH, also referred to as GRF) and its analogs; Somatomedin and derivatives thereof, including IGF-1 and IGF-2 such as somatokin-insulin-like growth factor-1 and recombinant fusions thereof, BP-3, alpha-2-adrenergic agents such as clonidine , Xylazine, detomidine and metomimidine or serotonin 5HTID agonists such as sorbitriptan, or substances that inhibit or inhibit the release of somatostatin, such as physostigmine and pyridostigmine, ThGRF 1-44 (Theratechnologies ); L 165166 from Merck &Company; Dipeptide derivatives disclosed in WO 98/58947; Inhibitors of dipeptidyl peptidase IV, such as the amino-acylpyrrolidine nitriles disclosed in US Pat. No. 652164, and WO 95/15309 and WO 98/19998; Beta-amino heterocyclic dipeptidyl peptidase inhibitors, such as those disclosed in US Patent Application No. 20030100563 and WO 2003/082817, US Patent Application Nos. 20030055261 and 20030040483, European Patent No. 18072 and 83864, International Patent Application Publication Nos. WO 89/07110, WO 89/01711, WO 89/10933, WO 88/9780, WO 83/02272, WO 91/18016, WO 92/01711, WO 93/04081 and WO 9514666, European Patent No. 0923539, US Patent Nos. 5,206,235, 5,283,241, 5,284,841, 5,310,737, 5,317,017, 5,374,721 5,430,144, 5,434,261, 5,438,136, 5,494,919, 5,494,920, 5,492,916, 5,536,716 and 5,578,593, and 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 , The number WO 95/17422, WO 95/17423 claim, 1 - WO 95/34311) and (GH release the compounds disclosed in WO 96/02530 call; Piperidine, pyrrolidine and hexahydro-1H-azepine disclosed in US Pat. Nos. 5,578,78 and 5783582, and WO 04/007468; Amido spiropiperidine, such as disclosed in WO 01/04119; 2-[(5,6-dimethyl-2-benzoimidazolyl) amino] -4-hydroxy-6-methyl-5-pyrimidine acetic acid (2) and 2-[(5, 6-dimethyl-2-benzoimidazolyl) amino] -4-hydroxy-6-methyl-5-pyrimidine acetic acid, 2-amino-5-pyrimidine acetic acid compound comprising ethyl ester; Benzimidazoles disclosed in European Patent No. 1155014; Cognate peptide compounds associated with GRF and peptides of US Pat. No. 4,411,890; WO 01/70228, WO 01/70227, WO 01/70228, WO 00/69433, WO 00/04013, WO 99/5156, WO 99 / Antagonists of gonadotropin releasing hormones such as those disclosed in 51595, WO 99 / 51231-4, WO 99 / 41251-2, WO 99/21557 and WO 99/21553; And WO 00/53602, WO 00/53185, WO 00/53181, WO 00/53180, WO 00/53179, WO 00/53178 and US Patent Nos. 6-azaindole compounds disclosed in 6288078; IGF-1 secretagogues; Insulin-like growth factor-2- (IGF-2 or somatomedin A) and IGF-2 secretagogues; Miyostatin antagonist; And compounds that inhibit fibroblast growth factor receptor-3 (FGFR-3) tyrosine kinases.

The polymeric material included is also preferably water soluble at room temperature. Non-limiting lists of such polymers include poly (alkylene oxide) homopolymers such as poly (ethylene glycol) or poly (propylene glycol), poly (oxyethylenated polyols), copolymers and block copolymers thereof, including block The water solubility of the copolymer should be maintained.

As an alternative to PEG-based polymers, materials that effectively exhibit non-antigenicity can be used, such as dextran, poly (vinyl pyrrolidone), poly (acrylamide), poly (vinyl alcohol), carbohydrate-based polymers, and the like. have. Indeed, activation of the α-terminal and ε-terminal groups of these polymeric materials can be carried out in a manner similar to that used to convert poly (alkylene oxides), and will therefore be apparent to those skilled in the art. will be. Those skilled in the art will appreciate that the above list is exemplary only and all polymeric materials having the qualities described herein may be considered. For the purposes of the present invention, "substantially non-antigenic" means any substance that is recognized in the art as being nontoxic in mammals and not eliciting a detectable immunogenic response.

Justice

The following definitions are a list of abbreviations and corresponding meanings used interchangeably herein:

-g grams

mg mg

-Ml or ml milliliters

RT room temperature

PEG poly (ethylene glycol)

The entire contents of all publications, patents, and patent applications cited herein are hereby incorporated by reference as if each individual publication, patent or patent application was specifically and individually described as being incorporated by reference.

Although the present invention has been described in some detail by way of explanation and illustration for clarity, it is easy for those skilled in the art to make changes and modifications without departing from the spirit and scope of the present invention. Will be self explanatory. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention described in the broad sense of the terms.

In the examples below, hGH is hGH of SEQ ID NO: 1. It will be appreciated that other hGH polypeptides may be PEGylated in a manner similar to that exemplified in the examples below.

Example  One

Glycerol Branch chain  43 K PEG  Aldehyde hGH

Where

(CH ₂ CH ₂ O) _n has an average molecular weight of about 3 Kd, and (CH ₂ CH ₂ O) _m each has an average molecular weight of about 20 Kd (GL3-400AL2).

This example demonstrates the production of N-terminal mono-PEGylated hGH by reductive alkylation. By using the difference between the relative pK _a value of the primary amine at the N-terminus versus the relative pK _a value of the primary amine at the ε-amino position of the lysine residue, about 43,000 MW of glycerol branched PEG aldehyde reagent (GL3 -400AL2, NOF corporation) was coupled to the N-terminus of hGH. 25 mM MES (Sigma Chemical, St. Louis, MO) or pH of pH 5.8 by adding the reagent so that the relative PEG: hGH molar ratio is 1.5: 1, 2: 1, 3.4: 1, 4: 1 or 5: 1 The hGH protein dissolved in 7.0 20 mM HEPES in an amount of 4 mg / ml, 7 mg / ml or 10 mg / ml was reacted with glycerol branched chain 43K PEG aldehyde. Pyridine borane (Sigma Chemical, St. Louis, Mo.) was added to a final concentration of 10 mM to facilitate the reaction. The reaction was carried out in the dark at 4 ° C. for 16-87 hours. The reaction was stopped by diluting with a buffer suitable for purification.

Table 1 shows the final purification of glycerol branched chain 43K PEG aldehyde hGH reacted for 63 hours at a ratio of multi-PEGylated species, mono-PEGylated conjugate and unreacted hGH, and a pH of 5.8 and a 1.5: 1: 1 molar ratio. Show yield.

Example  2

PEG anger hGH Tablets

PEGylated hGH species were purified from the reaction mixture to greater than 95% using a single ion exchange chromatography step (SEC analysis, FIG. 1).

Anion exchange chromatography

PEGylated hGH species were purified to greater than 95% from the reaction mixture using a single anion exchange chromatography step (SEC analysis, FIG. 1). Single-PEGylated hGH was purified from unmodified hGH and multi-PEGylated hGH species using anion exchange chromatography. As described above, a typical glycerol branched 43K PEG aldehyde hGH reaction mixture (80 mg or 1500 mg of protein) was Q-Sepharose heatlab column (5 ml) equilibrated with 25 mM HEPES (buffer A) at pH 7.3 (Amersham). Purification on Pharmacia Biotech, Piscataway, NJ) or Q-Sepharose column (26/20, 70 mL bed volume) (Amersham Pharmacia Biotech, Piscataway, NJ). The reaction mixture was diluted 7 × with Buffer A and loaded onto the column at a flow rate of 2.5 ml / min. The column was washed with 3 column volumes to 10 column volumes of Buffer A. Thereafter, various hGH species were eluted from the column using 20 column volumes of Buffer A and a linear NaCl gradient of 0 mM to 100 mM. The eluate was monitored by absorbance at 280 nm (A ₂₈₀ ) and fractions of appropriate size were collected. Degree of PEGylation Fractions were collected (e.g. evaluated in Example 3) for single-PEG, di-PEG, tri-PEG, and the like. The pool was then concentrated to 0.5 mg / ml to 5 mg / ml in a Centrirep YM10 concentrator (Amicon, Technology Corporation, Northborough, Mass.) Or by diafiltration. The protein concentration of the pool was measured by A ₂₈₀ using an extinction coefficient of 0.78.

Example  3

Biochemical Characterization

Purified PEGylated hGH pools were characterized by non-reducing SDS-PAGE, non-denaturing size exclusion chromatography, and peptide mapping.

Size Exclusion High Performance Liquid Chromatography SEC - HPLC )

The reaction mixture of glycerol branched 43K PEG aldehyde and hGH was evaluated by anion exchange purification pool and final purification product by non-modified SEC-HPLC. Analytical non-denaturing SEC-HPLC was performed on a column TSK G4000PWXL (Tosohaas) or Shodex KW-804 (Waters Corp) (if desired) in 20 mM phosphate pH 7.2, 150 mM NaCl at a flow rate of 0.5 ml / min. Superdex) 200 7.8 mm × 30 cm (Amersham Bioscience, Piscataway, NJ)). PEGylation significantly increases the hydrodynamic volume of the protein, resulting in shorter residence times. Novel species were observed in PEG aldehyde hGH reaction mixtures with unmodified hGH. This PEGylated and non-PEGylated species were separated on Q-Sepharose chromatography and the purified final single-PEGylated-aldehyde hGH species was later found to elute as a single peak on non-modified SEC (95 Purity greater than%, FIG. 1). The Q-Sepharose chromatography step effectively removed free PEG, hGH and multi-PEGylated hGH species from single-PEGylated hGH.

SDS - PAGE

SDS-PAGE was used to evaluate the reaction of glycerol branched chain 43K PEG aldehyde with hGH and the purified final product. SDS-PAGE was performed on 1-mm thick 10-NuPAGE gels (Invitrogen, Carlsbad, Calif.) Under reducing and non-reducing conditions, and the Novex Colloidal Coomassie ^™ G-250 staining kit (Invitrogen) , Carlsbad, Calif.).

N-terminal sequence

Automated Edman degradation chemistry techniques were used to determine the NH ₂ -terminal protein sequence. Applied Biosystems Model 494 Procise sequencer (Perkin Elmer, Wellesley, Mass.) Was used for digestion. Each PTH-AA derivative was identified by on-line RP-HPLC analysis using an Applied Biosystem Model 140C PTH analyzer equipped with a Perkin Elmer / Brownlee 2.1 mm inner PTH-C18 column.

Peptide Mapping

Trypsin digestion was performed at a concentration of 1 mg / ml and typically 25 μg of material per digest was used. Trypsin was added so that the trypsin to PEG-hGH ratio was 1:30 (weight / weight). Tris buffer was present at 30 mM, pH 7.5. Samples were incubated for 16 ± 0.5 hours at room temperature. The reaction was quenched by adding 50 μl of 1 N HCl per ml of digestion solution. After diluting the sample to a final concentration of 0.25 mg / ml in 6.25% acetonitrile, the sample was placed in an autosampler. Acetonitrile was added first (up to 19.8% acetonitrile) and mixed lightly, then water was added to the final volume (four times the starting volume). Excess decomposition solution can be removed and stored at -20 ° C for up to one week.

The Waters Alliance 2695 HPLC system was used for the analysis, but similar results can be obtained with other systems. An Astec C-4 polymer 25 cm × 4.6 mm column with 5 μm particles was used. Typically 50 μg of protein per sample was loaded and the experiment was performed at ambient temperature. Buffer A was 0.1% trifluoroacetic acid in water and Buffer B was 0.085% trifluoroacetic acid in acetonitrile. Samples were eluted with a linear gradient of Buffer B 0% to 45% over 90 minutes.

Peaks were detected using a Waters 996 PDA detector, which collected data between 210 nm and 300 nm. Samples were analyzed using extracted chromatograms at 214 nm.

Trypsinization maps were performed for hGH and glycerol branched 43K PEG aldehyde reacted at a molar ratio of 2: 1 (PEG: hGH) (FIG. 2). N-terminal trypsin fragment was referred to as T-1. The proportion of T-1 present when compared to non-PEGylated hGH suggests that at least 99% of the PEG modifications are at the N-terminus and the remaining modifications are explicitly bound to one of several possible lysine residues.

Example  4

In vitro  Biological analysis

Using a Biacore 3000 device in an assay configured to assess specific interactions between conjugates and hGH receptors (hGHR) (28kDa extracellular domain), a glycerol branched chain 43K PEG aldehyde hGH was used to recognize human receptors. The ability was tested. The results obtained from the surface plasmon resonance (SPR) experiments are shown in Table 3 below.

k _a (binding-rate) and k _d (dissociation-rate) were determined using an hGH binding protein (28 kDa, extracellular domain) labeled on a CM5 chip via amine coupling chemistry at ΔRU = 3000-5000 It was measured at 37 ° C. at a flow rate of 50 μl / min in HEP-BES buffer (0.01 M Hepes, pH 7.4, plus 0.15 M NaCl, 3 mM EDTA and 0.005% surfactant P20). K _a , denoted as M ⁻¹ s (sec) ^{− 1} and k _d , denoted as s ^{− 1} , are both average values of three or more measurements for one chip ± standard deviation. The data was evaluated to determine hGH binding to high binding site 1 to GHBP at a 1: 1 ratio.

Example  5

Pharmacokinetic Studies

In vivo  Efficacy-11 days Rat  Efficacy in the analysis (weight gain, tibia growth, serum BUN  descent)

Rat  Weight gain

Pituitary female Sprague Dawley rats purchased from Harlan Labs were prescreened for growth rates for 4-10 days. Rats were divided into six groups. Beginning at day 0, control rats were subcutaneously injected once daily with 0.3 mg / kg of hGH or vehicle for 11 consecutive days. The test group received 1.8 mg / kg single (once per week) doses of glycerol branched chain 43K PEG aldehyde hGH on days 0 and 6. Animal weights were measured daily. 4 shows the effect of hGH and glycerol branched chain 43K PEG aldehyde hGH on weight gain in a representative study.

Combine the data obtained by the study shown in FIG. 4 with data from an 11-day growth study for hGH (control) treated rats and a further growth study with glycerol branched 43K PEG aldehyde hGH conjugate, 1.8 mg. The average weight gain for animals treated once per week with / kg conjugate was 109% of the average weight gain achieved after daily hGH administration (3.3 mg / kg total).

Rat  Tibia growth

In an 11-day weight gain study, animals were sacrificed on day 11, the left tibia was removed, X-rayed, and bone length was measured using a caliper. 5 shows tibial length measurements for animals treated with glycerol branched chain 43K PEG aldehyde hGH.

Rat BUN  level

As a biomarker for metabolic effects after hGH-treatment, blood urea nitrogen levels were measured from 11 days of blood samples. FIG. 6 shows that blood urea nitrogen was significantly reduced both when treated with daily hGH and when treated with glycerol branched chain 43K PEG aldehyde hGH once a week.

In a six-day dose escalation study Rat  Weight gain

Pituitary gland rats were treated with various single doses of glycerol branched chain 43K PEG aldehyde hGH or daily with hGH and weight gain was monitored for 6 days. 7 shows the weight gain obtained for the various treatment groups. Blood samples were taken at the indicated time points and serum IGF-1 levels were measured by ELISA. Mean +/- SEM was constructed. A group (n = 6) mean was used to calculate the IGF-1 response using a one-way analysis of the deviations to the measured values, a series of 0.067 mg / kg, 0.2 mg / kg, 0.6 mg / kg and 1.8 mg AUCdO-6 (ng-hr / ml) values 20,040, 22,958, 28,129 and 37,839 were measured for each / kg dose.

In the 11-day dose escalation study Rat  Weight gain

In a second study, animals were treated with 1.8 mg / kg or higher doses, ie 5.1 mg / kg of glycerol branched 43K PEG aldehyde hGH conjugate, on days 0 and 6. Table 4 shows the dose-related weight gains at days 6 and 11 when compared to the dose-related weight gains achieved after daily (QD 11) administration of vehicle or hGH (0.3 mg / kg / d). .

* P <0.05 for vehicle, † p <0.05 for hGH; Mean weight gain (g) BW, body weight measured from day 0; hGH, human growth hormone.

Values represent mean ± SEM (mean standard error).

Values in parentheses represent the change from day 0 as mean ± SEM.

IGF -1 study

Animals used in the 6 day weight gain study were used. Blood samples were taken at various time points during the study and serum IGF-1 levels were measured by ELISA as shown in FIG. 8. Rat IGF-1 levels were monitored with an immunoassay kit (Diagnostic System Laboratories).

Example  6

Pharmacokinetic Studies

Pharmacokinetic studies were performed in normal Sprague-Dawley male rats with cannula. Six rats per group were injected with hGH or glycerol branched 43K PEG aldehyde hGH as 1.0 mg / kg single intravenous dose or 1.8 mg / kg single subcutaneous bolus. Blood samples were taken over days 1-5 as appropriate for the evaluation of the relevant PK parameters. Immunoassay was used to monitor hGH and glycerol branched 43K PEG aldehyde hGH blood levels at each sampling. Table 4 shows rat PK parameters for glycerol branched 43K PEG aldehyde hGH. The effect of PEGylation is evident in the half-life observed for elimination, with the parameter exceeding 6 hours for conjugates but the data reported for hGH from similar studies were 1.35 ± 0.2 hours (Clark ibid), 0.77- For 1.7 hours (Jorgensen et. Al., "Polyethylene glycol-conjugated proteins", PSTT 1 (8), November 1998), or 1 hour (Genotropin ^® (PNU-180307) Investigator Brochure).

hGH  Immunoassay

hGH AutoDELFIA kit fluorescence immunoassay (Perkin-Elmer) was used to measure hGH and glycerol branched chain 43K PEG aldehyde hGH protein concentration levels in rat plasma.

The relationship between plasma drug levels and the IGF-1 response was also directly identified in a comprehensive study design after subcutaneous administration of a single dose of glycerol branched chain 43K PEG aldehyde hGH (1.8 mg / kg) to pituitary female rodents.

Claims (12)

Polyethylene glycol-human growth hormone (PEG-hGH) conjugate of Formula I: Formula I

Where n is an integer from 60 to 75; m is an integer from 450 to 460; R is human growth hormone (hGH). The method of claim 1, PEG-hGH conjugate, wherein the (CH ₂ CH ₂ O) _n residues have an average molecular weight of about 3 Kd, and the (CH ₂ CH ₂ O) _m residues each have an average molecular weight of about 20 Kd. The method according to claim 1 or 2, PEG-hGH conjugate wherein hGH comprises the amino acid sequence of SEQ ID NO: 1. The method of claim 3, wherein PEG-hGH conjugate wherein PEG is conjugated to the N-terminal phenylalanine of SEQ ID NO: 1. The method of claim 4, wherein Single-PEGylated PEG-hGH conjugate. The method of claim 4, wherein PEG-hGH conjugate wherein at least 80% of the PEG is conjugated to the alpha amino group of the N-terminal phenylalanine of SEQ ID NO: 1. The method of claim 5, PEG-hGH conjugate wherein at least 90% of the PEG is conjugated to the alpha amino group of the N-terminal phenylalanine of SEQ ID NO: 1. The method of claim 5, PEG-hGH conjugate wherein at least 95% of the PEG is conjugated to the alpha amino group of the N-terminal phenylalanine of SEQ ID NO: 1. The method of claim 5, PEG-hGH conjugate wherein at least 98% of the PEG is conjugated to the N-terminal phenylalanine of SEQ ID NO: 1. A method of treating a patient with a growth or developmental disorder, comprising administering a therapeutically effective amount of the PEG-hGH conjugate of any one of claims 1 to 9 to a patient with a growth or developmental disorder. The method of claim 10, The growth or developmental disorder is selected from the group consisting of growth hormone deficiency (GHD), Turner's syndrome, chronic renal failure and short term pregnancy (SGA). The method of claim 11, Growth or developmental disorders include erectile dysfunction, HIV dyslipidemia, fibromyalgia, osteoporosis, memory disorders, depression, Crohn's disease, skeletal dysplasia, traumatic brain injury, subarachnoid hemorrhage, Noonan's syndrome, Down's syndrome, idiopathic short stature ), End-stage renal disease (ESRD), ultra-low birth weight (VLBW), bone marrow stem cell rescue, metabolic syndrome, glucocorticoid myopathy, short stature due to glucocorticoid therapy in children, and short premature children The method is selected from the group consisting of failure to keep up with growth.

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