EP1789092A2 - Conjugués de l'hormone de croissance humaine avec du polyéthylèneglycol ramifié à l'aide de glycérol, procédé de sa préparation, et méthode de son utilisation. - Google Patents

Conjugués de l'hormone de croissance humaine avec du polyéthylèneglycol ramifié à l'aide de glycérol, procédé de sa préparation, et méthode de son utilisation.

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Publication number
EP1789092A2
EP1789092A2 EP05784225A EP05784225A EP1789092A2 EP 1789092 A2 EP1789092 A2 EP 1789092A2 EP 05784225 A EP05784225 A EP 05784225A EP 05784225 A EP05784225 A EP 05784225A EP 1789092 A2 EP1789092 A2 EP 1789092A2
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EP
European Patent Office
Prior art keywords
hgh
peg
growth hormone
conjugate
growth
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EP05784225A
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German (de)
English (en)
Inventor
Rory F. Pfizer Global Res. & Development FINN
Ned R. Pfizer Global Res. & Development SIEGEL
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Pharmacia and Upjohn Co LLC
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Pharmacia and Upjohn Co LLC
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Application filed by Pharmacia and Upjohn Co LLC filed Critical Pharmacia and Upjohn Co LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/06—Antiabortive agents; Labour repressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to PEGylation, of human Growth Hormone (hGH) by which the chemical and/or physiological properties of hGH can be changed.
  • the PEGylated hGH conjugate may have an increased plasma residency duration, decreased clearance rate, improved stability, decreased antigenicity, decreased PEGylation heterogeneity or a combination thereof.
  • the present invention also relates to processes for the modification of hGH.
  • 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.
  • Native human growth hormone is a protein comprising a single chain of 191 amino acids cross-linked by two disulphide bridges and the monomeric form has a molecular weight of 22 kDa.
  • Human GH is secreted by the pituitary gland and which also can be produced by recombinant genetic engineering. hGH will cause growth in all bodily tissues that are capable of growth.
  • hGH plays an important role not only in promoting growth in the growing phase in human beings but also in maintaining normal body composition, anabolism, and lipid metabolism (K. Bameis. And U. Keller, Baillieres Clin. Endocrinlo. Metab. 10:337 (1996)).
  • Recombinant hGH has been commercially available for several years.
  • Two types of therapeutically useful recombinant hGH preparations are present on the market: the authentic one, e.g. GenotropinTM, or NutropinTM and an analogue with an additional methionine residue at the N- terminal end, e.g. SomatonormTM.
  • hGH is used to stimulate linear growth in patients with hypo pituitary dwarfism also referred to as Growth Hormone Deficiency (GHD) or Turner's syndrome but other indications have also been suggested including long-term treatment of growth failure in children who were born short for gestational age (SGA), for treatment of patients with Prader-Willi syndrome (PWS), chronic renal insufficiency (CRI), AIDS wasting, and Aging.
  • GDD hypo pituitary dwarfism
  • PWS Prader-Willi syndrome
  • CRI chronic renal insufficiency
  • aGHD adult GH deficiency
  • aGHD have various problems, such as characteristic changes in body composition including increase in fat mass, decrease in lean body mass and extracellular fluid, and reduction of bone mineral density, metabolic abnormalities of lipids, and cardiovascular dysfunction. Many of those problems are improved by hGH replacement therapy (J.
  • GH growth hormone
  • the organ systems affected include the skeleton, connective tissue, muscles, and viscera such as liver, intestine, and kidneys. Growth hormones exert their effect through interaction with specific receptors on the target cell's membrane.
  • hGH is a member of a family of homologous hormones that include placental lactogens, prolactins, and other genetic and species variants or growth hormone (Nicoll, C. S., et al. (1986) Endocrine Reviews 7: 169).
  • hGH is unusual among these in that it exhibits broad species specificity and binds to either the cloned somatogenic (Leung, D. W., et al. [1987] Nature 330; 537) or prolactin receptor (Boutin, J. M., et al. [1988] Cell; 53: 69).
  • the cloned gene for hGH has been expressed in a secreted form in Escherichia coli (Chang, C. N., et al. [1987] Gene 55:189), and its DNA and amino acid sequence has been reported (Goeddel, et al. [1979) Nature 281 : 544; Gray, et al. [1985] Gene 39:247).
  • hGH Human growth hormone
  • hGH In adults, as well as in children, hGH maintains a normal body composition by increasing nitrogen retention and stimulation of skeletal muscle growth, and by mobilization of body fat. Visceral adipose tissue is particularly responsive to hGH. In addition to enhanced lipolysis, hGH decreases the uptake of triglycerides into body fat stores. Serum concentrations of IGF-I (insulin-like growth factor-l), and IGFBP3 (insulin-like growth factor binding protein 3) are increased by hGH. [007] hGH is a potent anabolic agent, especially due to retention of nitrogen, phosphorus, potassium, and calcium. Treatment of hypophysectomized rats with GH can restore at least a portion of the growth rate of the rats.
  • IGF-I insulin-like growth factor-l
  • IGFBP3 insulin-like growth factor binding protein 3
  • hGH causes a variety of physiological and metabolic effects in various animal models including linear bone growth, lactation, activation of macrophages, insulin-like and diabetogenic effects, and others (R. K. Chawla et al., Annu. Rev. Med. 34:519 (1983); 0. G. P. lsaksson et al., Annu. Rev. Physiol. 47, 483 (1985); C. K. Edwards et al., Science 239, 769 (1988); M. 0. Thomer and M. L. Vance, J. Clin. Invest. 82:745 (1988); J. P. Hughes and H. G. Friesen, Ann. Rev. Physiol. 47:469 (1985)).
  • homologous receptors contain a glycosylated extracellular hormone binding domain, a single transmembrane domain, and a cytoplasmic domain, which differs considerably in sequence and size.
  • One or more receptors are assumed to play a determining role in the physiological response to hGH.
  • physiologically active proteins administered into a body can show their pharmacological activity only for a short period of time due to their high clearance rate in the body. Furthermore, the relative hydrophobicity of these proteins may limit their stability and/or solubility.
  • poly(ethylene glycol) For poly(ethylene glycol), a variety of means have been used to attach the poly(ethylene glycol) molecules to the protein. Generally, poly(ethylene glycol) molecules are connected to the protein via a reactive group found on the protein. Amino groups, such as those on lysine residues or at the N-terminus, are convenient for such attachment. For example, Royer (U.S. Pat. No. 4,002,531 , above) states that reductive alkylation was used for attachment of poly(ethylene glycol) molecules to an enzyme.
  • Chamow et al., Bioconjugate Chem. 5: 133-140 (1994) report the modification of CD4 immunoadhesin with monomethoxypoly(ethylene glycol) aldehyde via reductive alkylation. U.S.
  • 5,824,784 demonstrates PEGylating G-CSF including at the N-terminus under reductive alkylation conditions.
  • WO 93/00109 relates to a method for stimulating a mammal's or avian's GH responsive tissues comprising, maintaining a continuous, effective plasma GH concentration for a period of 3 or more days.
  • One way of achieving such plasma concentration is stated to be by use of GH coupled to a macromolecular substance such as PEG (polyethylene glycol).
  • the coupling to a macromolecular substance is stated to result in improved half-life.
  • PEGylated human growth hormone has been reported in WO 93/00109 using mPEG aldehyde-5000 and mPEG N-hydroxysuccinmidyl ester(mPEG-NHS-5000) to achieve a hydrodynamic volume greater than the 7OK molecular weight cut-off of the kidney filtration as described (Knauf, M.J. et al, J. Biol. Chem. 263:15064-15070,1988).
  • 93/00109 also discloses the use of mPEG-maleimide to PEGylate cysteine hGH variants.
  • WO 99/03887 discloses a cysteine variant growth hormone that is PEGylated. Designated as BT-005, this conjugate is purported to be more effective at stimulating weight gain in growth hormone deficient rats and to have a longer half-life than hGH.
  • PEGylated human growth hormone has also been reported in Clark et al. using succinimidyl ester of carboxymethylated PEG (Journal of Biological Chemistry 271 :21969-21977,
  • Clark et al. describes derivates of hGH of increasing size using mPEG-NHS-5000, which selectively conjugates to primary amines. Increasing levels of PEG modification reduced the affinity for its receptor and increased the EC 50 in a cell-based assay up to 1500 fold. Olson et al., Polymer
  • WO 94/20069 prophetically discloses PEGylated hGH as part of a formulation for pulmonary delivery.
  • US 4,179,337 discloses methods of PEGylating enzymes and hormones to obtain physiologically active non-immunogenic, water-soluble polypeptide conjugates.
  • GH is mentioned as one example of a hormone to be PEGylated.
  • EP 458064 A2 discloses PEGylation of introduced or naturally present cysteine residues in somatotropin.
  • EP 458064 A2 further mentions the incorporation of two cysteine residues in a loop termed the omega loop stated to be located at residues 102-112 in wild type bovine somatotropin, more specifically EP 458064 A2 discloses the substitution of residues numbered 102 and 112 of bovine somatotropin from Ser to Cys and Tyr to Cys, respectively.
  • WO 95/11987 suggests attachment of PEG to the thio group of a cysteine residue being either present in the parent molecule or introduced by site directed mutagenesis.
  • WO 95/11987 relates to PEGylation of protease nexin-1 , however PEGylation in general of hGH and other proteins is suggested as well.
  • WO 99/03887 discloses, e.g., growth hormone modified by replacement serine at position 25 with a cysteine residue and attachment of PEG to the introduced cysteine residue.
  • WO 00/42175 relates to a method for making proteins containing free cysteine residues for attachment of PEG.
  • WO 00/42175 discloses the following muteins of hGH: T3C, S144C and T148C and the cysteine PEGylation thereof.
  • WO 97/11178 (as well as 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 the introduction or replacement of lysine (e.g. K168A and K172R).
  • WO 9711178 also discloses the substitution G 120K.
  • WO 03/044056 discloses a variety of PEGylated hGH species including a lysine branched 4OK PEG aldehyde hGH conjugate.
  • US 2004/0127417 discloses lysine branched PEG butyraldehyde hGH conjugates.
  • WO 04/46222, US 2005/0058620, JP 08-059818, JP 11 -228685, and JP 2000-001541 disclose polyalkylene glycol derivatives having a reactive group at the primary carbon at the 1 -position of a glycerol skeleton and having polyalkylene glycol chains at the 2- and 3-positions.
  • Currently administration of rhGH is daily for a long period of time, and therefore a less frequent administration would be highly desirable.
  • An hGH molecule with a longer circulation half-life would decrease the number of necessary administrations and potentially provide more optimal therapeutic hGH levels with concomitant enhanced therapeutic effect.
  • the present invention provides PEG-hGH conjugates having a single PEG attached predominately at the N-terminal phenylalanine of hGH, which provides advantages over other PEG-hGH conjugates.
  • hGH with nine lysines may have some molecules having ten PEGs attached, some with nine, some with eight, some with seven, some with six, some with five, some with four, some with three, some with two, some with one and some with zero. And, among the molecules with several, the PEG may not be attached at the same location on different molecules. This resulting heterogeneity is disadvantageous when developing a therapeutic product making conjugation, purification, and characterization difficult, costly, and highly irreproducible.
  • Another approach (WO 00/42175) has been to use hGH variants containing free cysteine residues for attachment of PEG.
  • the present invention relates to PEGylated hGH using a glycerol branched poly(ethylene glycol) moiety which may have at least one improved chemical or physiological property selected from but not limited to; decreased clearance rate, increased plasma residency duration, increased stability, improved solubility, and decreased antigenicity.
  • the present invention has a number of aspects relating to chemically modifying hGH using a glycerol branched poly(ethylene glycol) moiety.
  • the present invention may also have one or more improved properties compared to lysine based branched PEG human growth hormone conjugates including but not limited to: a) increased stability of the glycerol skeleton, b) increased receptor binding, c) decreased cost, d) increased N-terminal selectivity of attachment, e) increased solubility, f) decreased immunogenicity, g) increased stability of the conjugate, h) increased manufacturability, and i) decreased proteolysis.
  • the present invention also relates to methods of producing the PEGylated hGH. Particularly, the present invention relates to a method of producing a PEGylated hGH using a glycerol branched PEG.
  • the present invention also relates to compositions comprising the PEGylated hGH alone or in combination with another therapeutic agent.
  • the present invention also relates to the use of the 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.
  • Figure 1 is a size exclusion HPLC tracing showing the elution profile of the purified monoPEGylated glycerol branched 43K PEG aldehyde hGH reaction product on a TSK G4000PWXL column.
  • Figure 2 is a HPLC tracing of tryptic map analysis of hGH and glycerol branched 43K
  • PEG aldehyde hGH The top panel is the tryptic map of hGH.
  • the lower panel is the tryptic map of glycerol branched 43K PEG aldehyde hGH.
  • T1 is the N-terminal tryptic fragment.
  • Figure 3 shows the amino acid sequence of human growth hormone (SEQ ID NO:1).
  • Figure 4 shows the glycerol branched 43K PEG aldehyde hGH efficacy in an eleven-day
  • Rat Weight Gain Assay Hypophysectomized female Sprague-Dawley rats were purchased at the age of 4-5 weeks (85-11 Og) from Harlan Labs. Upon entering animal facilities, the animals were maintained at a constant room temperature of 8O 0 F. After 3 days' acclimation, the rats were weighed daily for 4-10 days in order to establish basal growth rates. Starting at day 0, rats ( ⁇ 100g) in control groups then received one daily subcutaneous injection of -0.3 mg/kg hGH (A), or PBS vehicle ⁇ for eleven consecutive days.
  • A mg/kg hGH
  • the glycerol branched 43K PEG aldehyde hGH test group (*) received single doses of 1.8 mg/kg of glycerol branched 43K PEG aldehyde hGH on days 0 and 6. There were 6 animals per group. Plotted values represent average weight gain ⁇ SEM.
  • Figure 5 shows eleven-day tibia growth in response to glycerol branched 43K PEG aldehyde hGH. Animals were those treated in Figure 4. Animals were sacrificed after taking day 11 weights, the left tibias were X-rayed, and the bone length measured using a caliper. Average length +/- SEM is plotted. Asterisks denote significant differences from control group (P ⁇ 0.05). There were 6 animals per group.
  • Figure 6 shows eleven-day blood urea nitrogen levels in response to glycerol branched 43K PEG aldehyde hGH. Blood samples were taken from animals treated in Figure 4. Serum was prepared and urea nitrogen levels were measured. Average + SEM is plotted (6 animals per group). Asterisks denote significant differences from control group (P ⁇ 0.05).
  • Figure 7 shows a six-day dose escalation efficacy study for glycerol branched 43K PEG aldehyde hGH.
  • This growth study was performed in a similar manner to that described in Figure 4 except that varied single doses of glycerol branched 43K PEG aldehyde hGH were administered only on day 0 and the study was run for 6 days.
  • Control groups received once-daily subcutaneous injections of either 0.3 mg/kg hGH ( ⁇ ) or PBS vehicle (o) for six consecutive days.
  • the glycerol branched 43K PEG aldehyde hGH test groups received a single dose of glycerol branched 43K PEG aldehyde hGH on day 0.
  • FIG. 8 shows serum IGF-1 levels for six-day efficacy study. Animals were treated as described in Figure 7. Blood samples were taken at the various times plotted and the serum IGF-1 levels determined by ELISA.
  • Figure 9 shows the PK/PD assessment following single dose administration of glycerol branched 43K PEG aldehyde hGH to hypophysectomized female rats.
  • the present invention relates to glycerol branched polyethylene glycol-human growth hormone conjugates.
  • the glycerol branched polyethylene glycol derivative has an aldehyde reactive group and optionally a linker between the polyethylene glycol and the reactive functional group at the primary carbon at the 1 -position of a glycerol skeleton and having polyalkylene glycol chains at the 2- and 3-positions as described in WO 04/46222 or US 2005/0058620 (incorporated by reference) to create hGH conjugates.
  • the linker is not particularly limited as far as it is a covalent bond, but preferably includes an alkylene group and an alkylene group containing an ester bond, a urethane bond, an amide bond, an ether bond, a carbonate bond, or a secondary amino group.
  • Preferable alkylene group includes a methylene group, an ethylene group, a trimethylene group, a propylene group, an isopropylene group, a tetramethylene group, a butylene group, an isobutylene group, a pentam ethylene group, and a hexamethylene group.
  • a specific embodiment of the present invention is a human growth hormone-PEG conjugate having the structure of the Formula:
  • R is a human growth hormone polypeptide.
  • n is between about 64 and about 72.
  • the (CH 2 CH 2 O) n moiety has an average molecular weight between about 2.6 and about 3.5Kd, and particularly the average molecular weight is about 3Kd
  • each (CH 2 CH 2 O) n moiety has an average molecular weight between about 17.6 and about 22Kd, and particularly the average molecular weight is about 20Kd.
  • the (CH 2 CH 2 O) n moiety has an average molecular weight of about 3Kd and each (CH 2 CH 2 O) n , moiety has an average molecular weight of about 20Kd.
  • the term "about" when used in connection with the molecular weight of a PEG moiety means that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight and the stated molecular weight refers to the average molecular weight. It is understood that there is some degree of polydispersity associated with polymers such as poly(ethylene glycol). It is preferable to use PEGs with low polydispersity. In a specific embodiment one of the terminal polymer hydroxyl end-groups is converted or capped with a methyl group. As used herein, the term “mPEG” refers to a PEG, which is capped at one end with a methyl group. The mPEG can be represented structurally as CH 3 O-(CH 2 CH 2 O) n -H
  • human growth hormone polypeptide encompasses all hGH polypeptides, characterized by promoting growth in the growing phase and in maintaining normal body composition, anabolism, and lipid metabolism.
  • hGH polypeptide refers to the hGH polypeptide of SEQ ID NO:1
  • the hGH polypeptides of the present invention can be prepared in any suitable manner.
  • hGH polypeptides and fragments thereof may be purified from natural sources, chemically synthesized, produced by recombinant techniques including in vitro translation techniques or expression in a recombinant cell able to express hGH cDNA, or a combination of these methods, using techniques known to those skilled in the art (See, for example, "Methods in Enzymology, Academic Press, 1993” for a variety of methods 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.
  • polypeptides of the present invention are preferably provided in an isolated form, and may be partially or preferably substantially purified.
  • a specific embodiment of the present invention is a human growth hormone-PEG conjugate wherein greater than 80%, more preferably 81%, more preferably 82%, more preferably 83%, more preferably 84%, more preferably 85%, more preferably 86%, more preferably 87%, more preferably 88%, more preferably 89%, more preferably 90%, more preferably 91%, more preferably 92%, more preferably 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97, and more preferably 98% of the polyethylene glycol is conjugated to the amino-terminal phenylalanine of the human growth hormone of SEQ ID NO:1.
  • Another embodiment of the present invention is a substantially homogenous preparation of N-terminally PEGylated hGH optionally in a pharmaceutically acceptable diluent, carrier or adjuvant, said preparation being essentially free of hGH PEGylated at sites other than the N-terminus.
  • substantially homogenous preparation means a preparation where greater than 80%, more preferably 81%, more preferably 82%, more preferably 83%, more preferably 84%, more preferably 85%, more preferably 86%, more preferably 87%, more preferably 88%, more preferably 89%, more preferably 90%, more preferably 91 %, more preferably 92%, more preferably 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97, and more preferably 98% is monoPEGylated.
  • secondary amine linkages are formed between the N- terminal primary ⁇ - amino group of a hGH polypeptide and a glycerol branched chain PEG aldehyde by reductive alkylation as described in 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 with a suitable reducing agent such as NaCNBH 3 , NaBH 3 , Pyridine Borane etc.
  • a suitable reducing agent such as NaCNBH 3 , NaBH 3 , Pyridine Borane etc.
  • the glycerol branched PEG aldehyde is incubated with an hGH polypeptide resulting in the addition of the PEG moiety to amino groups via Schiff's base formation. These linkages are converted to stable secondary amines by reduction with a reducing agent.
  • the reductive alkylation process is depicted in the scheme below (from Chamow et al.).
  • Conjugation reactions were historically carried out in solution with molar excess of polymer and without regard to where the polymer will attach to the protein. Such general techniques, however, have typically been proven inadequate for conjugating bioactive proteins to non-antigenic polymers while retaining sufficient bioactivity.
  • One way to maintain the hGH bioactivity is to substantially avoid the conjugation of those hGH reactive groups associated with the receptor binding site(s) in the polymer coupling process.
  • Another aspect of the present invention is to provide a process of conjugating poly( ethylene glycol) to hGH maintaining high levels of retained activity.
  • the chemical modification through a covalent bond may be performed under any suitable condition generally adopted in a reaction of a biologically active substance with the activated poly(ethylene glycol).
  • the conjugation reaction is carried out under relatively mild conditions to avoid inactivating the hGH. Mild conditions include maintaining the pH of the reaction solution in the range of 3 to 10 and the reaction temperatures within the range of from about 0°-37°C.
  • suitable buffers pH 4 to 10
  • suitable buffers including phosphate, MES, citrate, acetate, succinate or HEPES, for 1-48 hrs at 4° -37 0 C.
  • the activated poly(ethylene glycol) may be used in about 0.01-100 times, preferably about 0.01-2.5 times, the molar amount of the number of free amino groups of hGH.
  • reaction conditions described herein can result in significant amounts of unmodified hGH, the unmodified hGH can be readily recycled into future batches for additional conjugation reactions.
  • the processes of the present invention generate surprisingly very little, i.e. less than about 20% and more preferably, less than about 10%, of high molecular weight species and species containing more than one polymer strand per hGH.
  • These reaction conditions are to be contrasted with those typically used for polymeric conjugation reactions wherein the activated polymer is present in several-fold molar excesses with respect to the target.
  • the conjugation reactions of the present invention initially provide a reaction mixture or pool containing mono- PEG-hGH conjugates, unreacted hGH, unreacted polymer, and less than about 20% high molecular weight species.
  • the high molecular weight species include conjugates containing more than one polymer strand and/or polymerized PEG-hGH species. After the unreacted species and high molecular weight species have been removed, compositions containing primarily mono- PEGylated-hGH conjugates are recovered. Given the fact that the conjugates for the most part include a single polymer strand, the conjugates are substantially homogeneous.
  • modified hGH have at least about 0.1% of the in vitro biological activity associated with the native or unmodified hGH as measured using standard FDC-P1 cell proliferation assays, (Clark et al. Journal of Biological Chemistry 271 :21969-21977, 1996), receptor binding assay (US 5,057,417), or hypophysectomized rat growth (Clark et al. Journal of Biological Chemistry 271 :21969-21977, 1996).
  • the modified hGH have about 25% of the in vitro biological activity, more preferably, the modified hGH have about 50% of the in vitro biological activity, more preferably, the modified hGH have about 75% of the in vitro biological activity, and most preferably the modified hGH have equivalent or improved in vitro biological activity.
  • the processes of the present invention preferably include rather limited ratios of polymer to hGH.
  • the hGH conjugates have been found to be predominantly limited to species containing only one strand of polymer.
  • the attachment of the polymer to the hGH reactive groups is substantially less random than when higher molar excesses of polymer linker are used.
  • the unmodified hGH present in the reaction pool, after the conjugation reaction has been quenched, can be recycled into future reactions using ion exchange or size exclusion chromatography or similar separation techniques.
  • a poly(ethylene glycol)-modified hGH may be purified from a reaction mixture by conventional methods which are used for purification of 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.
  • the mono PEGylated-hGH species is isolated from the reaction mixture to remove high, molecular weight species, and unmodified hGH.
  • Separation is effected by placing the mixed species in a buffer solution containing from about 0.5-10 mg/mL of the hGH-polymer conjugates.
  • Suitable solutions have a pH from about 4 to about 10.
  • the solutions preferably contain one or more buffer salts selected from KCI, NaCI, 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.
  • the hGH polymer conjugate solution may first have to undergo buffer exchange/ultrafiltration to remove any unreacted polymer.
  • the PEG- hGH conjugate solution can be ultrafiltered across a low molecular weight cut-off (10,000 to 30,000 Dalton) membrane to remove most unwanted materials such as unreacted polymer, surfactants, if present, or the like.
  • the fractionation of the conjugates into a pool containing the desired species is preferably carried out using an ion exchange chromatography medium.
  • Such media are capable of selectively binding PEG-hGH conjugates via differences in charge, which vary in a somewhat predictable fashion.
  • the surface charge of hGH is determined by the number of available charged groups on the surface of the protein. These charged groups typically serve as the point of potential attachment of poly(alkylene oxide) polymers. Therefore, hGH conjugates will have a different charge from the other species to allow selective isolation.
  • Strongly polar anion or cation exchange resins such as quaternary amine or sulfopropyl resins, respectively, are used for the method of the present invention.
  • Anion exchange resins are especially preferred.
  • a non-limiting list of included commercially available cation exchange resins suitable for use with the present invention are SP-hitrap®, SP Sepharose HP® and SP Sepharose® fast flow. Other suitable cation exchange resins e.g. S, and CM resins can also be used.
  • a non- limiting list of anion exchange resins, including commercially available anion exchange resins, suitable for use with the present invention are Q-hitrap®, Q Sepharose HP®, and Q sepharose® fasHlow.
  • the anion or cation exchange resin is preferably packed in a column and equilibrated by conventional means.
  • a buffer having the same pH and osmolality as the polymer conjugated hGH solution is used.
  • the elution buffer preferably contains one or more salts selected from KCI, NaCI, 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 .
  • the conjugate-containing solution is then adsorbed onto the column with unreacted polymer and some high molecular weight species not being retained.
  • a gradient flow of an elution buffer with increasing salt concentrations is applied to the column to elute the desired fraction of polyalkylene oxide-conjugated hGH.
  • the eluted pooled fractions are preferably limited to uniform polymer conjugates after the cation or anion exchange separation step. Any unconjugated hGH species can then be back washed from the column by conventional techniques. If desired, mono and multiply pegylated hGH species can be further separated from each other via additional ion exchange chromatography or size exclusion chromatography.
  • the temperature range for elutio ⁇ is between about 4°C and about 25°C.
  • elution is carried out at a temperature of from about 4°C to about 22°C.
  • the elution of the PEG-hGH fraction is detected by UV absorbance at 280 nm. Fraction collection may be achieved through simple time elution profiles.
  • a surfactant can be used in the processes of conjugating the poly( ethylene glycol) polymer with the hGH moiety.
  • Suitable surfactants include ionic-type agents such as sodium dodecyl sulfate (SDS).
  • SDS sodium dodecyl sulfate
  • Other ionic surfactants such as lithium dodecyl sulfate, quaternary ammonium compounds, taurocholic acid, caprylic acid, decane sulfonic acid, etc. can also be used.
  • Non-ionic surfactants can also be used.
  • materials such as poly(oxyethylene) sorbitans (Tweens), poly(oxyethylene) ethers (Tritons) can be used.
  • the only limitations on the surfactants used in the processes of the invention are that they are used under conditions and at concentrations that do not cause substantial irreversible denaturation of the hGH and do not completely inhibit polymer conjugation.
  • the surfactants are present in the reaction mixtures in amounts from about 0.01-0.5%; preferably from 0.05-0.5%; and most preferably from about 0.075- 0.25%. Mixtures of the surfactants are also contemplated.
  • surfactants provide a temporary, reversible protecting system during the polymer conjugation process. Surfactants have been shown to be effective in selectively discouraging polymer conjugation while allowing lysine-based or amino terminal-based conjugation to proceed.
  • the present poly(ethylene glycol)-modified hGH has a more enduring pharmacological effect, which may be possibly attributed to its prolonged half-life in vivo.
  • Another embodiment of the invention relates to methods for the prevention and/or treatment of a disease or disorder in which use of GH, preferably hGH is beneficial, comprising administering to a patient in need thereof a therapeutically effective amount of a poly(ethylene glycol)- modified hGH of the invention or agonist variant thereof, alone or in combination with another therapeutic agent.
  • the invention also relate to the use of a poly(ethylene glycol)-modified hGH of the invention or agonist variant thereof in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder in which use of GH, preferably hGH is beneficial.
  • the invention also relates to a pharmaceutical composition comprising a poly(ethylene glycol)-modified hGH of the invention or agonist variant thereof for the prevention and/or treatment of a disease or disorder in which use of GH, preferably hGH is beneficial.
  • GFD growth hormone deficiency
  • aGHD adult growth hormone deficiency
  • Turner's syndrome growth failure in children who were born short for gestational age
  • PWS Prader-Willi syndrome
  • CRI chronic renal insufficiency
  • Aids wasting Aging, end-stage Renal Failure
  • Cystic Fibrosis Erectile dysfunction
  • HIV lipodystrophy Fibromyalgia
  • Osteoporosis Memory disorders
  • Depression Crohn's disease
  • Skeletal dysplasias Traumatic brain injury, Subarachnoid haemorrhage, Noonan's syndrome, Down's syndrome, Idiopathic short stature (ISS), End stage renal disease (ESRD), Very low birth weight (VLBW)
  • Bone marrow stem cell rescue Metabolic syndrome
  • Glucocorticoid myopathy Short stature due to glucocorticoid
  • the poly(ethylene glycol)-modified hGH of the invention or agonist variants thereof are used in the prevention and/or treatment of a disorders or diseases selected from the group consisting of GHD, aGHD, SGA, PWS, Turner's syndrome and CRI.
  • the poly(ethylene glycol)-modified hGH of the invention or agonist variants thereof are used in the prevention and/or treatment of a disorders or diseases selected from the group consisting of idiopathic short stature, very low birth weight, traumatic brain injury, metabolic syndrome, and Noonan's syndrome.
  • compositions comprising a poly(ethylene glycol)-modified hGH of the invention alone or in combination with another therapeutic agent, and at least one pharmaceutically acceptable excipient or carrier.
  • the present poly(ethylene glycol)-modified hGH may then be formulated into pharmaceuticals containing also a pharmaceutically acceptable diluent, an agent for preparing an isotonic solution, a pH-conditioner and the like in order to administer them into a patient.
  • the above pharmaceuticals may be administered subcutaneously, intramuscularly, intravenously, pulmonary, intradermal ⁇ , or orally, depending on a purpose of treatment.
  • a dose may be also based on the kind and condition of the disorder of a patient to be treated, being normally between 0.1 mg and 5 mg by injection and between 0.1 mg and 50 mg in an oral administration for an adult.
  • the poly(ethylene glycol)-modified hGH or agonist variants thereof of the present invention may be used in combination with another therapeutic agent.
  • the terms "co-administration”, “co-administered” and “in combination with”, referring to the compounds A and one or more other therapeutic agents is intended to mean, and does refer to and include the following : o simultaneous administration of such combination of A and therapeutic agent(s) to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said patient; o substantially simultaneous administration of such combination of A and therapeutic agent(s) to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said patient, whereupon said components are released at substantially the same time to said patient; o sequential administration of such combination of A and therapeutic agent(s) to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are
  • Suitable examples of other therapeutic agents which may be used in combination with A, their pharmaceutically acceptable salts and/or their derived forms include, but are by no mean limited to: aromatase inhibitors such as exemestane, formestane, atamestane, fadrozole, letrozole, vorozole and anastrozole; free fatty acid regulators including fibric acid derivatives (such as fenofibrate, clofibrate, gemfibrozil, bezafibrate and ciprofibrate) and nicotinic acid derivatives such as acipimox; insulin sensitizing agents including but not limited to biguanides such as metformin, PPAR gamma insulin sensitizing agents and thiazolodeniones such as troglitazone and rosiglitazone Troglitazone, 5- [[4-[3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-]-benz
  • I PPAR agonists under development include: Reglitazar (JTT 501 , PNU 182716, PNU 716) (Chemical Name: lsoxazolidien-3, 5-dione, i 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) ChemicalName: (/-)-5-[4-(5-Methoxy- 1 H benzimidazol-2- ylmethoxy)benzyl] thiazolin-2,4-dione; hydrochloride; 15 DRF 2189, Chemical Name: 5-[[4-[2-(1- lndolyl)ethoxy
  • Patent 4,411 ,890 antagonists of gonadotropin releasing hormone such as those described in WO0170228, WO0170227, WO0170228, WO0069433, WO0004013, W0995156, WO9951595, WO9951231-4, WO9941251-2, WO9921557, WO9921553 and 6-AZAI NDOLE COMPOUNDS as described in WO0053602, WO0053185, WO0053181 , WO0053180, WO0053179, WO0053178, US6288078; IGF- 1 secretagogues; insulin-like growth factor-2 (IGF- 2 or somatomedin A) and IGF-2 secretagogues; myostatin antagonists and compounds which inhibit fibroblast growth factor receptor-3 (FGFR-3) tyrosine kinase.
  • gonadotropin releasing hormone such as those described in WO0170228, WO0170227, WO0170228, WO
  • the polymeric substances included are also preferably water-soluble at room temperature.
  • a non-limiting list of such polymers include poly(alkylene oxide) homopolymers such as poly(ethylene glycol) or poly(propylene glycols), poly(oxyethylenated polyols), copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
  • PEG-based polymers effectively non-antigenic materials such as dextran, polyvinyl pyrrolidones), poly(acrylamides), polyvinyl alcohols), carbohydrate-based polymers, and the like can be used.
  • hGH is that of SEQ ID NO:1. It is understood that other hGH polypeptides could also be PEGylated in a similar manner as exemplified in the subsequent examples.
  • (CH 2 CH 2 O) n has an average molecular weight of about 3Kd and each (CH 2 CH 2 O) m has an average molecular weight of about 20Kd
  • This example demonstrates generation of N-terminally monoPEGylated hGH by reductive alkylation.
  • the glycerol branched PEG aldehyde reagent of approximately 43,000 MW (GL3-400AL2 NOF corporation) was coupled via reductive alkylation to the N-terminus of hGH by taking advantage of the difference in the relative pK a value of the primary amine at the N-terminus versus pK a values of primary amines at the ⁇ -amino position of lysine residues.
  • hGH protein dissolved at 4, 7, or 10 mg/mL in 25 mM MES (Sigma Chemical, St.
  • Table 1 shows the percent of multi-PEGylated species, mono-PEGylated conjugate, un- reacted hGH, and final purification yield for glycerol branched 43K PEG aldehyde hGH reacted for 63 hrs at a pH of 5.8 and a 1.5:1 :1 molar ratio.
  • the PEG hGH species were purified from the reaction mixture to >95% (SEC analysis Figure 1) using a single anion exchange chromatography step. Mono-PEGylated hGH was purified from unmodified hGH and multi-PEGylated hGH species using anion exchange chromatography.
  • a typical glycerol branched 43K PEG aldehyde hGH reaction mixture (80 or 1500 mg protein), as described above, was purified on a Q-Sepharose Hitrap column (5 ml_)(Amersham Pharmacia Biotech, Piscataway, NJ) or Q-Sepharose column (26/20, 70 ml_ bed volume)(Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in 25 mM HEPES, pH 7.3 (Buffer A).
  • the reaction mixture was diluted 7X with buffer A and loaded onto the column at a flow rate of 2.5 mL/min.
  • the column was washed with 3-10 column volumes of buffer A.
  • the various hGH species were eluted from the column in 20 column volumes of Buffer A and a linear NaCI gradient of 0-100 mM.
  • the eluant was monitored by absorbance at 280 nm (A 2 ⁇ o) and appropriate size fractions were collected.
  • Fractions were pooled as to extent of PEGylation, e.g., mono, di, tri etc. (as assessed in example 3).
  • the pool was then concentrated to 0.5-5 mg/mL in a Centriprep YM10 concentrator (Amicon, Technology Corporation, Northborough, MA) or by diafiltration. Protein concentration of pool was determined by A 280 using an extinction coefficient of 0.78.
  • the 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)
  • PEGylation greatly increases the hydrodynamic volume of the protein resulting in a shift to an earlier retention time.
  • New species were observed in the PEG aldehyde hGH reaction mixtures along with unmodified hGH.
  • These PEGylated and non-PEGylated species were separated on Q-Sepharose chromatography, and the resultant purified mono PEG-Aldehyde hGH species were subsequently shown to elute as a single peak on non-denaturing SEC (> 95% purity, Figure 1).
  • the Q-Sepharose chromatography step effectively removed free PEG, hGH, and multi PEGylated hGH species from the mono-PEGylated hGH.
  • SDS-PAGE was used to assess the reaction of glycerol branched 43K PEG aldehyde with hGH and the purified final products. SDS-PAGE was carried out on 1 mm thick 10-NuPAGE gels (Invitrogen, Carlsbad, CA) under reducing and non-reducing conditions and stained using a Novex Colloidal CoomassieTM G-250 staining kit (Invitrogen, Carlsbad, CA.
  • Tryptic digests were performed at a concentration of 1 mg/mL and typically 25 ug of material was used per digest. Trypsin was added such that the trypsin to PEG-hGH ratio was 1:30
  • Tris buffer was present at 30 mM, pH 7.5. Samples were incubated at room temperature for
  • Peaks were detected using a Waters 996 PDA detector collecting data between 210 and
  • T- 1 Tryptic maps were performed for hGH, and glycerol branched 43K PEG aldehyde reacted at a molar ratio of 2:1 (PEG:hGH), ( Figure 2).
  • the N-terminal tryptic fragment was referred to as T- 1.
  • the percent of T- 1 present compared to unPEGylated hGH suggests that greater than 99% of the
  • PEG modification is at the N-terminus with remainder apparently linked to one of several possible lysine residues.
  • k a expressed as per M per second
  • k d expressed as per second both are an average value of at least 3 measurements on 1 chip + standard deviation. The data assumes to measure hGH binding to the high affinity site 1 on GHBP at 1 :1 ratio.
  • BW body weight
  • hGH human growth hormone
  • Values in parenthesis represent change from Day 0 in mean ⁇ SEM.
  • hGH and glycerol branched 43K PEG aldehyde hGH protein concentration levels in rat plasma were determined using the hGH AutoDELFIA kit fluorescence immunoassay (Perkin-Elmer). Table 5.

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Abstract

La présente invention concerne la pégylation d'hormone de croissance humaine (hGH) au moyen d'un glycérol de PEG ramifié. La présente invention concerne aussi des processus de pégylation de hGH. Cette invention concerne aussi des compositions pharmaceutiques comprenant cette hormone de croissance humaine pegylatée. Un autre mode de réalisation de l'invention concerne l'utilisation de cette hormone de croissance humaine pegylatée destinée au traitement de troubles de la croissance et du développement.
EP05784225A 2004-08-31 2005-08-25 Conjugués de l'hormone de croissance humaine avec du polyéthylèneglycol ramifié à l'aide de glycérol, procédé de sa préparation, et méthode de son utilisation. Withdrawn EP1789092A2 (fr)

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Families Citing this family (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69838552T2 (de) 1997-07-14 2008-05-21 Bolder Biotechnology, Inc., Louisville Derivate des wachstumshormons und verwandte proteine
US7495087B2 (en) 1997-07-14 2009-02-24 Bolder Biotechnology, Inc. Cysteine muteins in the C-D loop of human interleukin-11
JP2002534119A (ja) 1999-01-14 2002-10-15 ボルダー バイオテクノロジー， インコーポレイテッド 自由システイン残基を有するタンパク質の生産方法
US7855279B2 (en) 2005-09-27 2010-12-21 Amunix Operating, Inc. Unstructured recombinant polymers and uses thereof
EP1834963A1 (fr) * 2006-03-13 2007-09-19 Siegfried Ltd. Conjugues de proteines et de dipolymeres et leurs procedes de preparation
US20090117077A1 (en) * 2006-05-12 2009-05-07 Dong-A Pharm. Co., Ltd. Polyethylene glycol-interferon alpha conjugate
JP2008069073A (ja) * 2006-09-12 2008-03-27 Yokohama Tlo Co Ltd ラクトフェリン複合体及びその製造方法
KR101079993B1 (ko) * 2006-11-17 2011-11-04 동아제약주식회사 폴리에틸렌글리콜 과립구 콜로니 자극인자 접합체
CL2008002399A1 (es) 2007-08-16 2009-01-02 Pharmaessentia Corp Conjugado sustancialmente puro que posee una porcion polimerica, una porcion proteica (interferon alfa 2b) y un ligante alifatico de 1 a 10 atomos de carbono, util en el tratamiento de las hepatitis b o c.
WO2010011096A2 (fr) * 2008-07-23 2010-01-28 Hanmi Pharmaceutical Co., Ltd. Complexe de polypeptide comprenant un polymère non-peptidylique ayant trois extrémités fonctionnelles
MY156568A (en) 2008-07-31 2016-03-15 Pharmaessentia Corp Peptide-polymer conjugates
SI2340271T1 (sl) * 2008-10-10 2019-08-30 Polyactiva Pty Ltd. Konjugati polimer-bioaktivnega sredstva
US8535655B2 (en) 2008-10-10 2013-09-17 Polyactiva Pty Ltd. Biodegradable polymer—bioactive moiety conjugates
CA2739078C (fr) * 2008-10-10 2017-01-03 The Bionic Ear Institute Conjugues polymere biodegradable - fractions bioactives
US8680050B2 (en) 2009-02-03 2014-03-25 Amunix Operating Inc. Growth hormone polypeptides fused to extended recombinant polypeptides and methods of making and using same
US8703717B2 (en) 2009-02-03 2014-04-22 Amunix Operating Inc. Growth hormone polypeptides and methods of making and using same
DK2393828T3 (en) 2009-02-03 2017-01-23 Amunix Operating Inc Extended recombinant polypeptides and compositions comprising same
US9849188B2 (en) 2009-06-08 2017-12-26 Amunix Operating Inc. Growth hormone polypeptides and methods of making and using same
CN102612376A (zh) 2009-08-06 2012-07-25 诺沃-诺迪斯克保健股份有限公司 具有延长的体内功效的生长激素
CN101831067A (zh) * 2010-05-31 2010-09-15 王二新 聚乙二醇脂类缀合物及其在制备药物中的应用
EP2446898A1 (fr) 2010-09-30 2012-05-02 Laboratorios Del. Dr. Esteve, S.A. Utilisation de l'hormone de croissance pour améliorer la réponse immunitaire chez des patients immunodéprimés
CN102367290B (zh) * 2011-04-26 2013-05-08 厦门赛诺邦格生物科技有限公司 链官能化的多级支化聚乙二醇及其合成方法
WO2014110867A1 (fr) * 2013-01-17 2014-07-24 厦门赛诺邦格生物科技有限公司 Polyéthylèneglycol ramifié monofonctionnel et substance de type biologique modifiée par celui-ci
CN104877127B (zh) 2015-06-23 2017-11-10 厦门赛诺邦格生物科技股份有限公司 一种八臂聚乙二醇衍生物、制备方法及其修饰的生物相关物质
EA037151B1 (ru) 2014-11-06 2021-02-11 Фармаэссентия Корпорейшн Способ лечения с использованием пегилированного интерферона
CN111727063A (zh) * 2017-12-29 2020-09-29 豪夫迈·罗氏有限公司 用于提供聚乙二醇化蛋白质组合物的方法
JP7137625B2 (ja) * 2017-12-29 2022-09-14 エフ．ホフマン－ラ ロシュ アーゲー Ｐｅｇ化タンパク質組成物を提供するための方法
ES2905105T3 (es) 2017-12-29 2022-04-07 Hoffmann La Roche Procedimiento para proporcionar una composición de proteína PEGilada

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2147192A (en) * 1991-06-28 1993-01-25 Genentech Inc. Method of stimulating immune response using growth hormone
JP3092531B2 (ja) * 1996-11-05 2000-09-25 日本油脂株式会社 コハク酸イミジル基置換ポリオキシアルキレン誘導体の製造方法
DE69838552T2 (de) * 1997-07-14 2008-05-21 Bolder Biotechnology, Inc., Louisville Derivate des wachstumshormons und verwandte proteine
JP3921781B2 (ja) * 1998-02-12 2007-05-30 日本油脂株式会社 カルボキシル基含有ポリオキシアルキレン化合物
JP2002534119A (ja) * 1999-01-14 2002-10-15 ボルダー バイオテクノロジー， インコーポレイテッド 自由システイン残基を有するタンパク質の生産方法
JP2005525302A (ja) * 2001-11-20 2005-08-25 ファルマシア・コーポレーション 化学的に修飾されたヒト成長ホルモンコンジュゲート
BR0314172A (pt) * 2002-09-09 2005-07-26 Nektar Therapeutics Al Corp Polìmero solúvel em água, composição, forma de hidrato ou acetal, composto,composição, usos do composto e do conjugado, e, processo para preparar o conjugado
JP4412461B2 (ja) * 2002-11-20 2010-02-10 日油株式会社 修飾された生体関連物質、その製造方法および中間体
MXPA05007165A (es) * 2002-12-31 2005-09-21 Nektar Therapeutics Al Corp Reactivos polimericos que comprnden una cetona o un grupo funcional relacionado.
EA011351B1 (ru) * 2003-05-23 2009-02-27 Нектар Терапеутикс Ал, Корпорейшн Полимерные реагенты, способы их получения, а также содержащие их конъюгаты и фармацевтические препараты

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2006024953A2 *

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WO2006024953A3 (fr) 2007-01-18
NL1029828A1 (nl) 2006-03-01
PE20060654A1 (es) 2006-08-12
MX2007002441A (es) 2007-05-04
TW200621291A (en) 2006-07-01
CN101010105A (zh) 2007-08-01
NL1029828C2 (nl) 2006-10-20
BRPI0515118A (pt) 2008-07-01
UY29088A1 (es) 2006-03-31
CR8942A (es) 2007-08-16
IL181085A0 (en) 2007-07-04
AP2007003919A0 (en) 2007-02-28
MA28908B1 (fr) 2007-10-01
AU2005278903A1 (en) 2006-03-09
ZA200701802B (en) 2008-08-27
KR20070042567A (ko) 2007-04-23
WO2006024953A2 (fr) 2006-03-09
GT200500235A (es) 2006-03-21
CA2577999A1 (fr) 2006-03-09
AR050851A1 (es) 2006-11-29
JP2008511610A (ja) 2008-04-17
TNSN07078A1 (fr) 2008-06-02

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