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WO1992017200A2 - Formulations stables a base d'ion metal et d'hormone de croissance - Google Patents

Formulations stables a base d'ion metal et d'hormone de croissance Download PDF

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Publication number
WO1992017200A2
WO1992017200A2 PCT/US1992/002365 US9202365W WO9217200A2 WO 1992017200 A2 WO1992017200 A2 WO 1992017200A2 US 9202365 W US9202365 W US 9202365W WO 9217200 A2 WO9217200 A2 WO 9217200A2
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WIPO (PCT)
Prior art keywords
hgh
formulation
zinc
metal ion
concentration
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PCT/US1992/002365
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English (en)
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WO1992017200A3 (fr
Inventor
Brian C. Cunningham
Michael G. Mulkerrin
James A. Wells
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Genentech, Inc.
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Publication of WO1992017200A2 publication Critical patent/WO1992017200A2/fr
Publication of WO1992017200A3 publication Critical patent/WO1992017200A3/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin

Definitions

  • hGH human growth hormone
  • a divalent metal ion such as zinc and hGH.
  • the use of such hGH formulations having improved stability and therapeutic efficacy are described.
  • Description of the Background Art Human growth hormone (hGH) is synthesized and secreted into storage granules prior to its release from the anterior pituitary (for review see W. F. Daughaday, in Textbook of Endocrinology, seventh edition, Chapter 18, ed. J.D. Wilson and D.W. Foster, (W. B. Saunders Co., Philadelphia, 1985), p. 577; U.J. Lewis, Ann. Rev. Physbl.46, 33-42 (1984)). Histochemical analysis of the anterior pituitary indicates that zinc is present in high concentrations in growth hormone secretory granules (0. Thorlacius-Ussing, Neuroendocrinol. 45, 233-242 (1987)).
  • GH growth hormone
  • Metal ions such as zinc have been shown to be useful in the prolonged parenteral release of somatotropins in an oil formulation (EP 177,478, published 04.10.84; EP 343,696, published 29.11.89). Similar slow release formulations of bovine growth hormone complexed with metal ion in an oil vehicle have been shown (EP 216,485, published 01.04.87). Metal ions have been used to recover somatotropin from dilute aqueous solutions by forming a precipitate (EP 277,043, published 03.08.88). Prolactin has been examined as a regulatory hormone for zinc uptake by the prostate gland (Leake etal., J. of Endocrinology 102(1), p73-76, 1984).
  • Zinc deficiency has been associated with a tendency to hyperprolactinemia (Koppelman, Medical Hypotheses, 25(2), p65-68, 1988). A review of the zinc requirement in humans can be found in Prasad, Special Topics in Endocrinology and Metabolism, vol 7, p45-76, 1985. Zinc somatostatin complexes have been shown to inhibit growth hormone release (U. S. Patents 3,998,795 and 3,926,937).
  • hGH Human growth hormone
  • 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., (I986) 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., era/., [I987] Nature 330. 537) or prolactin receptor (Boutin, J. M.,ef al., [I988] C I52, 69).
  • the cloned gene for hGH has been expressed in a secreted form in Eschericha 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, era/., [I985] Gene 39. 247).
  • the three-dimensional structure of hGH is not available. However, the three-dimensional folding pattern for porcine growth hormone (pGH) has been reported at moderate resolution and refinement (Abdel-Meguid, S. S., etai, [I987] Proc. Natl. Acad. Sci. USA 84. 6434).
  • Human growth hormone's receptor and antibody epitopes have been identified by homolog-scanning mutagenesis (Cunningham et al., Science 243: 1330, 1989).
  • the structure of novel amino terminal methionyl bovine growth hormone containing a spliced-in sequence of human growth hormone including histid iel ⁇ and histidine 21 has been shown (U.S. Patent 4,880,910)
  • Human growth hormone (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 era/., Annu. Rev. Med.34, 519 (1983); 0. G. P.
  • the organ systems affected include the skeleton, connective tissue, muscles, and viscera such as liver, intestine, and kidneys.
  • Growth hormone exert its action through interaction with specific receptors on the target cell's membrane.
  • formulations must be prepared. Such formulations must maintain activity for appropriate extended periods of time, they must be acceptable in their own right for easy and rapid administration, and they must be readily formulated.
  • Human growth hormone has been formulated in a variety of ways as described in U.S Pat. No. 5,096,885. In many cases pharmaceutical formulations are provided in frozen or in lyophilized form. In such a case, the composition must be thawed or reconstituted prior to use.
  • the frozen or lyophilized form is often used to maintain biochemical integrity and the bioactiv ' ity of the medicinal agent contained in the compositions under a wide variety of storage conditions, as it is recognized by those skilled in the art, lyophilized preparations often maintain activity better than their liquid counte ⁇ arts.
  • lyophilized preparations are reconstituted prior to use by the addition of suitable pharmaceutically acceptable diluent(s), such as sterile water for injection or sterile physiological saline solution, or an appropriate physiologically acceptable diluent.
  • a pharmaceutical formulation can be provided in liquid form appropriate for immediate use.
  • Desirable is a liquid formulation which maintains its activity in long term storage.
  • Current formulations of hGH lose activity due to the formation of inactive dimers and higher order aggregates during formulation processing as well as during storage and reconstitution. Other chemical changes, such as deamidation, oxidation and enzymatic hydrolysis may also occur upon storage.
  • Prior attempts to to prevent the formation of inactive dimers of hGH have not succeeded. The problems associated with inactive dimers are noted in Becker, G.W., Biotechnology and Applied Biochemistry 9. 478 (1987).
  • Another object of the present invention is the preparation of formulations of liquid hGH, stable at room temperature or body temperature, thereby facilitating administration.
  • Still another object is to provide a formulation which can be aerosolized for pulmonary use, or used in a needleless jet injector for subcutaneous injection.
  • Yet another object of the invention is to provide an hGH formulation with enhanced therapeutic characteristics, such as the more even systemic delivery of hGH throughout the body, as opposed to a partial preference for the area surrounding the site of administration.
  • the present invention is based upon the discovery that the inclusion of a divalent metal ion such as zinc, cobalt or copper, preferably zinc, into an hGH formulation results in the formation of stable zinc:hGH dimers that exhibit unexpected stability to denaturation and maintain the activity of hGH for long periods at temperatures up to and beyond 37°C.
  • Additional components of the stabilizing formulation may be a buffer, an amino acid, a bulking agent or a nonionic surfactant.
  • Other helical cytokines may be formulated using divalent metal ions, such as zinc, cobalt and copper. Among those cytokines formulations stabilized by divalent metal ions are are human prolactin and human placental lactogen. Optimum stability of helical cytokines is achieved when the molar ration of divalent metal ton to helical cytokine is 1:1.
  • Fig.1 Equilibrium dialysis and Scatchard analysis for binding of ⁇ Zn * to hGH. From these Scatchard values the concentrations of bound and free zinc were calculated and it was determined that zinc forms a 1 :1 complex with hGH whereby a dimer containing two zinc ions and two hGH is produced. Error bars indicate standard deviations from triplicate determinations.
  • Fig.2 Gel filtration chromatography of hGH in the presence (A) or absence (B) of ZnCI 2 . Size standards (bovine ⁇ -globulin, ovalbumin and myoglobin) were run separately to calibrate the column, and their peak locations are indicated in the standard curve at the top of the chromatogram. In the absence of zinc (B) the hGH is a monomer, while in the presence of zinc (A) a dimer is formed which partially dissociates resulting in an apparent molecular weight that is 1.85 times the monomeric hGH peak.
  • A zinc
  • Fig.3. Sedimentation equilibrium analysis of hGH in the presence ( • ) or absence ( _. ) of 20 ⁇ M Z ⁇ Cfe.
  • Panel A shows the concentration of hGH (mg/ml) versus radial distance.
  • Panel B shows In [hGH concentration] vs r 2 which gives a linear plot in the absence of ZnCfe ( C ), as expected for a monomeric species. The plot is curvalinear in the presence of ZnCfe ( • ) showing self association.
  • Fig. 4A UV difference spectra for binding of Zn 2+ ( • —) and Co 2+ (— -) to hGH. Both zinc and cobalt induce hGH dimer formation in the same manner.
  • Fig. 4B Visible difference spectra for binding of increasing concentrations of Co 2+ to hGH. Titration of hGH with Co 2+ produces a visible abso ⁇ tion spectrum with a maximum at 525 nanometers and molar abso ⁇ tivity at 260 cn 1 M- 1 . The wavelength maximum and molar abso ⁇ tivity are characteristic of Co 2+ in a tetrahedral environment coordinated by nitrogen and oxygen containing ligands.
  • Visible difference spectra are calculated from the before mixing spectrum of the tandem cells (1.0 cm) containing on one side 23 ⁇ M hGH and on the other side Co 2 - 1 - ranging from 10 to 2500 ⁇ M, and the spectrum after mixing the components.
  • Fig.5. Dimerization of hGH (wild type) and hGH mutants by addition of zinc.
  • Panels A-D show elution profiles for hGH and the designated hGH mutants. Mutants are indicated by the single- letter amino acid for the wild-type residue followed by its position in mature hGH and then the mutant residue. Within each panel are superimposed gel filtration chromatograms run in buffers containing a fixed concentration of either 10 ⁇ M ZnCI 2 (shaded peaks) or 1 mM EDTA (unshaded peaks).
  • Fig.6 Panel A. Folding model for hGH based upon a 2.8 A structure of porcine GH (25) showing the putative location of the Zn 2+ ligands (starred) and the extent of the prolactin receptor (dashed ellipse) and hGH receptor (solid circle).
  • Panel B One of three possible bridged ligand models that can account for the involvement of His18, His21 , and Glu174 in coordinating 2 zinc atoms per hGH dimer. A salient feature of this model is the two -fold symmetry axis through the center of the "twinned" metal site. Glu174 from each hormone is shown coordinating both Zn 2+ atoms simultaneously. It is also possible that either His18 or His21 can bridge both zinc atoms instead of Glu174.
  • Fig.7 Stability of hGH to denaturation by Gu-HCI in the presence (• ) or absence (O ), of 25 ⁇ M ZnCI 2 .
  • a solution containing 30 ⁇ g hGH, 0.01 M Tris buffer pH 8.0, with or without 25 ⁇ M ZnCI 2 and the indicated GuHCI concentration was equilibrated for 2 min at 25'C prior to measuring the ⁇ 222 in a 1 cm CD cell.
  • the Zn 2+ was added before the GuHCI.
  • the fraction folded was calculated from the fractional change in ⁇ 222 in the absence of Gu-HCI (folded) relative to that in 6.5 M Gu-HCI (unfolded).
  • Novel formulations of zinc and hGH result in a stable hGH formulation suitable for prolonged storage, and for therapeutic .administration.
  • Therapeutic formulations containing the Zn 2+ -hGH dimer are extremely stable, even at body temperature, while still allowing therapeutic administration of the formulation.
  • the presence of zinc induces hGH to dimerize cooperatively by coordinating the same set of ligands used to bind hGH to the extracellular domain of the human prolactin receptor (hPRLbp) (B. C. Cunningham, S. Bass, G. Fuh, J. A. Wells Science 250, 1709-1712 (1990)).
  • hPRLbp human prolactin receptor
  • the present invention is based upon the discovery that the inclusion of a divalent metal ion, preferably zinc, into an hGH formulation results in the formation of stable zinc:hGH dimers that exhibit unexpected stability to denaturation, maintain the activity of hGH for periods at temperatures between -76°c and 60°C, particularly for long periods about 37°C, and are not susceptible to undesirable reactions otherwise encountered during processing, reconstitution and storage.
  • processing includes filtration, filling into vials, and lyophilization.
  • Therapeutic formulations of an hGH:metal ion formulation for therapeutic administration are prepared for storage by mixing hGH and metal ion having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's
  • Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyviny Ipy rrolidone ; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose.
  • chelating agents such as EDTA: sugar alcohols such as mannitof or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween.
  • the invention is thus directed to such formulations, and to all associated formulations and as a means for effectively stabilizing human growth hormone.
  • the formulation contains at least a divalent metal ion selected from zinc, cobalt or copper and substantially pure hGH free of contaminating proteins or infectious agents found in humans.
  • Formulations of the present invention may additionally contain a pharmaceutically acceptable buffer, amino acid, bulking agent and/or non-ionic surfactant. These include, for example, buffers, chelating agents, antioxidants, preservatives, cosolvents, and the like; specific examples of these could include, trimethylamaine salts ("Tris buffer”), and disodium edetate.
  • Tris buffer trimethylamaine salts
  • human growth hormone or "hGH” denote human growth hormone produced, for example, from natural source extraction and purification, and by recombinant cell culture systems.
  • the native sequence of hGH and its characteristics are set forth, for example, in Hormone Drugs, Gueriguigan et al., U.S.P. Convention, Rockville, MD
  • the terms likewise cover biologically active human growth hormone equivalents; e.g., differing in one or more amino acid(s) in the overall sequence. Further, the terms as used in this application are intended to cover substitution, deletion and insertion amino acid variants of hGH, or post translational modifications. Examples of such variants are described in PCT Pub. WO90/04788, published 3 May 1990.
  • Another hGH modification is that created by the covalent addition of polyethylene glycol to reactive hGH amino acids (Davis et al. U.S. patent 4,179,337).
  • the hGH used in the formulations of the present invention is generally produced by recombinant means as previously discussed.
  • This formulation recombinant hGH is substantially pure, free of other human proteins, free of infectious agents such as the human immunodeficiency virus (HIV) and it is soluble, both as a monomer and as a metal ion containing dimer.
  • substantially pure hGH means hGH that is free of vesicular proteins with which it ordinarily is associated in pi tuitary cells.
  • substantially pure means hGH which is greater than about 95% pure by weight of total protein, and preferably greater than 98% pure by weight.
  • a "pharmaceutically effective amount" of hGH refers to that amount which provides therapeutic effect in various administration regimens.
  • the compositions hereof may be prepared containing amounts of hGH at least about 0.1 mg/ml, upwards of about 20 mg/ml or more, preferably from about 1 mgml to about 10 mg/ml.
  • these compositions may contain from about 0.1 mg ml to about 10 mg/ml, corresponding to the currently contemplated dosage rate for such treatment.
  • the divalent metal ion suitable for use in the formulations of the present invention may be selected from zinc, cobalt or copper.
  • the preferred metal ion for therapeutic administration is zinc. Cobalt is less preferred due to toxicity when administered in large amounts. However, for use in stabilizing hGH in non therapeutic formulations, cobalt use is indicated.
  • the following discussion of zinc and zinc salts may be equally applied to cobalt and copper ions.
  • Zinc is present in the formulation as the divalent Zn+ 2 ion which may be formulated using any pharmaceutically acceptable zinc salt .
  • zinc chloride, zinc acetate, zinc carbonate, zinc citrate, and the like Most preferred is zinc chloride with a molecular weight of about 136.29 daltons (hydrated).
  • Zinc ion is optimally present at a level sufficient to form stable zinc-hGH dimers each containing two zinc ions and two hGH molecules, when stability is the principle concern.
  • the zinc may be present at a molar concentration greater than 5% of the hGH molar concentration, advantageously greater greater than 50% of the hGH molar concentration, and most preferred is greater than 90% of the hGH molar concentration.
  • the metal ion is zinc and the zinc:hGH molar ratio is 1:1. At this molar ratio the hGH:zinc dimer is soluble and when released into bodily fluids is rapidly distributed.
  • the molar concentration of zinc may exceed the molar concentration of hGH, however at pH ranges above 7.0 the solubility of zinc hydroxide becomes a limiting factor. Therefore, the excess zinc ion in pH 7.0-7.4 formulations normally is less than 100 ⁇ M, more commonly less than 50 ⁇ M. Buffer and pH
  • the formulation of the present preferably contains a buffer.
  • the buffer may be any pharmaceutically acceptable buffering agent such as phosphate, tris-HCI, citrate and the like.
  • the preferred buffer is a phosphate buffer and the molar ratio of hGH:phosphate buffer is 1 :25- 250, advantageously 1:50-200, most advantageously 1 :75-125.
  • a buffer concentration greater than or equal to 2.5mM and less than 20mM is preferred, most advantageously 5-1 OmM. In this concentration range of buffer, minimal inactivation of hGH occurs.
  • a sodium phosphate or tris or buffer is used.
  • Suitable pH ranges, adjusted with buffer, for the preparation of the formulations hereof are from about 4 to about 8, advantageously about 6 to 8, most advantageously about 7.4.
  • the formulation pH should be less than 7.5 to reduce deamkJation. Above pH 7.0 the solubility of any free zinc+ 2 is reduced due to the formation of zinc hydroxide. pH values below 7.0 may result in paniculate formation upon reconstitution from a lyophilized state.
  • a pharmaceutically acceptable amino acid for example glycine
  • glycine is added to the hGH:metal ion formulation, preferably a zinc:hGH formulation.
  • the molar ratio of hGH:glycine is 1 :5-200.
  • Glycine may inhibit the formation of inactive hGH dimers or other inactive complexes when it is added in these ratios.
  • amino acids such as alanine, glutamine, asparagine, arginine or lysine or derivatives of such amino acids may be used in the subject formulation. Such amino acids are particularly advantageous when lyophilizing the formulation to create a sufficient mass to form a stable, dry caked formulation Formulation Bulking Agents
  • a bulking agent may be added to the formulation.
  • the bulking agent may be any pharmaceutically acceptable agent such as a carbohydrate, for example, glucose, mannose, or dextrins; sugar alcohols such as mannitol or sorbitol.
  • the molar ratio of hGH:bulking agent may be between 1 :50-8000.
  • mannitol other sugars or sugar alcohols are used such as sucrose, maltose, fructose, lactose and the like.
  • mannitol, glycine and a non-ionic surfactant in an hGH formulation and as a lypholization bulking matrix is described in U.S. Pat. No.
  • Non-Ionic Surfactant in another embodiment a non-ionic surfactant is added to the hGH-zinc formulation.
  • the formulation of the subject invention may optionally include one of several types of non-ionic surfactants, such as the potysorbates (e.G. polysorbate 20, 80 etc.) and the poloxamers (e.g. poloxamer 188).
  • the potysorbates e.G. polysorbate 20, 80 etc.
  • the poloxamers e.g. poloxamer 188
  • Advantageously polysorbate 80 is used, and the molar ratio of hGH:polysorbate 80 may be 1 :0.03-30.
  • polysorbate 80 is added in amounts of about 0.001 to about 2% (w/v), in order to enhance further the stability of the hGH.
  • Polysorbate 80 in concentrations above 0.01% (w/v) may reduce the amount of inactive hGH aggregates forming upon lyophilization and reconstitution.
  • non-ionic surfactants improves formulation stability when exposed to shear and surface stresses without causing denaturing of the protein.
  • surfactant containing hGH formulations may be employed in aerosol devices such as those used in a pulmonary dosing, and needleless jet injector guns. Such delivery formulations may be improved by the addition of non-ionic surfactants in the range of 0.1-5% (w/v). hGH Formulation. Storage and Administration
  • hGH formulations created by the formulations of the present invention permits a wider use of hGH formulations that may be more dilute, or alternatively, more concentrated, then those commonly in use in the absence of divalent metal ions, such as zinc.
  • the presence of zinc-hGH dimers also reduces the creation of surface induced denaturation of hGH that occurs during aerosolization or needleless injection of an hGH formulation.
  • Further optimal dispensing of the hGH formulations may be made wherein the hGH formulations of the present invention are dispensed into vials at at 1-50 mg/vial, preferably 2-25 mg/vial, and most preferably 3-10 mg/vial.
  • hGH:metal ion formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. hGH:metal ion formulations ordinarily will be stored in lyophilized form or in solution.
  • Therapeutic hGH:metal ion compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • hGH:metal ion formulations are in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional routes, or by sustained release systems as noted below.
  • hGH:metal ion formulations is administered continuously by infusion or by bolus injection.
  • sustained-release preparations include semipermeabie matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels [e.g., poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J. Biomed. Mater. Res., 15: 167-277 [1981] and Langer, Chem. Tech., 12: 98-105 [1982] or poly(vinylalcohol)], polylactides (U.S.
  • hGH:metal bn formulations compositions also include liposomalty entrapped hGH:metal ion formulations. Liposomes containing hGH:metal ion formulations are prepared by methods known perse: DE 3,218,121; Epstein etai., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang era/., Proc. Natl. Acad. Sci.
  • the liposomes are of the small (about 200-
  • the increased stability of the hGH formulations of the present invention permits dispensed vials containing hGH to be stored at temperatures above freezing, particularly where refrigeration is not available, or has been interrupted. Stability at room and body temperatures facilitates administration by allowing the hGH formulations of the present invention to be warmed prior to use, and to be stored in this warmed state without substantial loss of activity prior to such administration. This permits hGH administration in clinical situations where hGH formulations must be stored, transported, and used in the absence of optimal refrigeration.
  • a preservative may be added where the anticipated use of the vial may compromise the sterility of the formulation.
  • the preferred pharmaceutically acceptable preservatives are phenol, benzylalcohol, and para or metacresol.
  • the improved stability of hGH-zinc formulations of the present invention facilitates the distribution, storage and administration of hGH not previously available for any hGH formulation.
  • the formulation of the present invention may be prepared by well known procedures. For example, a solution of hGH in the final formulation is prepared by buffer exchange on a gel filtration column.
  • the elution buffer contains the zinc, buffer and any other components of the desired formulation.
  • the concentration of the hGH protein is obtained by dilution of this resulting gel filtration eluate to a desired protein concentration.
  • the formulation is sterile filtered, and can be stored for more than several weeks at 5°C and above., or filled into sterile vials. The vials may then be stored at 5° C, frozen, or freeze-dried using an appropriate lyophilization cycle.
  • the formulation of the subject invention comprises the following components at pH 7.4:
  • a stabilized pharmaceutically acceptable formulation of human growth hormone comprising: a) substantially pure hGH, and b) divalent metal ion.
  • This hGH formulation contains a divalent metal ion selected from zinc, cobalt or copper.
  • the hGH formulation contains the divalent metal ion and hGH are in about equal molar amounts. In an one embodiment, the hGH molar concentration is in excess of the divalent metal ion molar concentration.
  • the formulation of hGH and zinc may contain hGH wherein the concentration is greater than 0.5 micromolar and less than 2.0 millimolar. More preferably, the hGH concentration is greater than 40 micromolar and less than 1.0 millimolar.
  • the hGH concentration is greater 0.2 millimolar and less than 0.5 millimolar.
  • the hGH;zinc dimer of the present invention may contain a zinc concentration greater than 0.5 micromolar and less than 2.0 millimolar. More preferably, the zinc concentration is greater than 40 micromolar and less than 1.0 millimolar. Most preferably, the zinc concentration is greater than 0.1 millimolar and less than 0.6 millimolar. In one preferred example, the hGH concentration is about 0.45 millimolar (10 mg/ml) and the zinc concentration is about 0.45 millimolar.
  • the hGH:zinc dimer formulation may contain zinc from any pharmacologically acceptable salt, for example zinc chloride, zinc acetate, zinc carbonate, or zinc citrate.
  • the hGH:zinc formulation of the present invention may contain one or more additional formulation components selected from the following: c) a buffer, d) an amino acid, e) a bulking agent, or f) nonionic surfactant.
  • the buffer may be any pharmacologically acceptable buffer, for example a phosphate or a tris buffer.
  • a preferred method of stabilizing human growth hormone is combining substantially pure hGH and zinc in about equal molar amounts.
  • a preferred method for the stable storage of a human growth hormone (hGH) and zinc formulation comprises: (a) formulating hGH and zinc in a molar ratio of about 1:1 in a pharmaceutically acceptable vehicle; (b) dispensing into a pharmaceutically acceptable storage container the formulation of step (a); and, (c) storing said container at a temperature between -76°C. and 60°C for a period of at least about 3 days.
  • a preferred method of preventing the formation of human growth hormone inactive aggregates is accomplished by adding divalent metal ion to an hGH formulation in an amount sufficient to prevent the formation of inactive hGH aggregates. This method of preventing inactive hGH aggregates may be accomplished using divalent metal ions such as zinc, cobalt or copper.
  • One method of administering an aqueous human growth hormone comprises the steps of administering an aqueous formulation with an aerosol device or needleless injector gun, wherein the hGH formulation comprises (a) substantially pure hGH, and (b) zinc.
  • This hGH formulation used in the aerosol device or needleless injector gun may contain one or more additional formulation components selected from the following: c) a buffer, d) an amino acid, e) a bulking agent, or f) nonionic surfactant.
  • This formulation may contain a phosphate or a tris buffer, it may contain one of the following amino acids: glycine, alanine, glutamine, asparagine, arginine or lysine, it may contain one of the following bulking agents: mannitol, sorbitol, sucrose, maltose, fructose, lactose, glucose or mannose, and it may contain a nonionic surfactant is selected from the following: polysorbate 80 or poloxamer.
  • the methods of the present invention may be used to create stabilized formulations of helical cytokines, such as human prolactin and human placental lactogen.
  • the formulation of a stabilized pharmaceutically acceptable formulation of human prolactin contains (a) substantially pure human prolactin, and (b) divalent metal ion.
  • the formulation of a stabilized pharmaceutically acceptable formulation of human placental lactogen contains (a) substantially pure human placental lactogen, and (b) divalent metal ion.
  • the formulation of prolactin or human placental lactogin may contain a divalent metal ion is selected from zinc, cobalt or copper, preferably zinc.
  • Titration of hGH with Co 2+ produces a Co 2+ visible abso ⁇ tion spectrum with a maximum at 525 nm and molar abso ⁇ tivity of 260 cm- 1 M “1 (Fig. 4B).
  • the wavelength maximum and molar abso ⁇ tivity is characteristic of a Co 2+ in a tetrahedral environment coordinated by a mixture of nitrogen and oxygen containing ligands (I. Bertini and C. Luchinat, Adv. Inorg. Biochem. ⁇ >, 71-111 (1984); M. T. Martin, B. Holmquist, J. F. Riordan, J. Inorg. Chem. 36, 27-37 (1989)).
  • Zinc typically coordinates four ligands in proteins (B. L. Vallee and D. S. Auld,, Proc. Natl. Acad. Sci. U. S. A. 87, 220-224 (1990)). Sedimentation equilibrium and zinc binding studies data indicate that Asp171 is not the fourth zinc ligand even though it is close by in the folded model of hGH. There are no other nearby side chains that could coordinate zinc. However, it is possible that the fourth zinc ligand is a water molecule. Alternatively, it is possible that His18, His21 , or Glu174 from each hGH molecule bridge the two Zn 2+ atoms in the dimer (for example see Fig, 6B).
  • each monomer of hGH in the dimer is related by a two-fold symmetry axis with the zinc atoms sandwiched at the interface.
  • the highly cooperative nature of Zn 2+ binding suggests that the two sites are interdependent.
  • Such a ligand bridged bi-metal site is not a natural precedent.
  • Cu-Zn superoxide dismutase contains a His residue that coordinates to both the copper and zinc (J. A. Tainer, E. D. Getzoff, J. S. Richardson, D. C.
  • the Zn + -hGH dimer produced here in vitro is the major storage form of hGH in vivo.
  • a stable formulation of hGH and zinc therefore results in a therapeutic complex analogous to what the pituitary releases.
  • Normal human pituitary glands (-1 ml volume) contain 2 to 4 mgs of hGH (W. F. Daughaday, in Textbook of Endocrinology, seventh edition, Chapter 18, ed. J. D. Wilson and D. W. Foster, (W. B. Saunders Co., Philadelphia, 1985), p. 577).
  • the zinc content in rat pituitary has been estimated to be 75 to 100 ng/mg dry weight (O.
  • This concentration is roughly 50-fold above that necessary to saturate the Zn 2+ site in hGH (-1 ⁇ M).
  • the total concentration of hGH in vesicles is about 2 to 4 mM. This concentration is about 1000-fold greater than the dissociation constant for the Zn 2+ -hGH dimer (2.6 ⁇ M).
  • zinc and hGH are localized in somatotropic vesicles in roughly equimolar amounts and with high enough affinity that virtually all of the hGH would exist as a (Zn 2+ -hGH) 2 complex.
  • the Zn 2+ -hGH dimer serves at least two important functions. Firstly, the Zn 2+ -hGH dimer is significantly more stable to denaturation than monomeric hGH as shown by its much greater resistance to unfolding in Gu-HCI (Fig. 7). In the presence of 25 ⁇ M ZnCI 2 , the concentration of Gu-HCI at which 50% of the hGH is denatured increases from 4.1 M to 4.8 M. hGH is released in pulses from the pituitary every 4 to 12 h depending upon the age, sex and health of the individual (A. L. Taylor, J. L. Finster, D. H. Mintz, J. Clin. Invest. 48, 2349 (1969); R. G. Thompson, A.
  • hGH is structurally similar to the family of helical cytokines including interieukin-2, colony stimulating factor, interferon- ⁇ and others (for review see D. A. D. Parry, E. Minasian, S. J. Leach, J. Mol. Recog ⁇ , 107-110 (1988)). It is believed that some of these proteins may be stored as complexes with zinc prior to their release from cells.
  • the formulations of the present invention may be prepared with human prolactin or human placental lactogen in place of the hGH. Both of these human helical cytokines contain a divalent metal binding site, preferentially zinc. Those cytokines which do not contain such a zinc complexing site can be modified to contain an hGH- like zinc binding site using site specific mutagenisis procedures well known in the art, thereby allowing the formation of stable zinc:helical cytokine complexes suitable for stable formulation.
  • a zinc binding site may be constructed by placing two histidines and one Glutamate between two polypeptide helicies adjacent in the same structural arrangement as in hGH.
  • a zinc binding site for example a site analogous to hGH amino acids His18, His21 and Asp174, can be introduced into the three dimensional structure of other helical cytokines.
  • helical cytokines are the interieukins, the interferons, polypeptide hormones, such as growth hormones, prolactin, and placental lactogen.
  • the actual location of such zinc binding amino acids need not be in the same linear position as in hGH. Rather, amino acids on the helical cytokine surface would need to specify analogous functional positions for binding the divalent metal ion between two helical regions of the cytokine polypeptides.
  • the helical cytokine amino acid sequence may be modified to create a zinc binding site so that two of the helical cytokines complex with two zinc (or other divalent metal ions), to form a stable dimer.
  • hGH was mixed with dialysis buffer containing 20 mM Tris (pH 7.5), 140 mM NaCI and 10 mM MgCI 2 .
  • the MgCI 2 was added to reduce non-specific binding of Zn 2+ to the dialysis cell membrane and hGH.
  • the MgCI 2 neither promoted nor inhibited dimerization of hGH.
  • Aliquots (100 ⁇ l) of the solution containing hGH (3.3 or 4.8 ⁇ M) was added to one side of the dialysis cell.
  • Serial dilutions (in triplicate) of cold ZnCI 2 containing 0.3 ⁇ M 65 ZnCI 2 (8 dilution steps ranging from 0 to 40 ⁇ M final) were added to the other side of the dialysis cell.
  • Figure 2 shows the gel filtration chromatography of hGH in the presence (A) or absence (B) of ZnCI 2 .
  • A ⁇ M hGH
  • 20 mM Tris (pH 7.5) 100 mM NaCI
  • 50 ⁇ M ZnCI 2 or 1 mM EDTA was applied to a Superose 12 column and eluted at 0.35 ml/min with the same buffer without hGH.
  • Size standards bovine ⁇ -globulin, ovalbumin and myogiobin
  • Figure 3 shows the sedimentation equilibrium analysis of hGH in the presence (• ) or absence ( _ J ) of 20 ⁇ M ZnCI 2 .
  • Samples of hGH (9.1 ⁇ M, 6.8 ⁇ M or 4.5 ⁇ M) in 10 mM Tris (pH 8.0), 0.1 M NaCI with or without 20 ⁇ M ZnCI 2 were loaded into a 6-channel Yphantis cell (26), and centrifuged in a Beckman Model E centrifuge at 20,000 ⁇ m for 48 h at 20'C.
  • Panel A shows the concentration of hGH (mg/ml) versus radial distance.
  • Tandem cells contained on one side hGH (45 ⁇ M final) and buffer (25 mM Tris (pH 7.5), 140 mM NaCI) plus either 50 ⁇ M ZnCi 2 or 50 ⁇ M CoCI 2 .
  • the other side contained just buffer plus either 50 ⁇ M ZnCI 2 or 50 ⁇ M CoCI 2 .
  • Titration of hGH with Co 2+ produces a Co 2+ visible abso ⁇ tion spectrum with a maximum at 525 nm and molar abso ⁇ tivity of 260 cm *"1 M-1 (Fig. 4B).
  • the wavelength maximum and molar abso ⁇ tivity is characteristic of a Co + in a tetrahedral environment coordinated by a mixture of nitrogen and oxygen containing ligands.
  • the circular dichroic spectrum of the Co 2+ -hGH dimeric complex is virtually flat indicating that the sum of all bound cobalt is in a symmetrical environment.
  • each cobalt site in the dimer is highly symmetrical, or more likely that the two cobalt sites are asymmetric but pseudo-mirror images of each other so that the dichroic effect of one is equal and of opposite sign thus canceling each other.
  • Shown in Figure 4B is the visible difference spectra for binding of increasing concentrations of Co 2+ to hGH. The experiment was identical to that in panel A except that the [hGH] was 23 ⁇ M and [Co 2+ ] ranged from 5 to 1250 ⁇ M.
  • the molar abso ⁇ tivity (260 cm- 1 M- 1 ) was calculated from the maximal change in absorbance at 525 nm (6 x 10 "3 cm- 1 ) divided by the concentration of hGH in the cuvett (23 ⁇ M).
  • FIG. 5 shows dimerization of hGH (wild type) and hGH mutants by addition of zinc.
  • Gel filtration analysis was performed as described for Figure 2 except that hGH and ZnCl 2 were applied at a concentration of 10 ⁇ M.
  • Panels A-D show elution profiles for hGH and the designated hGH mutants. Mutants are indicated by the single-letter amino acid for the wild-type residue followed by its position in mature hGH and then the mutant residue. For example, H18A designates a mutant where His18 is changed to Ala. Mutants of hGH were expressed in Escherichia coli and purified as previously described.
  • Zinc typically coordinates four ligands in proteins (B. L. Vallee and D. S. Auld, D. S. Proc. Natl. Acad. Sci. U. S. A. 87, 220-224 (1990)). Sedimentation equilibrium and zinc binding studies data indicate that Asp171 is not the fourth zinc ligand even though it is close by in the folded model of hGH. There are no other nea ⁇ y side chains that could coordinate zinc. However, it is possible that the fourth zinc ligand is a water molecule. Alternatively, it is possible that His18, His21. or Glu174 from each hGH molecule bridge the two Zn 2+ atoms in the dimer (for example see Fig.6B).
  • each monomer of hGH in the dimer is related by a two-fold symmetry axis with the zinc atoms sandwiched at the interface.
  • Panel B shows one of three possible bridged ligand models that can account for the involvement of His18, His21 , and Glu174 in coordinating 2 zinc atoms per hGH dimer.
  • a salient feature of this model is the two-fold symmetry axis through the center of the "twinned" metal site.
  • Glu174 from each hormone is shown coordinating both Zn 2+ atoms simultaneously.
  • Figure 7 shows the stability of hGH to denaturation by Guanidine-HCl in the presence (• ) or absence ), of 25 ⁇ M ZnCI 2 .
  • a solution containing 30 ⁇ g hGH, 0.01 M Tris buffer pH 8.0, with or without 25 ⁇ M ZnCI 2 and the indicated GuHCI concentration was equilibrated for 2 min at 25 ' C prior to measuring the ⁇ 222 in a 1 cm CD cell.
  • the Zn 2 ⁇ was added before the GuHCI.
  • the fraction folded was calculated from the fractional change in ⁇ 222 in the absence of Gu-HCI (folded) relative to that in 6.5 M Gu-HCI (unfolded).
  • hGH FORMULATION A stable formulation of hGH is prepared by first combining a zinc chloride solution with an hGH sample, dialyzing the hGH:zinc dimer sample against formulation buffer (5mM phosphate, pH 7.4, 50 ⁇ M zinc cloride), diluting and dispensing into vials. A solution of zinc chloride (1mM) is slowly added directly with stirring to the hGH sample (20mg/ml) to achieve a 1 :1 molar ratio, while maintaining the pH at 7.0-7.4 using phosphate buffer (1.0 M, pH 7.4).
  • formulation buffer 5mM phosphate, pH 7.4, 50 ⁇ M zinc cloride
  • the hGH:zinc dimer is dialyzed (8 hr, 4° C, hGH:buffer (v/v) 1:1000) against an aqueous formulation buffer containing zinc chloride (50 ⁇ M) and a sodium phosphate buffer (5 mM) equilibrated at pH 7.4 .
  • the hGH:zinc dimer mixture is then diluted by formulation buffer to a concentration of 5 mg/ml hGH.
  • the solution is sterile filtered, and stored at 4°C until needed, then 1.0 ml is dispensed into sterile vials.

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Abstract

Cette invention concerne des complexes stables d'hormone de croissance humaine (hGH) et d'ion métal bivalent permettant d'obtenir des formulations stabilisées de hGH par formation de dimères ion métal-hGH. Les dimères stables sont caractérisés et les formulations thérapeutiques de zinc et hGH sont également décrites.
PCT/US1992/002365 1991-03-28 1992-03-25 Formulations stables a base d'ion metal et d'hormone de croissance WO1992017200A2 (fr)

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Cited By (18)

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WO1993019776A1 (fr) * 1992-04-03 1993-10-14 Kabi Pharmacia Ab Formulation de proteines contenant une hormone de croissance
WO1997003692A1 (fr) * 1995-07-14 1997-02-06 Novo Nordisk A/S Formulation pharmaceutique stabilisee comprenant une hormone de croissance pre-traitee avec des ions zinc et facultativement lysine ou calcium
US5763394A (en) * 1988-04-15 1998-06-09 Genentech, Inc. Human growth hormone aqueous formulation
WO1998027980A3 (fr) * 1996-12-20 1998-08-13 Takeda Chemical Industries Ltd Procede de production d'une preparation a liberation prolongee
US5981485A (en) * 1997-07-14 1999-11-09 Genentech, Inc. Human growth hormone aqueous formulation
US6022858A (en) * 1991-12-20 2000-02-08 Novo Nordisk A/S Pharmaceutical formulation of human-growth hormone pretreated with zinc salt
US6087324A (en) * 1993-06-24 2000-07-11 Takeda Chemical Industries, Ltd. Sustained-release preparation
EP0905143A3 (fr) * 1997-09-26 2000-09-06 Takeda Chemical Industries, Ltd. Complexe d'hormone de croissance humaine et de zinc et utilisation
US6280959B1 (en) * 1997-10-31 2001-08-28 Smithkline Beecham Corporation Metal complexes
WO2001078683A3 (fr) * 2000-04-19 2002-05-30 Genentech Inc Formulations a liberation lente
EP1032388A4 (fr) * 1997-10-31 2002-10-02 Smithkline Beecham Corp Nouveaux complexes metalliques
WO2003000282A1 (fr) * 2001-06-21 2003-01-03 Genentech, Inc. Formulation a liberation lente
US6569406B2 (en) 2000-08-07 2003-05-27 Nektar Therapeutics Inhaleable spray dried 4-helix bundle protein powders having minimized aggregation
US6605594B2 (en) * 1996-12-31 2003-08-12 Monsanto Technology, Llc Aqueous glycerol formulations of somatotropin
US7824700B2 (en) 2001-02-23 2010-11-02 Genentech, Inc. Erodible polymers for injection
US7855066B1 (en) 1999-06-28 2010-12-21 Genentech, Inc. Methods for making Apo-2 ligand using divalent metal ions
EP2348043A1 (fr) 2001-10-02 2011-07-27 Genentech, Inc. Variantes du ligand APO-2 et leurs utilisations
EP2500032A1 (fr) 2002-06-24 2012-09-19 Genentech, Inc. Variantes du ligand/Trail APO-2 et leurs utilisations

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GB885798A (en) * 1958-12-06 1961-12-28 Organon Labor Ltd Preparations of the growth hormone
ATE62598T1 (de) * 1984-10-04 1991-05-15 Monsanto Co Verzoegerte freisetzung biologisch aktiver somatotropine.
IL79681A (en) * 1985-08-12 1991-06-10 Int Minerals & Chem Corp Transition metal complexes of growth hormones and their prolonged release compositions
US5096885A (en) * 1988-04-15 1992-03-17 Genentech, Inc. Human growth hormone formulation

Cited By (33)

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US6448225B2 (en) 1988-04-15 2002-09-10 Genentech, Inc. Human growth hormone aqueous formulation
US5763394A (en) * 1988-04-15 1998-06-09 Genentech, Inc. Human growth hormone aqueous formulation
US6022858A (en) * 1991-12-20 2000-02-08 Novo Nordisk A/S Pharmaceutical formulation of human-growth hormone pretreated with zinc salt
US5567677A (en) * 1992-04-03 1996-10-22 Pharmacia Ab Protein formulation comprising growth hormone
WO1993019776A1 (fr) * 1992-04-03 1993-10-14 Kabi Pharmacia Ab Formulation de proteines contenant une hormone de croissance
US6087324A (en) * 1993-06-24 2000-07-11 Takeda Chemical Industries, Ltd. Sustained-release preparation
WO1997003692A1 (fr) * 1995-07-14 1997-02-06 Novo Nordisk A/S Formulation pharmaceutique stabilisee comprenant une hormone de croissance pre-traitee avec des ions zinc et facultativement lysine ou calcium
AU715997B2 (en) * 1995-07-14 2000-02-17 Novo Nordisk A/S A stabilized pharmaceutical formulation comprising a growth hormone pre-treated with zinc and optionally lysine or calcium ions
US6197350B1 (en) 1996-12-20 2001-03-06 Takeda Chemical Industries, Ltd. Method of producing a sustained-release preparation
US6399103B1 (en) 1996-12-20 2002-06-04 Takeda Chemical Industries, Inc. Method of producing a sustained-release preparation
WO1998027980A3 (fr) * 1996-12-20 1998-08-13 Takeda Chemical Industries Ltd Procede de production d'une preparation a liberation prolongee
US6605594B2 (en) * 1996-12-31 2003-08-12 Monsanto Technology, Llc Aqueous glycerol formulations of somatotropin
US5981485A (en) * 1997-07-14 1999-11-09 Genentech, Inc. Human growth hormone aqueous formulation
US6191107B1 (en) 1997-09-26 2001-02-20 Takeda Chemical Industries, Ltd. Complex of human growth hormone and zinc
EP0905143A3 (fr) * 1997-09-26 2000-09-06 Takeda Chemical Industries, Ltd. Complexe d'hormone de croissance humaine et de zinc et utilisation
US6429296B2 (en) 1997-09-26 2002-08-06 Takeda Chemical Industries, Ltd. Complex of human growth hormone and zinc and use
US6280959B1 (en) * 1997-10-31 2001-08-28 Smithkline Beecham Corporation Metal complexes
EP1032388A4 (fr) * 1997-10-31 2002-10-02 Smithkline Beecham Corp Nouveaux complexes metalliques
EP2339003A2 (fr) 1999-06-28 2011-06-29 Genentech, Inc. Polypeptides variants substitutionnels du ligand APO-2
EP2311956A1 (fr) 1999-06-28 2011-04-20 Genentech, Inc. Methodes de production de ligand à APO-2 utilisant des ions métalliques divalents
US7855066B1 (en) 1999-06-28 2010-12-21 Genentech, Inc. Methods for making Apo-2 ligand using divalent metal ions
WO2001078683A3 (fr) * 2000-04-19 2002-05-30 Genentech Inc Formulations a liberation lente
US6992065B2 (en) 2000-04-19 2006-01-31 Genentech, Inc. Sustained release formulations
US6838075B2 (en) 2000-08-07 2005-01-04 Nektar Therapeutics Inhaleable spray dried 4-helix bundle protein powders having minimized aggregation
US6569406B2 (en) 2000-08-07 2003-05-27 Nektar Therapeutics Inhaleable spray dried 4-helix bundle protein powders having minimized aggregation
US8501216B2 (en) 2001-02-23 2013-08-06 Genentech, Inc. Bioerodible polymers for injection
US7824700B2 (en) 2001-02-23 2010-11-02 Genentech, Inc. Erodible polymers for injection
WO2003000282A1 (fr) * 2001-06-21 2003-01-03 Genentech, Inc. Formulation a liberation lente
US7318931B2 (en) 2001-06-21 2008-01-15 Genentech, Inc. Sustained release formulation
US8067020B2 (en) 2001-06-21 2011-11-29 Genetech, Inc. Sustained release formulation
AU2002320122B2 (en) * 2001-06-21 2007-07-26 Genentech, Inc. Sustained release formulation
EP2348043A1 (fr) 2001-10-02 2011-07-27 Genentech, Inc. Variantes du ligand APO-2 et leurs utilisations
EP2500032A1 (fr) 2002-06-24 2012-09-19 Genentech, Inc. Variantes du ligand/Trail APO-2 et leurs utilisations

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