Classifications

• • 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
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • 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
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • 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
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers

Definitions

• the " present invention relates to novel solutions of somatotropins which remain clear for an extended period of time and which remain clear when subjected to mechanical agitation, and to kits for making the solutions.
• Somatotropins also known as growth hormones, are polypeptide hormones secreted by the pituitary glands of many animal species. These hormones are valuable for a number of therapeutic uses, and compositions comprising somatotropin can be administered in the treatment of pituitary deficiency in humans and gastrointestinal bleeding or to promote the healing of bone fractures and accelerate the healing of contusions and other wounds. Somatotropins also are useful in promoting meat and milk production in animals when administered through various drug-releasing devices or by injection. (See E.J. Tur an, "Some Effects of Pituitary Anterior Growth Factor" Thesis: Purdue
• aggregation of adsorbed somatotropin molecules can take place to give soluble or insoluble polymeric forms. This aggregation will manifest itself, for example, as turbidity of the solution or as biological inactivation of the somatotropin protein on stirring or shaking of the aqueous solutions (See A.F. Henson et al., J. Colloid Interface Sci. 32: 162 (1970)).
• somatotropin solutions which remain clear for extended periods and which do not become cloudy or precipitate when subjected to mechanical agitation.
• the somatotropin solutions contain a lyophilized somatotropin composition containing about 1 part somatotropin per 2 to 8 parts arginine HCl on a weight basis, the pH of the composition prior to lyophilization within the range of about 7.2 to about 8.5, which is dissolved in a diluent. If the pH of the somatotropin composition prior to lyophilization was less than 7.8, the diluent comprises EDTA, a nonionic surfactant and a buffer.
• the diluent comprises EDTA and a nonionic surfactant and optionally, further can comprise a suitable buffer or nonbuffering agent if desired. If a non-buffering agent is added, desirably a buffer is not also added to the diluent, although both agents can be used.
• kits for making the solutions include a vial containing the lyophilized somatotropin composition containing about 1 part somatotropin per 2 to 8 parts arginine HCl on a weight basis.
• the kits also include a vial containing a biocompatible diluent comprising EDTA and a nonionic surfactant.
• the diluent also can comprise a buffer or non-buffering agent. If the pH of the somatotropin composition prior to lyophilization was less than 7.8, the vial containing the diluent further comprises a buffer.
• the vial optionally can contain a buffer or nonbuffering agent.
• the diluent can be added to the lyophilized somatotropin composition and the resultant mixture then shaken to dissolve the somatotropin.
• This invention is directed to somatotropin solutions which remain clear for extended periods and which do not become cloudy or precipitate when subjected to mechanical agitation such as shaking or vortex-mixing.
• a somatotropin solution which remains "clear” is a solution in which the somatotropin does not precipitate after it is dissolved in the diluent.
• These somatotropin solutions will remain clear after several days of storage, typically at least about 5 days.
• the somatotropin solutions maintain clarity for at least about 5 days when stored, typically, for example, at about 25°C. At slightly higher storage temperatures, the somatotropin solutions maintain clarity, but possibly for a shorter period of time.
• a solution which remains clear will have an apparent absorbance measurement at 360 n (O.D.
• the somatotropin solutions contain a lyophilized somatotropin composition, the pH of which prior to lyophilization was at least about 7.2, generally within the range of from about 7.2 to about 8.5. If the pH of the somatotropin composition prior to lyophilization was " less than 7.8, the diluent comprises EDTA, a nonionic surfactant and a suitable buffer to stabilize the final somatotropin solution. If the pH of the somatotropin composition prior to lyophilization was 7.8 or higher, the diluent comprises EDTA and an nonionic surfactant and, optionally, a buffer or nonbuffering agent.
• a non-buffering agent is added to the diluent, desirably a buffer is not also added to the diluent, although both agents can be used. Neither the buffer nor nonbuffering agent need be used if the pH of the . somatotropin composition prior to lyophilization was at least 7.8, but either can be provided to adjust or maintain the isotonicity of the resultant somatotropin solution such that it is less hypertonic. This is desirable if the somatotropin solution will be injected into animals.
• the choice of a suitable additive can be made based on a number of factors, including, for example, cost, presence on the FDA GRAS list, autoclavability, chemical stability, lack of interaction with the somatotropin and bioco patibility.
• the lyophilized somatotropin composition is prepared by dissolving arginine HCl with somatotropin, adjusting the pH of the solution to at least 7.2, preferably from 7.2 to 8.5, and removing any undissolved material by filtration or centrifugation.
• the somatotropin and arginine HCl solution then is lyophilized using standard procedures known in the art.
• the solution to be lyophilized generally contains about 1 part somatotropin per 2 to 8 parts arginine HCl on a weight basis and preferably comprises about 1 part somatotropin per 3 parts arginine HCl.
• the solution preferably contains 10 to 150 mg/ml somatotropin and 30 to 450 mg/ml arginine HCl and has a final pH of at least 7.2.
• the solution to be lyophilized contains 30 mg/ml somatotropin and 90 mg/ml arginine HCl and has a final pH of from 7.8 to 8.5.
• the somatotropin which may be employed in the lyophilized somatotropin composition of this invention can be any somatotropin, including natural or recombinant bovine, porcine, human, avian, ovine, or equine somatotropin.
• the somatotropin employed is porcine somatotropin.
• somatotropin is intended to include the full length natural or recombinant somatotropin as well as derivatives thereof that have growth-promoting capabilities.
• Derivatives include biologically active fragments or analogs >of the polypeptide hormone, such as delta 7 porcine somatotropin, which has an amino acid sequence corresponding to that of porcine somatotropin, less the first seven amino acids of the mature, full length hormone (described in European Patent Application Publication No. 0 104 920 to Biogen N.V. ) .
• biologically active as used herein means a polypeptide that, following its administration to a living being, has a demonstrable effect on a biological process of that living being.
• the somatotropins employed in the compositions of this invention can be metal-associated somatotropins.
• Metal-associated somatotropin is produced by the addition of salts of transition metals to an aqueous solution containing the somatotropin.
• the salts of the transition metals form insoluble complexes with the somatotropin, thus precipitating the somatotropin out of the solution.
• the metal-associated somatotropins comprise the somatotropin molecules and metal ions such as Zn +2 , Cu +2 , Co +2 , Mn +2 , Fe +2 or Fe +3 .
• metal-associated somatotropins contain ligand bonds between the metal ion and the nitrogen atoms of some of the amino acid residues in the somatotropin molecule.
• the metal-associated somatotropins are used as starting materials for making the lyophilized somatotropin compositions.
• the metals likely are not associated with the somatotropin once the metal-associated somatotropin is lyophilized, although they are present in the lyophilized somatotropin composition.
• the presence of the transition metal in the product has been shown to have no significant adverse effect on the bioactivity of the somatotropin when the product is administered to a living being.
• the somatotropin solutions of this invention are made by dissolving the lyophilized somatotropin composition with a diluent such that the pH of the final solution is at least about 7.2, typically from about 7.2 to about 8.5, and preferably from about 7.2 to about 8.2. If the pH of the solution is greater than about 8.5, " degradation of the protein can occur. Typically, about 0.5 to about 40 mg lyophilized somatotropin composition are provided per ml of diluent.
• the final concentration of the somatotropin solution is about 10 to 30 mg lyophilized somatotropin composition per ml of diluent and most preferably about 20 mg lyophilized somatotropin composition per ml of diluent.
• the weight of the lyophilized somatotropin composition will be the total of the weight of the arginine HCl and the weight of the somatotropin contained in the lyophilized somatotropin composition.
• the diluent comprises EDTA, a nonionic surfactant and a buffer.
• the diluent comprises EDTA and a nonionic surfactant and optionally also comprises a buffer or nonbuffering agent.
• the pH of the final somatotropin solution will be the same as the pH of the diluent.
• the pH of the lyophilized somatotropin composition dissolved in the diluent will determine the pH of the final somatotropin solution.
• the concentration of EDTA provided in the diluent is at least 1.5 mM, and is preferably from about 1.5 mM to about 10 mM.
• the concentration of EDTA used in the diluent desirably is at least 1.0 mM, and is preferably from about 1.0 mM to about 10 mM.
• Surfactants which are suitable for use in the diluent include polyoxyethylene-23 lauryl ether (Brij 35), Tween 80, polyoxyethylene-20 cetyl ether (Brij 58), and other polyoxyethylene nonionic surfactants having similar hydrophilic/hydrophobic balance (HLB).
• Such nonionic surfactants have been noted in the prior art as stabilizing and preserving activity in purified enzymes. (See T. Kitani et al., Eur. J. Biochem. 119: 177-181 (1981); M. Pritchard et al., Biochem. Biophvs. Res. Commun. 100: 1597-1603 (1981); Seely et al.. Biochemistry, Vol. 21, No.
• the surfactant is present in a concentration ranging from about 0.08% to 2.0%. If the surfactant utilized is Brij 35, the concentration of the Brij 35 is at least 0.08%, and preferably is from about 0.1% to about 0.2%. If the surfactant is Tween 80 or Brij 58, the concentration preferably is from about 0.1% to about 1.0%.
• a buffer is provided in the diluent to increase the stability of the somatotropin solution.
• the buffer can be Tris HCl, phosphate, or some other neutral, host- compatible pH buffer.
• the buffer is present in a concentration ranging from about 0.2 M to about
• Tris HCl generally has a concentration of at least 0.2 M, and is preferably from about 0.2 M to about 0.3 M.
• a buffer as described above or a non-buffering agent can be added to the diluent, if desired, to adjust or maintain the isotonicity of the resultant somatotropin solution.
• the non-buffering agent also is host-compatible. Such agents include sucrose, trehalose, or NaCl.
• the non- buffering agent if present, generally is provided at a concentration from about 0.05 M to about 0.5 M. When sucrose or trehalose is utilized as the non-buffering agent, preferred concentrations are within the range of from about 0.1 to about 0.3 M. When NaCl is utilized as the non-buffering agent a concentration of from 0.05 M to 0.15 M generally is preferred.
• the somatotropin compositions of this invention can be made using kits containing a vial of the lyophilized somatotropin composition as discussed above and a vial containing the diluent discussed above.
• the diluent can be added to the lyophilized somatotropin composition and the resultant product shaken to dissolve the somatotropin.
• the somatotropin solutions of this invention can be used for further processing or can be administered directly to animals.
• the solution generally can be stored for at least about 5 days at ambient temperatures, typically about 25°C, without becoming cloudy.
• the solutions also can be stored at slightly higher temperatures, although the solutions may maintain their clarity a shorter period of time.
• These solutions also can be subjected to mechanical agitation, such as shaking or mixing, without precipitation of the somatotropin from the solution.
• the somatotropin in these solutions maintains its biological activity and can be administered to animals in accordance with conventional techniques to promote growth.
• solution clarity was determined by visual inspection and/or by measuring the apparent absorbance at 360 nm (O.D. 360); a wavelength where somatotropins have no intrinsic absorbance.
• O.D. 360 apparent absorbance at 360 nm
• the somatotropin solution was placed in the sample cuvette and the O.D. 360 determined using a Shimadzu UVU160 spectrophotometer. A diluent solution which did not contain somatotropin was used in the reference cuvette. Quartz cuvettes were used in all studies.
• "clear" solutions are those which have an O.D. 360 of less than 0.10.
• Example 1 Thirty-three grams of Zn-associated porcine somatotropin (pST), made in accordance with the procedures disclosed in published European Patent Application No. 83300803.9, and 90 grams of arginine HCl were dissolved in one liter of sterile water. The pH was adjusted to 7.8 by the addition of aqueous HCl or NaOH and insolubles were removed by filtration through a polyvinylidene difluoride membrane. The clarified solution then was lyophilized.
• pST Zn-associated porcine somatotropin
• the resulting formulation (termed the "lyophilized porcine somatotropin (pST) composition") contained approximately 30 g pST per 90 g arginine HCl, due to the loss of about 10% of the original 33 g of pST as insoluble during the filtration step.
• Example 2 Solid arginine HCl (1.92 g) was added to a non-Zn complexed pST solution containing 0.639 g pST in 21.6 ml of a pH 9.8, 0.46 mM sodium carbonate buffer. The arginine was dissolved by stirring and the pH of the solution was adjusted to 7.8 by the dropwise addition of 1 M NaOH. The resulting solution contained approximately 30 mg/ml pST and 90 mg/1 arginine. 5 Aliquots (7.2 ml) of this solution were pipetted into three 50-ml vials, frozen at -80°C, and lyophilized.
• This lyophilized pST composition readily dissolved in 200 mM Tris HCl, 2 mM EDTA, 0.15% Brij 35 (pH 7.8) at 20 mg/ml (dissolution time ⁇ 3 minutes). The 0 resulting solution was clear by visual inspection.
• Zinc-associated pST Zn-pST
• non-metal complexed pST non-Zn pST
• lyophilized pST composition 0 made as described in Example 1
• Zinc-associated pST Zn-pST
• non-metal complexed pST non-Zn pST
• lyophilized pST composition 0 made as described in Example 1
• solution clarity was determined by visual observation arid by 5 measuring turbidity at 360nm.
• Table 1 shows that - only the lyophilized pST composition gave a clear solution after dissolution under these conditions. The results are shown in Table 1.
• Table 1 Table 1
• the somatotropin solutions containing a Brij 35 concentration of at least 0.08% resulted in a clear solution after vortexing the lyophilized pST composition in the diluent under these conditions.
• the somatotropin solutions containing a Brij 35 concentration of at least 0.08% resulted in a clear solution after vortexing the lyophilized pST composition in the diluent under these conditions.
• Brij 35 concentration of less than 0.08% did not remain clear following mechanical agitation by vortex mixing.
• Example 5 Forty mg aliquots of lyophilized pST composition were weighed into 2 dram glass vials and dissolved in the diluent containing 200 mM Tris HCl, 0.10% Brij 35 at the following EDTA concentrations and final pH:
• EDTA concentration of at least 1.5 mM resulted in a clear solution for at least 10 days after dissolution of the lyophilized pST composition in the diluent under these conditions.
• somatotropin solutions with a pH of 7.6 or greater, containing an EDTA concentration of at least 1.0 mM resulted in a clear solution for at least 5 days after dissolution of the lyophilized pST in the diluent under these conditions.
• Example 6 Forty mg aliquots of lyophilized pST composition were weighed into 16 two-dram glass vials. Two ml of the following diluents were added: 1. 300 mM Tris HCl, 0.15% Brij 35, 1.5 mM EDTA, pH 7.4
• a Tris HCl concentration of at least 200 mM must be employed in a diluent containing 0.15% Brij 35 and 1.5 mM EDTA at a pH of 7.4 in order for the somatotropin solution to remain clear for six days.
• Solutions 6-8 in Table 4 illustrated that an EDTA concentration of at least 1.5 mM was necessary, in addition to a Brij 35 concentration of 0.15% and a Tris HCl concentration of 200 mM at a pH of 7.2, in order for the somatotropin solution to remain clear for six days.
• the somatotropin solution remained clear after six days when the diluent comprised 0.15% Brij 35, 2 mM EDTA and either 1%, 5%, or 10% sucrose or 0.2 M, 0.1 M or 0.0 M NaCl and the final pH of the samples were approximately 7.8-7.9.

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• 1993
  • 1993-04-05 AU AU42783/93A patent/AU4278393A/en not_active Abandoned
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