WO1990008551A1 - Stabilisation de solutions de proteines hydrophobes a base aqueuse et substrat porteur de liberation continue - Google Patents

Stabilisation de solutions de proteines hydrophobes a base aqueuse et substrat porteur de liberation continue Download PDF

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Publication number
WO1990008551A1
WO1990008551A1 PCT/US1990/000256 US9000256W WO9008551A1 WO 1990008551 A1 WO1990008551 A1 WO 1990008551A1 US 9000256 W US9000256 W US 9000256W WO 9008551 A1 WO9008551 A1 WO 9008551A1
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WO
WIPO (PCT)
Prior art keywords
sustained release
protein
release vehicle
phase
solution
Prior art date
Application number
PCT/US1990/000256
Other languages
English (en)
Inventor
Wen-Ghih Tsang
Andrew S. Magee
Ann W. Shyr
Original Assignee
Damon Biotech, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Damon Biotech, Inc. filed Critical Damon Biotech, Inc.
Publication of WO1990008551A1 publication Critical patent/WO1990008551A1/fr

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Classifications

    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
    • 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
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin

Definitions

  • the present invention relates to the formation and stabilization of agueous-based solutions of hydrophobic proteins. Sustained release vehicles made using these stabilized solutions are also disclosed.
  • the first class of sustained release vehicles have potential problems with differential breakdown rates, thereby providing uneven release while the second class of vehicles are normally bio-incompatible objects which must be removed after exhausted, often surgically.
  • sustained release vehicles are the sodium alginate-based microcapsules described in United States Patent No. 4,690,682, filed September 1, 1987, on application of Dr. Franklin Lim, and United States Patent Application Serial No. 121,214, filed November 16, 1987, on application of Wen-Ghih Tsang and Andrew Magee, both assigned to " the assignee of the present application.
  • These vehicles use tortuous path-like pores of sodium alginate microcapsules as a "filtering" device whereby an osmotic gradient is set up between a high internal concentration of the material to be released and the large surrounding aqueous volume.
  • the proteins or other materials which have been encapsulated in this type of vehicle have been limited to hydrophilic materials which are easily dissolved in the aqueous solution used to make the capsules.
  • alginate-protein solutions One odd phenomenon of alginate-protein solutions is the ability, over time, to form stable two-phase solutions. Although other polymers form separate phases, these phases are sometimes unstable and/or denature the polymers. For example, Tolstoguzov, Antonov and their co-workers have shown that the casein-alginate-water and trypsin-alginate-water systems are useful for making protein spinneret fibers because of their ability to make two-phase solutions. However, these two-phase systems were investigated as alternatives for the denatured protein normally used to form these protein matrix fibers and denaturation was not considered a problem. Despite the ability to form stable two-phase systems, neither the casein nor trypsin system produced notably better results. The two-phase system formed is interesting, however, since both casein and trypsin are hydrophilic, easily soluble proteins.
  • Hydrophobic proteins e.g., proteins which are substantially insoluble or have low solubilities in aqueous solutions
  • sustained release vehicles which dispense the protein into primarily aqueous solutions.
  • an object of the invention is to provide a method stabilizing aqueous solutions of relatively high concentrations of hydrophobic proteins.
  • Another object of the invention is to provide a sustained release vehicle which is biocompatible and allows controlled release of proteins.
  • a further object of the invention is to provide sustained release systems usable for a broad variety of proteins, particularly hydrophobic proteins, without mechanical or electro-mechanical pumping systems.
  • the present invention features methods of producing stable, high concentration aqueous solutions of hydrophobic proteins and sustained release vehicles made from those solutions.
  • the invention is based, in part, on the discovery that aqueous solutions of certain polymers, e.g., alginic acid derivatives and other polysaccharides, will. when mixed with proteins, separate and form stable two-phase systems.
  • This two-phase system can be turned into microcapsules which permits higher concentrations of proteins, e.g., hydrophobic proteins, to be encapsulated than is normally possible. Controlling the rate of release over time is also achievable using this system.
  • the method of producing the stable, high concentration aqueous solutions of the hydrophobic proteins commences with the formation of a first aqueous solution of the polymer which has the ability to form a two-phase systems when mixed with the protein, preferably an alginic acid derivative, e.g, sodium alginate.
  • the hydrophobic protein is mixed or dissolved in the first polymeric solution, forming a polymer hydrophobic protein solution.
  • An alginic acid derivative-hydrophobic protein solution is a preferred first solution. This solution is allowed time to stabilize, preferably at slightly above its freezing point with nutation, until two distinct phases form; one phase having a high concentration of the hydrophobic protein and the other having a lower concentration of the hydrophobic protein but richer in the polymer.
  • the protein-rich phase is normally oily in consistency while the protein-poor phase is substantially aqueous.
  • the phases may be separated and the protein-rich phase can provide a stable, high concentration aqueous solution of the hydrophobic protein.
  • Preferred sodium alginate for use in this stabilization has a high mannuronic:guluronic acid ratio.
  • Hydrophobic proteins useful in the invention include growth hormones such as those selected from a group consisting of somatotropin, and derivatives and analogs thereof.
  • the sustained release vehicle can be made from the two-phases of the separated solution or in a preferred embodiment, from the protein-rich phase.
  • the initial aqueous solution should be at or near protein saturation.
  • the alginate or other polysaccharide is gelled, e.g., by contacting the phase with a multivalent cation, thereby forming discrete gel balls. If sodium alginate is used, the preferred cations are calcium ions.
  • the protein-rich phase forms pockets of protein in the gel ball.
  • the gel balls themselves may be used as sustained release vehicles but the formation of microcapsules from the gel balls is preferred.
  • the gel balls are reacted with a membrane forming material, e.g., a polycationic material, thereby forming microcapsules with a protective membrane.
  • the formed microcapsules may be further treated by putting a further protective coating thereon, e.g., by soaking the microcapsules in alginate solution to yield a negative surface charge.
  • Preferred polycationic polymers are selected from a group consisting of polyornithine, polylysine, polyglutamic acid, and co-polymers, derivatives and mixtures thereof. -7-
  • the invention includes not just the method of making this sustained release vehicle and the stabilization method but also the sustained release vehicle itself, either in the gel ball or microcapsule form. While any protein which forms "the two-phase system with the polymer can be used, an alginate acid-growth hormone combination such as sodium alginate-somatotropin, is preferred.
  • the present invention permits the production of stable aqueous solutions of hydrophobic proteins, e.g., growth hormones, in higher concentrations than can otherwise be obtained. Further, stabilized, high concentration protein solutions can be formed into sustained release vehicles which permit the protein to be released over time at a relatively steady controllable rate.
  • hydrophobic proteins e.g., growth hormones
  • the invention is based on the production of the protein-rich oily phase of a two-phase polymeric hydrophobic protein solution. If alginic acid derivatives are used as the polymer, this two-phase system does not appear immediately but rather develops over time. As will be evident from the following examples, the development and stabilization of the two-phase system may take several days. The same alginic acid derivative-hydrophobic protein solution does not provide the same sustained release properties sought unless the two-phase system has developed. The following examples more clearly delineate the advantages and methods used in the invention.
  • Bovine somatotropin (bST) was added to both neutral saline and a 1.4% sodium alginate (Kelco LV) solutions.
  • the bST was substantially insoluble in saline, at pH 7.4.
  • a 50 mg/ml solution was prepared relatively easily in the sodium alginate system.
  • the alginate-rich phase which was about 90% of the volume, had a protein concentration of about 20 mg/ml while the oily protein phase had a bST concentration of about 300 mg/ml, showing a pronounced concentration solubilization and stabilization effect.
  • Example 2 the sustained release effect of the making microcapsules from the two-phase is compared with using an unseparated sodium alginate-hydrophobic protein solution.
  • a 1.4% (w/v) sodium alginate (Kelco LV) solution was prepared and bovine somatotropin (bST) was mixed into the alginate solution at a concentration of 50 mg/ml. As noted from the results of Example 1, this is a higher concentration then could be obtained without the alginate.
  • One portion of the sodium alginate solution was encapsulated immediately, using standard techniques, by allowing drops of solution to fall into a 1.2% calcium chloride solution, thereby forming gel balls.
  • a jet-head droplet forming apparatus consisting of a housing having an upper air intake nozzle and an elongate hollow body friction fitted into a stopper.
  • a syringe e.g., a 10 cc syringe, equipped with a stepping pump is mounted atop the housing with a needle, e.g., a 0.01 inch I.D. Teflon-coated needle, passing through the length of the housing.
  • the interior of the housing is designed such that the tip of the needle is subjected to a constant laminar air-flow which acts as an air knife.
  • the syringe full of the solution containing the material to be encapsulated is mounted atop the housing, and the stepping pump is activated to incrementally force drops of the solution to the top of the needle.
  • Each drop is "cut off” by the air stream and falls approximately 2.5-3.5 cm into an encapsulation solution containing 1.2% CaCl, and 0.3% 80/20 polyornithine/polyglutamic acid copolymer where it is immediately gelled and coated into capsules.
  • the other portion of the sodium alginate-bST solution was held at 4 ⁇ C. for forty hours while undergoing nutation or gentle mixing. After forty hours, the solution separated into two distinct phases; an oily-phase containing most of the protein and a substantially aqueous phase containing most of the alginate. The entire solution containing the separated phases was used to make microcapsules, using the same procedure as previously described. A protein-rich phase acts as suspended pockets of high protein concentration, forming a visible spindle structure. Over time, the spindle structure disintegrates, releasing bST from the capsules.
  • the two sets of microcapsules were tested for sustained release by injection into Hypox rats.
  • the rats' rate of growth was measured by weighing them every day.
  • the rats which received the capsules made from the unseparated alginate-bST solution had a very rapid weight gain in days one and two and then a negative or substantially no weight gain thereafter, showing that all of the bST was released within two days.
  • This is similar to the results for rats receiving a single large dosage injection of bST, which show a high initial weight gain followed by weight decreases or substantially flat weight gain after day two.
  • rats which received microcapsules made from the separated solution show a high weight gain in days one and two and then substantially constant weight gain for days two through seven.
  • the sustained release results were similar to the results obtained by giving rats daily injections of bST, showing that the single injection of the microencapsulated bST acted as a reservoir, yielding a substantially continuous stream of bST to the rats.
  • microcapsules were prepared using a 40 mg/ml bST solution in 1.6% Kelco LV sodium alginate. The solution was nutated for approximately forty hours at 4°C. and formed into microcapsules as described in Example 2. A 3% 80/20 polyornithine/polyglutamic acid copolymer was used for membrane formation.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

On décrit un procédé permettant de produire des solutions stables à concentrations élevées de protéines hydrophiles, utiles pour obtenir des substrats porteurs qui libèrent de manière continue des protéines, par exemple des protéines hydrophobes, en milieux aqueux.
PCT/US1990/000256 1989-01-26 1990-01-10 Stabilisation de solutions de proteines hydrophobes a base aqueuse et substrat porteur de liberation continue WO1990008551A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US30241089A 1989-01-26 1989-01-26
US302,410 1989-01-26

Publications (1)

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WO1990008551A1 true WO1990008551A1 (fr) 1990-08-09

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EP (1) EP0457837A4 (fr)
JP (1) JPH04502768A (fr)
AU (1) AU5107490A (fr)
CA (1) CA2046324A1 (fr)
WO (1) WO1990008551A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040071A1 (fr) * 1995-06-07 1996-12-19 Neocrin Company Procede de fabrication de capsules d'un volume minimal contenant une substance biologique
WO1998046211A1 (fr) * 1997-04-17 1998-10-22 Amgen Inc. Gels d'alginate a liberation prolongee
EP0947201A1 (fr) * 1998-02-04 1999-10-06 Roche Diagnostics GmbH (HRB 3962 - vormals Boehringer Mannheim GmbH) Compositions pharmaceutiques contenant des protéines Hedgehog, et leur utilisation
EP1025861A1 (fr) * 1999-02-04 2000-08-09 Roche Diagnostics GmbH Compositions pharmaceutiques des Proteines Hedgehog rendues hydrophobes, et leur utilisation
JP2010196159A (ja) * 2009-02-02 2010-09-09 Jfe Steel Corp 高強度溶融亜鉛めっき鋼板およびその製造方法
US11932838B2 (en) 2014-11-25 2024-03-19 Corning Incorporated Cell culture media extending materials and methods

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4690682A (en) * 1983-04-15 1987-09-01 Damon Biotech, Inc. Sustained release

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1160892A (fr) * 1980-04-04 1984-01-24 Soliman Y. K. Shenouda Fibres artificielles a base de proteines
WO1985005029A1 (fr) * 1984-05-09 1985-11-21 Medaphore Inc. Insuline administree par voie orale et son procede de preparation
EP0193917A3 (fr) * 1985-03-06 1987-09-23 American Cyanamid Company Compositions dispersibles et solubles dans l'eau de polymères pour administration parentérale

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4690682A (en) * 1983-04-15 1987-09-01 Damon Biotech, Inc. Sustained release

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Volume 97, 1982 (Columbus, Ohio, USA ), ANTONOV, YU. A., See pages 466-467, Abstract No. 37761c. *
See also references of EP0457837A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040071A1 (fr) * 1995-06-07 1996-12-19 Neocrin Company Procede de fabrication de capsules d'un volume minimal contenant une substance biologique
WO1998046211A1 (fr) * 1997-04-17 1998-10-22 Amgen Inc. Gels d'alginate a liberation prolongee
US6656508B2 (en) 1997-04-17 2003-12-02 Amgen Inc. Sustained-release alginate gels
EP0947201A1 (fr) * 1998-02-04 1999-10-06 Roche Diagnostics GmbH (HRB 3962 - vormals Boehringer Mannheim GmbH) Compositions pharmaceutiques contenant des protéines Hedgehog, et leur utilisation
EP1025861A1 (fr) * 1999-02-04 2000-08-09 Roche Diagnostics GmbH Compositions pharmaceutiques des Proteines Hedgehog rendues hydrophobes, et leur utilisation
WO2000045848A1 (fr) * 1999-02-04 2000-08-10 Curis, Inc. Composition pharmaceutique de proteines hedgehog rendues hydrophobes et leur utilisation
JP2010196159A (ja) * 2009-02-02 2010-09-09 Jfe Steel Corp 高強度溶融亜鉛めっき鋼板およびその製造方法
JP4623233B2 (ja) * 2009-02-02 2011-02-02 Ｊｆｅスチール株式会社 高強度溶融亜鉛めっき鋼板およびその製造方法
US11932838B2 (en) 2014-11-25 2024-03-19 Corning Incorporated Cell culture media extending materials and methods

Also Published As

Publication number Publication date
EP0457837A4 (en) 1993-09-15
AU5107490A (en) 1990-08-24
CA2046324A1 (fr) 1990-07-27
JPH04502768A (ja) 1992-05-21
EP0457837A1 (fr) 1991-11-27

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