WO1990008551A1 - Stabilization of aqueous-based hydrophobic protein solutions and sustained release vehicle - Google Patents
Stabilization of aqueous-based hydrophobic protein solutions and sustained release vehicle Download PDFInfo
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- 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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- Prior art keywords
- sustained release
- protein
- release vehicle
- phase
- solution
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- 238000013268 sustained release Methods 0.000 title claims abstract description 45
- 239000012730 sustained-release form Substances 0.000 title claims abstract description 45
- 230000002209 hydrophobic effect Effects 0.000 title claims abstract description 37
- 239000012460 protein solution Substances 0.000 title claims description 11
- 230000006641 stabilisation Effects 0.000 title description 9
- 238000011105 stabilization Methods 0.000 title description 9
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 79
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 79
- 238000000034 method Methods 0.000 claims abstract description 33
- 239000000243 solution Substances 0.000 claims description 33
- 235000010443 alginic acid Nutrition 0.000 claims description 24
- 229920000615 alginic acid Polymers 0.000 claims description 24
- 239000003094 microcapsule Substances 0.000 claims description 21
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 18
- 239000007864 aqueous solution Substances 0.000 claims description 18
- 239000000661 sodium alginate Substances 0.000 claims description 18
- 235000010413 sodium alginate Nutrition 0.000 claims description 18
- 229940005550 sodium alginate Drugs 0.000 claims description 18
- 239000000463 material Substances 0.000 claims description 17
- 229960001126 alginic acid Drugs 0.000 claims description 16
- 239000000783 alginic acid Substances 0.000 claims description 16
- 150000004781 alginic acids Chemical class 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 9
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 claims description 8
- 229940072056 alginate Drugs 0.000 claims description 8
- 108010051696 Growth Hormone Proteins 0.000 claims description 7
- 102000018997 Growth Hormone Human genes 0.000 claims description 7
- 239000000122 growth hormone Substances 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 229920001282 polysaccharide Polymers 0.000 claims description 6
- 239000005017 polysaccharide Substances 0.000 claims description 6
- 150000004804 polysaccharides Chemical class 0.000 claims description 6
- 108010020346 Polyglutamic Acid Proteins 0.000 claims description 5
- 229920002643 polyglutamic acid Polymers 0.000 claims description 5
- 229920002714 polyornithine Polymers 0.000 claims description 5
- 108010055896 polyornithine Proteins 0.000 claims description 5
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 3
- 108010039918 Polylysine Proteins 0.000 claims description 3
- 229910001424 calcium ion Inorganic materials 0.000 claims description 3
- 150000001768 cations Chemical class 0.000 claims description 3
- 229920001577 copolymer Polymers 0.000 claims description 3
- 238000001727 in vivo Methods 0.000 claims description 3
- 229920000656 polylysine Polymers 0.000 claims description 3
- 239000011253 protective coating Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 229920002851 polycationic polymer Polymers 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 150000002500 ions Chemical class 0.000 claims 3
- 239000003431 cross linking reagent Substances 0.000 claims 2
- 239000012047 saturated solution Substances 0.000 claims 2
- 239000002253 acid Substances 0.000 claims 1
- 238000004132 cross linking Methods 0.000 claims 1
- 238000005191 phase separation Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 235000018102 proteins Nutrition 0.000 description 49
- 239000012071 phase Substances 0.000 description 38
- 108010006025 bovine growth hormone Proteins 0.000 description 17
- 241000700159 Rattus Species 0.000 description 9
- 229920000642 polymer Polymers 0.000 description 8
- 235000019786 weight gain Nutrition 0.000 description 8
- 230000004584 weight gain Effects 0.000 description 8
- 239000002775 capsule Substances 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000010412 perfusion Effects 0.000 description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 238000013270 controlled release Methods 0.000 description 2
- -1 e.g. Proteins 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000005063 solubilization Methods 0.000 description 2
- 230000007928 solubilization Effects 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- AEMOLEFTQBMNLQ-BZINKQHNSA-N D-Guluronic Acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-BZINKQHNSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000008425 Protein deficiency Diseases 0.000 description 1
- 239000004809 Teflon Substances 0.000 description 1
- 229920006362 Teflon® Polymers 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 235000021053 average weight gain Nutrition 0.000 description 1
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 1
- 239000007963 capsule composition Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
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- 230000001988 toxicity Effects 0.000 description 1
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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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Abstract
Disclosed is a method for producing stable, high concentration solutions of hydrophilic proteins. These methods are useful in producing vehicles which provide sustained release of proteins, e.g., hydrophobic proteins, into aqueous environments.
Description
STABILIZATION OF AQUEOUS-BASED HYDROPHOBIC PROTEIN SOLUTIONS AND SUSTAINED RELEASE VEHICLE
Background of the Invention
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.
Much of the interest in identification, genetic engineering and purification of proteins is related to the possibility of jLn vivo use of the proteins, e.g., as treatment for protein deficiencies. Proteins such as enzymes and hormones modulate reactions in the body and the lack, or an insufficient amount, of these proteins leads to a variety of deficiency problems. However, intravenous or subcutaneous injections of protein into the system are often insufficient for long term amelioration of many problems because of toxicity and feedback control problems unless relatively low levels are used on a frequent basis. In order to solve these problems and relieve patients, and potential patients, of the onerous task of frequent injections, a variety of different sustained release vehicles have been tried. These vehicles are in two general categories: those which release protein through a breakdown mechanism, e.g., collagen or deztran
degradation by the body; and those which use some type of pump-type mechanism, either osmotic or electro-mechanical, to release material over time. 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.
Among the more promising 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.
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, are particularly difficult to use in sustained release vehicles which dispense the protein into primarily aqueous solutions. There are several problems which contribute to this: first, it is difficult to obtain a meaningful concentration of hydrophobic proteins in the aqueous solution; second, to the extent that any concentration is obtained, it is relatively unstable; and third, there are surface effects at the interface between the phases.
None of the work done by the Tolstoguzov group appears to touch upon the problem of stabilizing hydrophobic protein systems. In fact, they did not report investigation of hydrophobic proteins in a two-phase system. Therefore, their
work provides no clues to solve the need for sustained release vehicles to provide constant, time-controlled release of hydrophobic molecules.
Accordingly, 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.
These and other objects and features of the invention will be apparent from the following description.
Summary of the Invention
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.
To make the sustained release vehicle of the invention, the same stabilization steps are followed. 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. After separation of the protein-rich phase, 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. To form microcapsules, 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.
Description of the Invention
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.
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.
Example 1.
This Example illustrates the solubilization and stabilization attributes of the two-phase system of 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. In contrast, a 50 mg/ml solution was prepared relatively easily in the sodium alginate system. When the sodium alginate bST solution was allowed to stand at 4°C. for forty hours with nutation, a two-phase system developed. 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.
In this Example, 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. In use, 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 4βC. 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. In contrast, 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.
The results with gel balls rather than formed microcapsules were not as good but still showed better results than the single injection form.
Example 3 .
In this Example, capsules made using the procedure previously described, including the nutation at 4° for forty hours, were perfused with Tris buffer to test sustained release characteristics. In vivo testing of bST formulations in Hypox rats, such as is described in Example 2, is limited in time duration due to an immune response by the animals after seven days. Therefore, perfusing experiments were performed in order to simulate the in vivo performance of these formulations over a longer time period.
The 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.
Three different samples were used in the
Example: a control group of capsules which was not perfused before injection, a first test group of microcapsules which were perfused for two days, and a second test group of microcapsules which were perfused for five days. Perfusion was carried out by flowing a Tris buffer, pH 7.4. at 37°C, past the capsules at a rate of 10 ml/hr.
After perfusion, the control and each of the test samples were injected into Hypox rats. Weight
gain was measured as an indication of bST release rate. Table 1 shows the weight gain at" day 2, days 2-4, days 4-7, and -days 7-10.
TABLE 1
Average weight gain in σrams
Day 2 2-4 4-7 7-10
Control 13.0 7.Ε 1.7 -1.1
2 days perfusion 12.6 7.3 S.3 -1.7
5 days perfusion 14.3 5.3 4..3 0.5
As is clear fxpm the results .shown in Table 1, the samples which have been perfused ϊor two or five days provide essentially identical growth rates and, therefore, release rates, as did ±he unperfused control sample. The performance of the partially depleted samples suggest that the capsule formulation is capable of delivering bST at a steady state for significant time periods, exceeding ewsn days.
The foregoing Examples are meant to be non-limiting and are here solely for ease in explanation of the invention. The invention is defined by the following claims.
What is claimed is:
Claims
1. A method of producing sustained release of proteins comprising the steps of:
forming an aqueous solution of a polymeric material and a protein, said polymeric material having the ability to form a two-phase system when mixed with said protein,
permitting said solution to separate into said two-phase system having a first phase containing a high concentration of said hydrophobic protein and a second phase containing a low concentration of said hydrophobic protein, and
forming said sustained release vehicle from said solution, whereby said first phase forms pockets of protein within said vehicle.
2. The method of claim 1 wherein said polymeric material comprises a polysaccharide.
3. The method of claim 2 wherein said polysaccharide comprises an alginic acid derivative.
4. The method of claim 3 wherein said alginic acid derivative comprises sodium alginate.
5. The method of claim 1 wherein said proteins comprise hydrophobic proteins.
6. The method of claim 5 wherein said aqueous solution is a substantially saturated solution of said hydrophobic protein.
7. The method of claim 1 wherein said separating step further comprises maintaining said aqueous solution at a temperature slightly above its freezing point until said two-phase system develops and is stabilized.
8. The method of claim 7 wherein said solution undergoes nutation while being held at the temperature slightly above its freezing point.
9. The method of claim 4 wherein said step of forming said sustained release vehicle comprises the step of gelling said first phase with a multivalent cation to form discrete gel balls.
10. The method of claim 9 wherein said step of forming said sustained release vehicle further comprises the step of reacting said gel balls with a polycationic material to form a membrane about said gel balls, thereby forming microcapsules.
11. The method of claim 10 further comprising the step of putting a protective coating about said microcapsules.
12. The method of claim 11 wherein said step of applying a protective coating comprises soaking said microcapsules in an alginate solution.
13. The method of claim 10 wherein said sodium alginate is gelled by contact with calcium ions.
14. The method of claim 12 wherein said polycationic polymer is selected from a group consisting of polyornithine, polylysine, and polyglutamic acid, and copolymers, derivatives, and mixtures thereof.
15. The method of claim 10 wherein said hydrophobic protein is a growth hormone.
16. The method of claim 15 wherein said growth hormone comprises somatotropin, a derivative, or an analog thereof.
17. The method of claim 3 wherein said alginic acid derivative has a high mannuronic:guluronic ratio.
18. The method of claim 9 wherein said method further comprises the step of separating said first phase from said second phase and forming said sustained release vehicle from said first phase.
19. A sustained release vehicle for providing in vivo sustained release of a hydrophobic protein, said sustained release vehicle comprising a cross-linked protein-rich phase of a mixture of a polymeric material and a protein, said polymeric material having the ability to form a two-phase system when mixed with said protein, said protein-rich phase being one of the phases of said two-phase system, said cross-linking being achieved by contacting said polymeric material with a cross-linking agent.
20. The sustained release vehicle of claim 19 wherein said polymeric material comprises a polysaccharide.
21. The sustained release vehicle of claim 20
5 wherein said polysaccharide comprises an alginic acid derivative.
22. The sustained release vehicle of claim 21 wherein said alginic acid derivative comprises sodium alginate.
1023. The sustained release vehicle of claim 21 wherein said cross-linking agent comprises a multivalent ion.
24. The sustained release vehicle of claim 21 wherein said protein comprises a hydrophobic protein.
1525. The sustained release vehicle of claim 24 wherein said mixture is a substantially saturated solution of said hydrophobic protein.
26. The sustained release vehicle of claim 19 wherein said phase separation takes place while
20 maintaining said mixture at a temperature slightly above its freezing point until said two-phase system develops and is stabilized.
27. The sustained release vehicle of claim 26 wherein said mixture undergoes nutation while being
25 held at the temperature slightly above its freezing point.
28. The sustained release vehicle of claim 23 wherein said separated protein-rich phase is in the form of pockets of protein contained within a gel ball after contacting with said multivalent ions.
529. The sustained release vehicle of claim 22 wherein said sodium alginate comprises sodium alginate with a high mannuronic:guluronic ratio.
30. The sustained release vehicle of claim 23 wherein said multivalent ion comprises a calcium ion.
1031. The sustained release vehicle of claim 30 wherein said sustained release vehicle further comprises a membrane formed about said gel ball, said membrane formed by reacting said gel ball with a polycationic material.
15 32. The sustained release vehicle of claim 31 wherein said polycationic material is selected from a group consisting of polyornithine, polylysine, and polyglutamic acid, and mixtures, copolymers, and derivatives thereof.
20 33. A method of producing stable, high concentration aqueous solutions of hydrophobic proteins comprising the steps of:
forming a first aqueous solution of an alginic acid derivative,
25 mixing said hydrophobic protein with said first alginic acid derivative solution, forming an alginic acid derivative-hydrophobic protein solution. allowing two distinct phases form in said alginic acid derivative-hydrophobic protein solution, one containing a high concentration of said hydrophobic protein and one containing a low concentration of said hydrophobic protein, and
separating said phase containing the high concentration of said hydrophobic protein to provide a stable, high concentration aqueous solution of said hydrophobic protein.
34. The method of claim 33 wherein said step of allowing said alginic acid derivative-hydrophobic solution to separate into two-phases is carried out at a temperature slightly above its freezing point.
35. The method of claim 33 wherein said alginic acid derivative comprises sodium alginate.
36. The method of claim 35 wherein said sodium alginate has a high mannuronicrguluronic acid ratio.
37. The method of claim 33 wherein said hydrophobic protein is a growth hormone.
38. The method of claim 39 wherein said growth hormone is selected from a group consisting of somatotropin, a derivative or an analog thereof.
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)
| Publication Number | Publication Date |
|---|---|
| WO1990008551A1 true WO1990008551A1 (en) | 1990-08-09 |
Family
ID=23167629
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1990/000256 WO1990008551A1 (en) | 1989-01-26 | 1990-01-10 | Stabilization of aqueous-based hydrophobic protein solutions and sustained release vehicle |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0457837A4 (en) |
| JP (1) | JPH04502768A (en) |
| AU (1) | AU5107490A (en) |
| CA (1) | CA2046324A1 (en) |
| WO (1) | WO1990008551A1 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996040071A1 (en) * | 1995-06-07 | 1996-12-19 | Neocrin Company | Method for the manufacture of minimal volume capsules containing biological material |
| WO1998046211A1 (en) * | 1997-04-17 | 1998-10-22 | Amgen Inc. | Sustained-release alginate gels |
| EP0947201A1 (en) * | 1998-02-04 | 1999-10-06 | Roche Diagnostics GmbH (HRB 3962 - vormals Boehringer Mannheim GmbH) | Pharmaceutical composition of hedgehog proteins and use thereof |
| EP1025861A1 (en) * | 1999-02-04 | 2000-08-09 | Roche Diagnostics GmbH | Pharmaceutical compositions of hydrophobically modified Hedgehog Proteins and their use |
| JP2010196159A (en) * | 2009-02-02 | 2010-09-09 | Jfe Steel Corp | High-strength hot-dip galvanized steel sheet and method for producing the same |
| US11932838B2 (en) | 2014-11-25 | 2024-03-19 | Corning Incorporated | Cell culture media extending materials and methods |
Citations (1)
| 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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1160892A (en) * | 1980-04-04 | 1984-01-24 | Soliman Y. K. Shenouda | Fabricated protein fibers |
| WO1985005029A1 (en) * | 1984-05-09 | 1985-11-21 | Medaphore Inc. | Oral insulin and a method of making the same |
| EP0193917A3 (en) * | 1985-03-06 | 1987-09-23 | American Cyanamid Company | Water dispersible and water soluble carbohydrate polymer compositions for parenteral administration |
-
1990
- 1990-01-10 WO PCT/US1990/000256 patent/WO1990008551A1/en not_active Application Discontinuation
- 1990-01-10 AU AU51074/90A patent/AU5107490A/en not_active Abandoned
- 1990-01-10 CA CA 2046324 patent/CA2046324A1/en not_active Abandoned
- 1990-01-10 EP EP19900903563 patent/EP0457837A4/en not_active Withdrawn
- 1990-01-10 JP JP50368490A patent/JPH04502768A/en active Pending
Patent Citations (1)
| 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)
| 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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996040071A1 (en) * | 1995-06-07 | 1996-12-19 | Neocrin Company | Method for the manufacture of minimal volume capsules containing biological material |
| WO1998046211A1 (en) * | 1997-04-17 | 1998-10-22 | Amgen Inc. | Sustained-release alginate gels |
| US6656508B2 (en) | 1997-04-17 | 2003-12-02 | Amgen Inc. | Sustained-release alginate gels |
| EP0947201A1 (en) * | 1998-02-04 | 1999-10-06 | Roche Diagnostics GmbH (HRB 3962 - vormals Boehringer Mannheim GmbH) | Pharmaceutical composition of hedgehog proteins and use thereof |
| EP1025861A1 (en) * | 1999-02-04 | 2000-08-09 | Roche Diagnostics GmbH | Pharmaceutical compositions of hydrophobically modified Hedgehog Proteins and their use |
| WO2000045848A1 (en) * | 1999-02-04 | 2000-08-10 | Curis, Inc. | Pharmaceutical composition of hydrophobically modified hedgehog proteins and their use |
| JP2010196159A (en) * | 2009-02-02 | 2010-09-09 | Jfe Steel Corp | High-strength hot-dip galvanized steel sheet and method for producing the same |
| JP4623233B2 (en) * | 2009-02-02 | 2011-02-02 | Ｊｆｅスチール株式会社 | High-strength hot-dip galvanized steel sheet and manufacturing method thereof |
| US11932838B2 (en) | 2014-11-25 | 2024-03-19 | Corning Incorporated | Cell culture media extending materials and methods |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH04502768A (en) | 1992-05-21 |
| EP0457837A4 (en) | 1993-09-15 |
| CA2046324A1 (en) | 1990-07-27 |
| AU5107490A (en) | 1990-08-24 |
| EP0457837A1 (en) | 1991-11-27 |
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