JPH04502768A - Stabilization and sustained release vehicle for hydrophobic protein aqueous solutions - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Stabilization and sustained release vehicle for hydrophobic protein aqueous solution l Bean peel 11 The present invention relates to the formation and stabilization of aqueous hydrophobic protein solutions. Also, these stabilizing solutions Also disclosed are sustained release vehicles made using liquids.

Much of the interest in protein identification, genetic engineering and purification is that these It concerns the in vivo utilization of proteins, for example the potential treatment of protein deficiencies.

Proteins such as enzymes and hormones regulate reactions in the body and Deficiency or scarcity leads to various scarcity problems. However, protein immobilization in one tissue Pulse or subcutaneous injections are recommended for long-term relief of many problems at relatively low levels. If not used frequently, toxicity and feedback control issues often Often inadequate. Resolve these issues and ensure that patients (and potential patients) receive frequent A variety of different sustained release vehicles have been tried to save patients from the hassle of injections.

These vehicles fall into two general categories, e.g. the body's breakdown of protein or dextran to release proteins; and Several types of pumping mechanisms (osmotic or is an electrical mechanism). The first class of sustained release vehicles are brake has the potential problem of discrepancies in down-down speeds, causing the supply to be released unevenly. , on the other hand, the second class of vehicles is usually exhausted (after which, often surgically It is a bioincompatible object that must be removed.

Among the more promising sustained release vehicles are US patent applications filed on September 1, 1987. U.S. Patent No. 4.690.682 (filed by Dr. Franklin Lim) ), and U.S. Patent Application No. 121,214, filed November 16, 1987. (Wen-GhihTsang and Andrew Magee) microencapsulated sodium alginate (both from the assignee of this application). ). These vehicles are sodium alginate microcapsules. tortuous path-1ike pore ) as a “filtration” device, thereby achieving a high internal concentration of the substance to be released. Set up an osmotic gradient with a large external water volume. In this type of vehicle, The encapsulated protein or other substance is added to the aqueous solution used to make the capsule. Limited to easily soluble hydrophilic substances.

One strange phenomenon of alginate-protein solutions is that over long periods of time, the stable 2 It is the ability to form phase solutions. Other polymers form separate phases, but these phases are sometimes unstable and/or denature the polymer. For example, Tolst Oguzov, Antonov and their co-workers, casein-alginate- water and the trypsin-alginate-water system due to their ability to form two-phase solutions. Therefore, protein fibers It was shown that it is useful for making. However, these two-phase systems usually Another of the denatured proteins used to form the fibers of these protein matrices It was studied as a means and degeneration was not considered a problem. Forms a stable two-phase system Despite their ability to It did not occur. However, casein and trypsin are both hydrophilic; The two-phase system formed is interesting because it is easily soluble.

Hydrophobic proteins, e.g., proteins that are substantially insoluble or have low solubility in aqueous solution, are White matter is particularly difficult to use in sustained release vehicles, where it is first formulated into an aqueous solution. Ru. There are several issues contributing to this: first, the presence of hydrophobic proteins in aqueous solution; It is difficult to obtain meaningful concentrations; secondly, it is difficult to obtain any concentration range that can be obtained. Even if it is, it is relatively unstable; third, there are surface effects at the phase boundaries. .

In the work of ToI Stoguzov's group, the stabilization of hydrophobic protein systems was investigated. If you open up to the problem, you won't see anything.

In fact, they did not report the study of hydrophobic proteins in a two-phase system. Therefore, he Their work developed a sustained release vehicle that provides a constant, time-controlled release of hydrophobic molecules. He doesn't give Kuru any clues to solve what he needs.

Therefore, the purpose of this invention is to stabilize an aqueous solution of a relatively high concentration of hydrophobic protein. The objective is to provide a method for

Another objective of this invention is that it is biocompatible and allows controlled protein release. The objective is to provide a sustained release vehicle.

A further object of the invention is that it can be used for a variety of proteins (especially hydrophobic proteins); Providing a sustained release vehicle without the use of mechanical or electrical pump systems Is Rukoto.

These and other objects and features of the invention will become apparent from the following.

L beans stand and 1 The present invention provides methods for producing stable, highly concentrated hydrophobic protein aqueous solutions, and solutions thereof. Describes a sustained release vehicle made from. This invention is based in part on certain types of Aqueous solutions of polymers (e.g. alginate derivatives and other polysaccharides) are mixed with proteins. It is based on the discovery that when combined, they separate to form a stable two-phase system. child The two-phase system is capable of encapsulating more proteins (e.g., hydrophobic can be placed into microcapsules that can be highly concentrated. long term Control of release rates across the spectrum can also be achieved using this system.

A method for producing a stable, highly concentrated hydrophobic protein aqueous solution is when mixed with protein. Starting with the formation of a first aqueous solution of a polymer that has the ability to form a two-phase system ( Preferably, alginic acid derivatives such as sodium alginate). hydrophobic protein, Mix or dissolve in a first polymer solution to form a polymer, hydrophobic protein solution do.

The alginate derivative-hydrophobic protein solution is a preferred first solution. This solution is , two distinct phases (one phase containing a high concentration of hydrophobic proteins, the other phase containing hydrophobic proteins) (preferably at its freezing point) until it forms (low concentration but rich in polymer) give time to stabilize (with some nutation). available. The protein-rich phase is usually always oily, whereas the protein-poor phase The phase is substantially aqueous. These phases can be separated, with the protein-rich phase containing hydrophobic proteins. Provides a stable, highly concentrated aqueous solution of white matter. Algin is preferred for use in this stabilization. Sodium mannuronic acid has a high mannuronic acid nigeruronic acid ratio. In this invention Useful hydrophobic proteins include somatotropin and its derivatives and analogs thereof. including growth hormones such as those selected from the group consisting of:

A similar stabilization step is followed to create the sustained release vehicle of this invention. Now. Sustained release vehicles can be made from two phases of one separate solution, or are preferably In a new embodiment, it can be made from a protein-rich phase. The first aqueous solution contains protein should be at or near saturation. After separation of the protein-rich phase, alginate salts or other polysaccharides, for example, by contacting the phase with polyvalent cations, thereby It is gelled by forming separate gel balls. if If sodium alginate is used, the preferred cation is calcium ion. be. The protein-rich phase forms protein pockets within the gel sphere.

The gel sphere itself can be used as a sustained release vehicle; Preferably, microcapsules are formed. to form microcapsules reacts the gel spheres with a membrane-forming substance, e.g. a polycationic substance, thereby Forms microcapsules with a protective film. The formed microcapsules are by placing a protective coating over them, e.g. It may be treated by immersion in a phosphate solution to create a negative surface charge. preferred Polycationic polymers include polyornithine, polylysine, and polyglutamic acid. , copolymers, derivatives thereof, and mixtures thereof.

This invention provides a method for making and stabilizing this sustained release vehicle, as well as a method for producing and stabilizing this sustained release vehicle. Also included is the release vehicle itself (in the form of gel spheres or microcapsules). Polymer and two-phase Although any protein forming the system can be used, sodium alginate-somatotropic Preferred are alginate salts such as chlorine, acid-growth hormone combinations.

theory 1 The present invention provides a method for producing stable aqueous solutions of hydrophobic proteins, such as growth hormone. allows at higher concentrations than would otherwise be obtained. In addition, stabilized high concentration A protein solution at a temperature that releases proteins at a relatively stable and controllable rate over an extended period of time. may be formulated into a sustained release vehicle.

The invention is based on the formation of a protein-rich oil phase in a two-phase polymeric hydrophobic protein solution. I'm there. If alginate derivatives are used as polymers, this two-phase system It does not appear immediately, but rather develops over time. It is clear from the example below. However, the development and stabilization of this two-phase system may take several days. Same alginate derivative -Hydrophobic protein solutions are claimed if this two-phase system did not develop. It has the same sustained release properties for a long time.

The following examples more clearly depict the advantages and methods used in this invention. Ru.

Kyou” [mei”] This example illustrates the solubilization and stabilization attributes of the two-phase system of this invention. Ushisoma Totropine (bST) was added to a neutral salt solution and 1.4% sodium alginate (Kel co LV) solution. bST is substantially salt-free at pH 7.4. It was insoluble in solution. In contrast, the 50 mg/ml solution is sodium alginate It could be prepared relatively easily in the system. Sodium alginate bST solution at 4℃ In the case of 40 hours of volition, a two-phase system developed. rich in alginate The phase (approximately 90% of the volume) had a protein concentration of approximately 20 mg/ml. , on the other hand, the oily protein phase has a bsTfi degree of about 300 mg/ml. , showed the solubilizing and stabilizing effects of the above-mentioned concentrations.

Five-year-old Yu In this example, the sustained release of making microcapsules from this two-phase Effects when using unseparated sodium alginate-hydrophobic protein solution compared with. 1.4% (W/V) Sodium alginate (Nielco L V) Prepare a solution and add 5% bovine somatotropin (bST) to the alginate solution. It was added at a concentration of 0 mg/ml. Recognized from the results of Example 1 As such, this is a higher concentration than that obtained without alginate. a A portion of the sodium ruginate solution was diluted with 1.2% potassium chloride using standard techniques. Capture immediately by dropping into a lucium solution, thereby forming gel spheres. It became a cell. A jet-head droplet forming device has an air intake nozzle at the top. A stepper consisting of an outer sheath and an elongated hollow body fitted with a friction stopper. A syringe (for example, a 10cc syringe) equipped with a Attach a long-penetrating needle (e.g., 0.201 inch 1.D, Teflon-coated needle). and place it on top of the jacket. Inside the jacket, the tips of five needles act as air knives. It is designed to be exposed to a constant laminar flow of air. When using the capsule Place the syringe filled with the solution containing the substance to be encapsulated on top of the envelope and step The droplets of solution are pumped into the tip of the needle, each droplet being moved by a stream of air. “Cut” and fall about 2.5-3.5 cm and 1.2% Ca Cl * and 0.3 Encapsulation solution containing %80/20 polyornithine/polyglutamic acid copolymer It falls into the liquid, where it immediately gels and becomes coated in a capsule.

The other portion of the sodium alginate-bST solution was stirred or gently stirred. The mixture was kept at 4° C. for 40 hours. After 40 hours, the solution separated into two distinct phases. ; an oil phase containing the majority of the protein and a substantially aqueous phase containing the majority of the alginate; . The entire solution, including the separated phases, is then microcapsulated using a procedure similar to that described above. used to make. The protein-rich phase acts as a floating pocket for highly concentrated proteins. , forming a visible spindle structure. long Over time, this spindle structure disassembles and releases bST from the capsule.

Two sets of microcapsules were injected into ) Iypox rats to test for sustained release. I tried it. The growth of the rats was determined by weighing them daily. separate Rats that received capsules made from alginate-bST solution without Negative or virtually no weight gain after ~2 days of very rapid weight gain No, indicating that all bST was released within 2 days. This is simply This is similar to the results in rats that received large doses of bST, which initially Severe weight gain, then weight loss or no substantial change after 2 days That is what it is. In contrast, microcapsules made from isolated solutions Rats that received the test showed severe weight gain for 1-2 days, and then for 2-7 days. There was a virtually constant weight gain per day over the period. This sustained release result is The results are similar to those obtained by daily injections of bST in rats, and a single injection of microencapsulated bST into the rat is essentially continuous injection of bST into the rat. It was shown to act as a reservoir to generate flow.

Although the results with gel spheres were not as good as those with formed microcapsules, However, the results were still better than the simple injection form.

迤褤danyu In this example, samples were made using the previously described procedure (including 40 hours of mold movement at 4°C). Capsules were perfused with Tris buffer to test sustained release properties. bST preparation In vivo studies (similar to those described in Example 2) in Hypox rats showed that 7 After days, the duration is limited due to the animal's immune response. Therefore, the perfusion experiment , to simulate the longer term in vivo performance of these formulations. I went to

Microcapsules are 40% in 1.6% Kelco LV Sodium Alginate. It was manufactured using mg/mlbsT.

The solution was incubated at 4° C. for approximately 40 hours and microcoated as described in Example 2. Formed into a capsule. 3% 80/20 polyornithine/polyglutamic acid copoly mer was used for film formation.

In this example, three different samples were used: a control without perfusion before injection; capsules, the first group of test microcapsules perfused for two days, and and a second group of test microcapsules perfused for 5 days. perfusion tris Buffer solution, pH 7.4, is flowed around the capsule at a rate of 10 ml/h at 37°C. This was done by doing this.

After perfusion, control and each test sample were injected into Hypox rats. body weight The increase was measured as a guide to bST release rate. Table 1 shows days 2, 2-4, 4 Weight gain is shown on day -7 and day 7-100.

・Two grams 2nd day 2-4 4-7 7-10 Control 13.0 7.2 1.7 -1.12 days perfusion 12.6 7 ．． 3 6.3 - 1.75 days perfusion 14.3 5.3 4.3 0.5 in Table 1 As is clear from the results shown, samples perfused for 2 or 5 days were not perfused. gave essentially the same growth rate compared to the control sample and therefore essentially give the same release rate. The performance of partially reduced samples was determined by this capsule. The formulation can deliver bST in a stable manner for a significant period of time exceeding 7 days. It suggests that there is.

The above embodiments are not intended to be limiting and are provided herein only to facilitate the description of the invention. It is for the purpose of The invention is limited by the scope of the following claims.

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Claims (38)

[Claims] 1. Steps below: forming an aqueous solution of a polymeric material and a protein, said polymeric material containing said protein; It has the ability to form a two-phase system when mixed with The above solution is mixed into a first phase containing a high concentration of the above hydrophobic protein and the above hydrophobic protein. and a second phase containing a low concentration of forming said sustained release vehicle from said solution, whereby said first phase is forming a protein pocket inside said vehicle; A method of producing sustained release of a protein, comprising: 2. 2. The method of claim 1, wherein said polymeric material comprises a polysaccharide. 3. 3. The method of claim 2, wherein the polysaccharide comprises an alginic acid derivative. 4. 4. The alginic acid derivative according to claim 3, wherein the alginic acid derivative comprises sodium alginate. Method. 5. 2. The method of claim 1, wherein said protein comprises a hydrophobic protein. 6. Claim 5, wherein said aqueous solution is a substantially saturated solution of said hydrophobic protein. Method described. 7. Said separation step is further carried out until said two-phase system has developed and stabilized. Claim 1 comprising maintaining said aqueous solution at a temperature slightly above its freezing point. The method described in. 8. While the solution is held at a temperature slightly above its freezing point, it undergoes nutation. 8. The method according to claim 7. 9. Said step of forming said sustained release vehicle comprises converting said first phase into a polyvalent 5. The method of claim 4, comprising gelling with cations to form discrete gel spheres. How to put it on. 10. Said step of forming said sustained release vehicle further comprises forming said sustained release vehicle. The gel spheres are reacted with a polycationic substance to form a membrane around the gel spheres, and 10. The method of claim 9, comprising forming microcapsules by. 11. further comprising applying a protective overcoat around the microcapsules. , the method according to claim 10. 12. The step of applying said protective coating comprises coating said microcapsules with alginic acid. 12. The method of claim 11, comprising soaking in a salt solution. 13. The above sodium alginate is gelled by contact with calcium ions. 11. The method according to claim 10. 14. The above polycationic polymers can be combined with polyornithine, polylysine and polycationic polymers. Consisting of glutamic acid and their copolymers, derivatives and mixtures thereof 13. The method according to claim 12, wherein the method is selected from the group. 15. 11. The method of claim 10, wherein the hydrophobic protein is growth hormone. 16. The growth hormone is somatotropin, a derivative thereof or an analog thereof. 16. The method of claim 15, comprising: 17. Claim wherein said alginic acid derivative has a high mannuron:guluron ratio. The method described in 3. 18. The method further comprises separating the first phase from the second phase, and 10. Forming a sustained release vehicle of from said first phase. Method described. 19. Sustained release to provide sustained release of hydrophobic proteins in vivo a vehicle, said sustained release vehicle being a mixture of a polymeric substance and a protein; a cross-linked protein-rich phase, said polymeric material being mixed with said protein. and the protein-rich phase is one of the two-phase systems. the crosslinking is achieved by contacting the polymeric material with a crosslinking agent. A sustained release vehicle. 20. 20. The sustained release vehicle of claim 19, wherein said polymeric material comprises a polysaccharide. Cru. 21. Sustained release according to claim 20, wherein the polysaccharide comprises an alginate derivative. Vehicle. 22. 22. The alginic acid derivative according to claim 21, wherein the alginic acid derivative comprises sodium alginate. sustained release vehicle. 23. 22. The sustained release vehicle of claim 21, wherein said crosslinking agent comprises a multivalent ion. Le. 24. 22. The sustained release vehicle of claim 21, wherein said protein comprises a hydrophobic protein. Cru. 25. Claim 2, wherein said mixture is substantially a saturated solution of said hydrophobic protein. 4. The sustained release vehicle according to 4. 26. said mixing until said phase separation develops and stabilizes said two-phase system. 20. Occurring while maintaining the object at a temperature slightly above its freezing point. Sustained release vehicle. 27. Nutation occurs while the mixture is held at a temperature slightly above its freezing point. 27. The sustained release vehicle of claim 26, wherein the sustained release vehicle receives: 28. The separated protein-rich phase forms a gel sphere after contact with the multivalent ions. 24. The sustained release vehicle of claim 23, which is in the form of a pocket of included protein. Le. 29. Alginic acid with high mannuron:guluron ratio as mentioned above sodium alginate 23. The sustained release vehicle of claim 22, comprising sodium. 30. 24. The persistent ions of claim 23, wherein said multivalent ions include calcium ions. Release vehicle. 31. The sustained release vehicle further comprises a membrane formed around the gel sphere. The membrane is prepared by reacting the gel spheres with a polycationic substance. 31. The sustained release vehicle of claim 30. 32. The above polycationic substances are combined with polyornithine, polylysine and polyglue. From the group consisting of tamic acid and mixtures thereof, copolymers and derivatives thereof 32. The sustained release vehicle of claim 31. 33. A method for producing a stable, highly concentrated aqueous solution of a hydrophobic protein, comprising the following steps: Tep: forming a first aqueous solution of an alginate derivative; mixed with an alginic acid derivative solution to form an alginic acid derivative-hydrophobic protein solution. , Two distinct phases were formed in the alginate derivative-hydrophobic protein solution, one of which was One contains the above hydrophobic protein at a high concentration, and the other contains the above hydrophobic protein at a low concentration. including, and The phase containing a high concentration of the hydrophobic protein is separated and the hydrophobic protein is isolated. Supplying a stable and highly concentrated aqueous solution A method for producing stable, highly concentrated aqueous solutions of hydrophobic proteins, including: 34. The above step of separating the alginate derivative-hydrophobic solution into two phases. 34. The method of claim 33, wherein the step is carried out at a temperature slightly above its freezing point. 35. 34. The alginic acid derivative according to claim 33, wherein the alginic acid derivative comprises sodium alginate. How to put it on. 36. The said sodium alginate has a high mannuron: guluron ratio, The method according to claim 35. 37. 34. The method of claim 33, wherein said hydrophobic protein is growth hormone. 38. The above-mentioned growth hormone is somatotropin, a derivative thereof or an analog thereof. 38. The method of claim 37, wherein the method is selected from the group consisting of: