CA2046324A1 - Stabilization of aqueous-based hydrophobic protein solutions and sustained release vehicle - Google Patents

Abstract

Abstract of the Invention 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

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STABILIZATIO~ OF AQUEOUS-~ASED HYDROPHOBIC
P~OTEI~LSOLUTI~NS A~D ~T~IWE~ R~L~E VEHI~E

~he present inventio~ r~late~ to the formation and ~ta~ za~ion of agueou~ bas~
solution~ o hy~rophobic protein~. ~u~ aine~ release vehicles made u~in~ these stabilize~l ~olutions are al~o disclo~ed.

Much of the interest in i~entification, genetic engineering ~nd purification of pro ein~ is related to ~he pos~ibility of i~ v QQ use of the -~
proteins, ~.~., as treatme~ for protein deficiencies. Prot~in~ ~uch 3S en2ymes 2nd hormon~s mo~ulate reaction~ in the body an~ the lack, or an insuf icient amount, o~ these proteins leads to a ~ariety ~ deficiency problems. However, intravenous or subrutaneous injec~ions of protein into ~he system are often insufficien~ or long tesm amelioratqon of many problems because of to~icity and feedback 20 control problems unless relatively low levels are used on a frequent ba8i8 . In order to ~olve these problems and relieve patients, and potential patients, of the onerous task of reguent ~njeetion~, a variety of d~fferent sustainea release vehicles 25 have been tried. These vehicle~ are in two general categori~: those which release protein through a br~ak~own mechan~sm, e.g., eollagen or ~e~tran ;' :
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~egradation by the boay, an~ those which U8e some type of pump-type mechanism, either osmotic or electro-mechanical, t~ release ~aterial over time.
~he fir t cla~ of ~ustained relea~e vehicles have pote~tial problem~ wi~h ~ erential break~ow~ rates~
thereby provi~in~ un~Yen release while the ~econa class o~ vehicles are normally bio-incompatibl@
~b~ects which muæt be remove~ after e~hau~te~ oten ~urg~cally.

Among the mo~e promi~ing ~ustaine~ relesse vehicles are the o~ium alginate-based microcGpsules ~escribed in United States Patent No. 4,690,682, iled September 1, 1987, on applicat:ion of Dr.
~rankl;n Lim, and United States Patent Applic~tion Serial No. 121,214, f~led November 16, 19879 on application of Wen-~.hih Tsang and Andrew Magee, both assigned to the ~ssi~nee of the prexent application.
These vehicles use ~or~uous path-like pores of sodium algina~e microcapsul~s as ~ ~filtering~ device 20 whereby an osmo~ic gradient is ~et up between a high internal concentration of the material to be released and the large surrounding aqueous volume. The proteins or other materials which ha~e bee~
encapsulated in this type of vehicle have been limited to hydrophilic materials which are easily dissolved in the aqueous ~olution used to make the capsules.

~ One o~d phenomenon of alginate-protein ; ~olutions i~ the ability, over time, to form ~table two-phas~ ~o~utions. Although other pol~mers form ~epar~te pha3es, these phases are sometimes unst~ble , . ~ ~

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and~or ~enature the polymers. For e~ample, Tolstoguzov, ~ntono~ a~d their co~wor~er~ ~ave ~hown that the ca~ein-alginate-water ~nd trypsin-alginate w~ter ~y~te~ ~re u~e~ul for ma~ing protein spinn~ret ibers ~ecause oP their ~bllity to make two-pha~e ~olutions. However~ these two-pha~e ~y~tems were investigate~ ~8 alternative~ ~or the denatured protein normally used to form these protein matris fibers ana denaturation wa~ not con~idere~ a 10 problem. Desp~te the ability to form ~t~bl~
two-phase ~ystems, ne~ther the casein nor tryp~in ~ystem produced notably better results. The two-pha~e ~y~tem orme~ is interesting, however, since both casein and trypsin ar~ hydrophilic, easily soluble protein~.

Hydrophobic proteins, eOg.~ proteins wbich are ~ubstantially insoluble or have low solub~lities in a~ueous ~olution~, are pasticularly difficult to u~e 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 ~oncentration of hy~rophobic proteins in the agueous ~olution; ~econd, to the e~tent that ~ny concentration is obtained, it is relati~ely unstable; and third, there are ~ur~ace efects at the ~ntsrf~cc bstween the phases.
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~on~ o~ the work dona by the Tolsto3uzov ~roup appear~ to touch upon the probl~m of ~tabili~inq hy~rophobi~ prote~n 6y8tem8. In act, they ~i~ not report ~nv~ti~tion o~ hy~ophob~c protein~ two-phase ~y~tem. Thereore, thelr '' .: .

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work provi~es no ~lues to solYe the neea ~or ~u~taine~ release ~ehlcle~ to prov~de constant, time-controllea release o~ hydrophobic molPcules, Accordingly~ a~ obj~ct 9~ ~he in~e~tion i~
5 to provi~ a me~hod ~tabilizin~ aqueous ~olut~on~ of relatively high concentration~ of hydrophob~c proteins.

Another object o~ the inv~ntion i8 to provide a ~ustaine~ relea~e vehicle which i~
10 biocompatible an~ allow~ controlle~ release of pro~eins.

A ~urther object o the invention ~ to provide sustaine~ release ~ystems usable ~or a broad variety of proteins, particularly hydrophobie 15 proteins, without merhanical or electro-mechanical pumping systems.

These ~na other object~ an~ ~eatures of the invention will be apparent ~rom the following description.

20 Summarv of the Inven~iQn The present ~nvention fe~tures methods of producing ~ta~le, high ~oncentration ~queouæ
~olution~ of hydrophobic proteinæ an~ ~u~taine~
releaæe vehicle~ made f rom thosa ~olutions. The 25 invention i~ ba~e~, in par~, on the diæeovery that aqU~OUB 801ution6 of cert~in polymer~, e.g., algirlic aei~ ~erivative~ an~ othar poly~acchar~ , will, . . . . .
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when mi~ed with protein~, sep~rate an~ form stable two-pha~e ~ys~ems. ~hi~ two-phase ~y~em can be turned ~nto microcapsules which permits higher concentrations of protei~, e.g., hydrophobi~
protein~ f to be encapsul~ted than 13 normally possibl~. Control~ing ~he rate of relea~e oYer time i~ al~o achievable u~ing thiæ ~y~tem.

The metho~ of pro~ucin~ the ~table, hi~h concentration aqueou~ ~olutions o the hydropho~ic protein~ commenc~ with the format~on of ~ fir~t aqueou~ solution of the polymer which ha~ the ability to form a twophase systems when mi~ed with the protein, preferably an ~lginic aci~ ~erivati~e, e.g, ~o~ium alginate. The hy~rophobic prot~in i~ mi~ed or dissolved in the first polymesic ~olutio~, ~orming a polymer hydrophobic protein solu~ion. An alginic acid derivative-hydrophobi~ protei~ ~olution is a preferr~ ~irst ~olutio~. Thi~ ~olution is allowed time to stabilize, preferably at slightly above its freezin~ point with nutat~on, until two distinct phases form; one phase baving ~ high concentration of the hy~rophobic protein an~ tha other having a lower co~centration of the hydrophobic protein but richer in the polymer. The protein-rich ~hase is normally oily in consistency while the protein-poor phase is substantially ~queous. The pha~es may be separated an~ thc protein-rl~h pha~e can provide a st~hle~ Ihigh conc~ntratio~ agueous ~vlution o~ thc h~drophobic prot~in. Pr~erred so~ium algin~te or use in thi~
~t~bili~tion ha~ a high mannuronic:~uluronic aci~
ratio. Hy~rophobic proteins u~Qful in the ~nvention inclu~e ~rowt~h hormones ~ucA ~ tho~e ~electe~ ~rom ` " ~`. -' ' . ' -6~ 3 2i.~

group consisting of 60matotropin, and derivatives and analogs thereof.

To make ~he sustained release vehicle of the invention, the same ~tabilization steps are 5 follow~. The ~ustained rel~ase vehicle can be mads from the two-phase~ o~ the 6epara~ed solution or ~n a preferred embo~iment, from the protein-rich phass.
The initial aqueous ~olution shoul~ be at or near pro~ein saturation. After separation of the 10 protein-rich phase, the 31ginate or other polysaccharide is gelled, e.g., by contacting the phase with a multivalent cation, thereby forming discrete gel ~alls. ~f sodium alginate is used, the preferred cations are calcium ions. The protein-rich 15 phase forms pockets of protein in the gel ball.

The gel balls themselves may be used as sustained relPase vehi~les but the formation of microcapsules from the 921 ~alls is preferred. ~o form microcapsu~es, the gel balls ar~ reacted with a 20 membrane forming material, e.g., a polycationic materi`al, 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 : 25 in alginate solution ~o yield a negative surface charge. Preferred polycationic polymers are selected from 3 group consisting of polyornithine, polylysine, polyglutamic ~cid, and ~o-polymer~, derivatives and ~i~tures thereo~.

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The in~e~tisn includes not jus~ the methoa of makinq thi~ ~u~tainea release vehi~le and the ~itabiliza~ion met~o~ ~ut ~ o the ~ustained release vehicle itsel~, either in the gel ball or microcapsule o~m. While any protein whi~h ~orms the two-pha~e ~iy~te~ ~ith ~he polymer can be u~ed, an alginate acid-g~wth ~ormone ~ombination ~u~h a~
~iodium alginate-som~totropi~, is preferred.

12escriDtion o~ h~ .YentiQn The present invention permits the production of stable aqueous sol~tions of hydrophobic proteins, e.g., growth hosm~n~s, i~ higher concentrations than can otherwise be ~bt~ined. ~urther, stabilized, bigh concentration proteiD solu~ions can be formed into sustained release vehicles which permit the pro~ein to be released ~ver ~ime at a relatively stea~y controllable ra~e.

The invention is ~ased on the production of the protein-rich ~ily phase of a two-phase polymeric hydrophobic protein ~olution. If alginic acid derivatives are u~e~ as the polymer, this two-pha!ie system does not appear immediately but rather develops over time. As will be evident from the fol}owing e~ample~, the ~evelopment and ~tabilization ~5 of the two-phase system may take ~ieveral ~ays. The ~ame al~nic aci~l ~lerivative-hyarophobic proteln ~olutlon does not pro~ide the same ~ustaine~ release p~operties ~ught ~nl~ss the two-phase system has develope~.

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The following e~ample~ more clearly delaneate ~he ad~antage~ and metho~s use~ in the invention.

Exampl~ 1.

Thi~ E2ample illu~trate~ the solubilization and s~abilization attr~butes o~ the ~wo-phase 8y8tem o~ the invention. ~ovine ~omatotropin (bST~ was added to both neutral salin~ and a 1.4~ ~o~ium alginate (Xelco LV) ~olutions. The bS~ was 10 substantislly insoluble in ~aline, ~t pH 7.4~ In contrast~ a 50 m~ml ~olution was prepared ~elatively aasily in the sodium alginate system. When tbe sodium alginate b~T solution was allowe~ to stand at 4C. for forty hours with nu~ation, a two-phas~
15 system de~eloped. The alginate-rich phase, which was about 90~ uf the volume, had a protein conc~ntration of about 20 mg/ml while the oily protein phase had A
bST concentration of ab~ut 300 mg~ml, showing a pronounced concentratio~ solubilization ~nd 20 stabili2ation effect~

Ex~m~le Zt ~ n this E~ample, the sustained release effect of ~he making microcapsules ~rom the two-phasa is compare~ with usin~ an unsep~rated ~odium alglnate-hy~rophobic protein solution. ~ 1.4~ (w~v) ~o~ium ~lginate (Kelco ~V) ~olution was prepared alnd bovine somatotropin ~bST) wa~ mi~d into the al~inat~
~olution ~t ~ concent~atlon of 50 mg~ml~ As note~
from the reæult~ of E~am~le 1, thi~ i8 a higher ., ,. . ~ ~ . ;. - . , ".
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concentration then cOula be o~tainea without the alginate. One portion of the sodium alginate solution w~s encapsulated immediately, usin~ standard techniques, ~y allowing ~rops of olution to ~all 5 into a 1.~% calc~um chlor;de s~lution, thereby forming gel balls. ~ jet-head droplet forming apparatus consistinq o~ a hous~ng having an upper air intake nozzle and an elongate ~ollow b~dy friction ~itte~ ~nto a ~topper. A ~yringe, e.g., a 10 cc 10 syringe, equippe~ with a stepping pump i~ mounted atop the housing with a needle, e.~ 0.01 inch I.~. Teflon-coatea needle, passing through the length of the housing. The interior of the hous;ng i~
designed such that the tip of th~ needle i~ ~ubjscted 15 to a const~nt laminar air-flow which aots as an air kni~e. In use, the syringe full of the solution containing ~he material to be eneapsulated is mounted atop the housing, and the stepping pump is activat~d to incrementally force drops of the solution to th~
20 top of the needle. Each drop is ~cut off~ by the air stream and falls ~pproximately 2.5-3.5 cm into an encapsulation solution containing 1.2~ CaC12 and O.3~ 80/20 polyornithine/polyglutamic acid copolymer where it is immediately gelled and coated into 25 capsules.

The other portion of the ~odium alginate-bST
solution was hel~ ~t 4cC~ for forty hours whil~
un~er~oing nutation or gentl2 tnlxing. After forty hour~, the ~olution separated into two ~i~tinct 30 phas2s; an oily phase containing most oE the protein and a substantially Ayueous phaæe ~ontaining mo~t of the alginate. Tha entire ~olution containing the .. ... ..

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~eparated phases wa~ u~e~ to make microcapsules, using the same procedur0 as previously describe~. A
protein-rich phase act~ as suspen~ed pockets ~ high prstein concen~ration, forming a vis1ble spinale ctructure. Over time, the ~pinal~ ~tructure disintegrate~, releasing b5T from the capsules.

The two sets of microcap~ules were teste~
for sustained relea~e by injection into Hypo~ ra~s.
The rats' rate o~ ~rowth was measure~ by weighing them every day. The rat~ which recelvea the capsules made from the unseparate~ alginate-bST ~olution had a very rapi~ weight gain ~n day one and two an~ then a neqative or substantially no weigh~ gain therea~ter, showing hat all of th~ bST was released within two days. ThiS is similar to the results or rat~
receiving a single large dosaye injec~ion of bST, which show a high initial weight gain followPd by weight decreases or substantially ~lat weight gain after day two. In contrast, rats which re~eived microcapsules mad~ from the separat~d solution ~how a hi~h 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 resul~s obtained by giving rats daily injections o bST, showing that the single injection o~ the microencapsulated bST w t~d as a reservoir, - yielaing a ~ubstantially continuous stream o bST to the rats.

The results with gel balls rathcr than ~orme~ microcapsule~ were not as ~oo~ but ~till showed better result~ than the ~ingle injection form.

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In thi~ E~ample, capsules made using the procedure previously described, including the nu~ation at 4 ~or forty hours, wese per~used wit~
5 ~ri~ buffer ~o t~st ~ustainea relea~
character~st~cs. ~n vivo test~ng of bST formu}ations ~n Hypos rats, such as i8 descri~ed in ~ample 2, is lim;ted in time duration due to an immune r~sponse by the animal~ after seyen day Thereeore, per~using 10 experiment6 were performed in ord~r to simulate the ia ~i~Q performance of these formulations over a longer time period.

The microcapsules were prepared using a 40 mg~ml bST solution in l.S% Kelco LV sodium 15 alginate. The solution was nutated or appro~imately orty hours a~ 4C~ ~nd formed into microcapsules as described in Example 2. ~ 3% BO/2~
polyornithin~polyglutamic acid copolymer was used for membra~e formation.

~o Three different samples were used in the Example: a control group of capsules which wa~ not perfused before injection, a first test group of mi~rocapsules which were perfused for two days, and a .-~econd test group uf microcapsules which were 25 perfused for five ~ays. Perfu~on was carried out by ~lowing ~ Tri~ bu~fe~, p~ 7.4. at 37C., past the cap~ulas at ~ rata o~ 10 mlJhr.

After per~usion, the control ~nd each of the ta~t s~mple~ were in~ected into Hypo~ rats. Weight ~&~

gain was measured as an indication of bST release rate. Table 1 shows the weiqht gain at ~ay 2, ~ays 2-4, days 4-7, and aa~s 7-10.

era~e weight Qain in qrams Day 2 2-4 4-7 7-10 Control 13.0 7.2 1.7 -1.1 2 days per~usion 12,6 7.3 6.3 -1.7 5 days perfusion 14.3 5.3 4.3 Q.5 As is clear from the results ~hown in Table 1, the samples which have been perfused for two or five days provide essentially identical growth rat~es and, therefore, rele~se rates, as did the unper~uæed control sample. The performance of the partially 15 depleted samples suggest that the capsule formulation is capable of ~elivering bS~ a~ a s~eady state for significant time periods, esceeding seven days.

The foregoing Examples are meant to be non-limiting and are here ~olely for ease in 20 explan~tion of the invention. The invention is defined by the following claims.

What is claimed is:

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Claims (38)

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 comprises 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 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 wherein said polysaccharide comprises an alginic acid derivative.

22. The sustained release vehicle of claim 21 wherein said alginic acid derivative comprises sodium alginate.

23. The sustained release vehicle of claim 21 wherein said protein comprises a hydrophobic protein.

24. The sustained release vehicle of claim 21 wherein said protein comprises a hydrophobic protein.

25. 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 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 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.

29. 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.

31. The sustained release vehicle further 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.

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.

33. A method of producing stable, high concentration aqueous solutions of hydrophobic proteins comprises the steps of:
forming a first aqueous solution of an alginic acid derivative, mixing said hydrophobic protein with said first alginic acid derivative solution, forming an alginic acid derivative-hydrophobic protein solution, allowing two distinct phase 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-phase 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 mannuronic:guluronic 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.