Note: Descriptions are shown in the official language in which they were submitted.
2 ~
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.
': ;
~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
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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
~&~
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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:
,
