Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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1 BACKGROUND 0~` T~E P~IOR ART
2 FqELD OF ~ INVENTION
3 Thlq lnvention relates to the use of liquid mem~
4 brane technology in preparlng medicinals. The medicinals
prepared by thls invention may be ingested and may be uti-
6 lized as traps ~or toxins present in the GI (gastrointes-
7 tinal) tract, or as slow release compositions of drugs, or
8 as reactors. In the trap embodiment the liquid membrane
9 encapsulated medicinal is an emulsion comprisin~ an ext ~ a
phase which is immiscible with the liquids present in the
11 GI tract and permeable to the toxins therein, and an in-
12 terior phase which comprises a reagent capable of convert-
j 13 ing said toxin to a nonpermeable form. In addition, hydro-
1~ philic adsorbents may be encapsulated such as a hydrophilic
carbon or a silica gel. When the compositions of the in-
16 stant invention are utilized as sloW release drugs, the
17 internal phase o~ the emulsion will comprise a drug which
18 is slightly soluble in the external phase of the emulsion
19 whereby said drug permeates through said exterior phase o~ `
the emulsion over a period of time lnto the GI tract. ~he
21 third method for utillzing the compositions of the instànt
22 invention comprises encapsulating a catalyst for a reaction
,.
23 whlch is desired to be ~arrled out in the~I tract. In
24 this embodiment the reactants present in the ~I tract
~ermeate through the external phase of the~emulsion into
26 an interior phase wherein said catalyst~ for example, an
27 enzyme is immobillzed and are converted to reaction prod-
28 ucts which then may permeate throu~h~the external phase
29 back into the GI tract. In all cases~the 11quid membrane
,
encapsulated mèdicinals may be administered by either oral
31 ingestion or injection anywhere else into the GI tract.
597
SUMMARY OF ~ PRIOR ART
2 It is known in the ~rt that solid microcapsul'es
3 may be utillzed to encapsul~te medlcinal~. For example,
4 in the December 20, 1~71 issue o~ "The Jo~rnal of the
American Medical Associatlon" in the "Medical News Section~', ' ~ '
6 a revlew o~ the microencapsulated medicinal art is presented.
7 In this article, a technique ~or treating uremic wastes in
- 8 the gastrointestinal tract with microencapsulated activated
9 carbon is disclosed. The microcapsule is permeable to the
uremic wastes and said activated carbon ls utilized to ab-
11 sorb some o~-the wastes. In thls technique, uric acid and
12 creatinine are removed. The above reference also teaches
13 a technique wherein urea is converted to ammonia and C02 by
14 the use o~ m1croencapsulated urease. The ammonia is then
reacted with and trapped by a microencapsulated ethylene
16 malelc acid copolymer while the carbon dioxide is exhaled
17 through the lungs.
18 It is known in the ark of slow release medicinals
19 that medicinals can be encapsulated by varlous solid ma-
~20 terials, for example, hydroxy alkyl cellulose ethers, as
' 21 taught in U.S. patent 39493,407, and'gelatin, as taught in
22 U.S. Patent 33526j682. In both o~ these patents, the
23 microencapsulated medicinal is released over a time period
24 into the GI tract by dissolution o~ the solid capsule
material. ~ '''''`' '''' '' ~
26 There are various problems known in the art in ~;
27 using solid microcapsules as reactors~as traps and as~slow
'~ 28 release compositions. One problem is that solid capsules -;
., ... ~ . ~ ,
29 are prone to swell ~ollowed by rupture~and indeed various ~ '
methods to solve thls problem have been utilized,'includ~
31 lng crenation, etc. Thls process increases the cost o~ '
; 32 microencapsulated systems and when long resldence times in
~(~45976
1 the GI tract are encountered, these crenated compounds or
2 compositlons stlll rupture to an undesirable extent.
3 Furthermore, the microencapsules which do not dissolve ln
4 the tract o~ten lead to ~ecal compactlon. In the composi-
5 tions of ~he instant invention, the encapsulating medium is
6 liquid; thus, expanslon of the internRl phase does no~ lead
7 to ruptue of tfie composltIon~as ~n tne solid~microencap-
8 sulated system disclosed above.
9 As pointed out in the patents cited above, when
gelatin is utilized to encapsulate ~edicinals to provide
11 slow release, various conditlons encountered during stor-
12 age can f~ect the rate of release in the GI tract, For
13 exampleg gelatin is very sensitive to temperature and
14 humidity, etc. In the emulsion systems o~ the instant
invention, storage conditions do not substantially a~ect
16 the rate of release of the compositions in the GI tract.
17 U.S. 3,5389216 describes an invention in which --
18 a thixotropic or gela~inous o~l containing a drug ~or
19 sustained release is injected into an anlmal. The instant
invention is quite di~ferent in that a suspendable emul-
21 sion is ingested.
22 SUMMARY OF_THE INVENTION
23 T~e instant invention relates to medicinal com-
24 pounds which comprise a medicinal emulsi~ied in a water
,
~ 25 immiscible external phase. me emulsion is designed to
; ~ ..
~l 26 be stable during pa~sage through the GI tract where said
sl 27 medicinals will be ut~lized. To prepare the composition
r 28 oP the~instant invention,-the medicinal is usually dis-
~` 29 solved in an aqueous ~edium and the solution thereof emul-
sified in an oil which is immiscible with the liquids
~ 31 present in the GI tract. The~ dl phase would generally
!f~ 32 contain~a sur~actant to enable the preparation of~an ~
., ~ : :. . . .
4 - :
~ .
~0~7 ~
1 emulsion which Will be stable dur~ng passage through the
2 GI tract. The'external phase of the emulsion thu~ acts
3 llke a l~quld membrane surroundlne the internal phase.
4 In a preferred embodiment, the emulsion described
5 above is ~urther dispersed in a liquid which is immiscible ~'
6 with the exterior phflse of the emulsion9 ~or exa~ple,
7 water. This preferred embodiment allows the u~e of the
8 compositions o~ the instant invention in a ~orm wherein
9' dispersion o~ the emulsion in the GI tract is increased.
10 Furthermore~ because of the well known unpalatabilit~ of
11 the usual oils which are used to form the emulsions o~
12 the instant inve~tion9 the continuous phase comprising
13 water or water and ~lavorlng agents is desirable.
14 The compositions of the lnstant invention can be
15 utilized in three dl~ferent manners. For examplej to re
16 move toxins9 reactants and adsorbents can be emulsified in
.
'' 17 the interior pha~e o~ an emulsion. The exterior phase o~-
18 th~s emulsion will be designed to allow the toxins present
19 in 'the GI tract to permeate through and react With the
20 reactant or be adsorbed on the adsorbent present in the
, ~
21 internal phase of the emulslon. In t'his manner, toxins
22 are continuously and irre~ersibly removed as the emulsion
23 pa~ses through the GI tract. In this te~ique, the mem-
24 brane is designed to be impermeable to the reaction prod-
25 ucts or adsorbed products formed in the interior phase o~
26 the emulsion.
27 In an alternate method, the liquid membrane is
- . , ~. .
' 28 utilized to encapsulate catalysts which will~be used in
29 carrying out reactions while passing through the GI tract.'
- . :
~ 30 The catalyst may be, for example9 an enzyme9 e.g. urease.
'1 31 Because of the liquid membrane encapsulating the catalysts,
:: . . ; . ,
b 32 the catalyst ltsel~ can be used under conditions where the ~ ;
1~45~7~
i catalyst in an uncapsulated state Would be destroyed. For
2 example, urease could be protected from the loW pH present
3 ln the ~tomach o~ the GI tract by designing the liquid
4 membrane to exclude the passage o~ ions includIng hydrogen
5 ion.
6 In the third use of the compositionB of the in-
7 stant in~ention9 medlcinals are released by perméating
8 through the exterior phase of the emulsion into the GI
9 tract during the passage of the emulsion through the GI
10 tract. In this embodlmentg the medic~nal compound is
11 emulsified in a liquid in which the medicinal is only
12 sparingly soluble. This low solubility in the external
13 phase o~ the emulsion allows passage o~ the medicinal into
14 the GI tract over lon~ time periods.
15 In preparing the compositions of the ~nstant in-
16 vention~ it is desirable to incorporate a sur~actant in
17 the external phase. ThiS surfactant may be present in
18 amounts from .01 to 90% wt. o~ said external phase. Pref-
, .
;~ 19 erably9 the surfacta~t will be present in amounts ~rom 1
to 5 wt. % ~ said extarnal phase. The external phase ~s
~.
, 21 generally made up of the sur~actant and an oil. The oil,
' .
22 o~ course9 is designed to be immiscible with the li~uids
23 present in the ~ tract. A ~urther quali~ication ~or~oils
?
" 24 wh~ch may be utilized in preparing the compositions of the
25 instant invention is that the oils must not be harm~ul to
; , .
26 the human body. These oils along with the surfactant
,~ 27 should also be ~airly inert so that they are not destroyed
28 by the envlronment in the GI tract. ~ ~
29 The body digests many o~ the natural animal and
vegetable oils. These readily digested oils such as tri-
31 glycerides cannot be ll~ed to form a large fraction of the ~
32 oil in the external phase. The natural~ digestive processes
!, .
~ 4~
1 would be expected to rcmo~re these oils f~om the emul~ion
2 as~it pass~d throug~ the G~ trRct~
3 . It is well known ln the art that ~'most artl~iclal
4 or natural emul~ions are broken ln the stomach", See, ~or
5 example, Physiology of ~he Di~estive ~ract, H. ~. Da~enport,
6 3rd Ed. 1g71 Y~ar Book Medical Publishers, Inc~, ChicagoJ
7 I11., page 197. It requires a special type o~ emulsion com-
8 position to pass through the GI tract intact. Some exam-
~ 9 ples o~ oils which can be utilized in form~ng the com
positions o the instant invention include hydrocarbon
11 oils, eOg. paraf~ins, isopara~fins, naphthenes, and aro-
; 12 matics, having molecular weigh~s up to 1,000. Parti-
13 cularly desirable are the mineral oils which have been
14 highly refined for use in human ingestion. Additionally,
oils or trea~ed oils from animal or vegetable sources may
16 be u6ed if they can pass through the GI tract substantial-
17 ly unconverted, for example, vegetable oils and animal
18 fats that are heavily hydrogen~ted so as to contain at least
19 10 wt. % more hydrogen than at normal saturation. Fur~her,
.
20 sillcone Muids containing the repeating unit CH3 can
21 t
22 -Si-0-
2~
2~ CH3
be used. P~r~luorinated hydrocarbons'may also be used.
26 Any o~ these oils should have a ~iscosity o~ 2 to 1000
.
27 cent~stokes at normal body t mperature. The pre~erable
28 range ls 10 to 150 centistokes. `
29 The surfactants must also be harmless to the ~ ;~
human body lf they are to be utiIlzed in the instant in-
31 vention. The specif~c sur~actants which can be used in
.-
i 32 preparing the emulsions above include sorbitan monooleate
.~ . ~ , , .
33 and other types o~ sorbitan`fatty acid esters, e.g. sorbi-
34 tan, sorbitan monolaurat~ sorbltan monopalmitate~ sorbi~n
; ~
,
~ 4 ~
stearate, sorbltan trlste~rate, ~orbitan trioleate; poly-
2 oxyethylene ~orbltan fatty acld esters; and mono- and di-
3 glycerides.
4 It may also be desirable to use strengthening
agents to improve the stability o~ the emulsions. Nonllmit-
6 ing examples of strengthening agents include; polyisobut~
7 i.e. especially the lower molecular weights, e.g. a mol~-
8 cular weight of about 900, polyisobutylene succinic anhy-
9 dride-pentaerythritol adducts, ethylene-vinyl acetate co-
polymers, sulfonated butyl rubber and decylmethacrylate-
11 vinyl pyridine copolymers.
12 EXAMPIE 1
,
13 A controlled releese medic~nal (sodium _allcylabe.
14 In experlment 19 a solut1~n o~ 8 wt. % sodium salicylate
and 8 wt. % sucrose in distilled water was used to form the
16 internal phase of the emulsionO Experiment 2 used 10 wt. %
17 sodium salicylate and 8 wt. % sucrose in water~ but was the
18 same as experiment l in all other respects.
19 These internal phases were added with ~igorous
agitation, in an amount su~c~ent to ~orm 33 wt. % o~ the
21 ~inal emulsion, to an vil phase consisting o~:
22 2.0 wt. ~ Sorbitan monooleate
0.5 wt. % o~ a high molecular weight polyamine
24 with the structure:
2~ CH3 ~ CH3 ~ H ~ 0 H 0
27H-C - C - C - C ' ' " ' ' -'
2~ ~ H , ~ ~ N-(CH2-CH2-N)I~-C-CH3
2390 3~ C~I3~ /~ O
31 H H
32 wherein m is an integer o~ about ~0
3~ 3.5 wt. % o~ fl polylsobutylene~with a molecular
3~ weight of about 900
94.0 wt. ~ o~ an isoparaffinic lubricating oil
36 stock with a viscoslty at 100F of about 100
37 Saybolt Uhiversal seconds.
38 200 grams o~ this emulslon was suspended in 600-grams o~ a
39 synthetic gut ~luid whlch comprised:
o.8 wt. % ~lbumin ~rom eggs
41 0.5 wt. ~ NaCl
' .
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~ o.4 wt. ~ NaHC03
2 98.3 wt. ~ distllled w~ter
3 with mlld agltation to slmul~te conditlon~ ln the small
4 intestine. The appearance o~ sodium salicylate and sucr~
,
In the bulk synthetlc gut fluld were monitored with time
6 by analysis. The resu~ts are shown in Ta~le l below
7 Table 1
.
8 Controlled Release o~ M~dicinals (godium Sali~te)
9 by Diffusion in Liquid Membrane
Concn. in % o~ Max. ~ ~ibrium
11 External Phase3 Concn. in
12 ~ tl) Outer Phase -
1~ Time so~m ~ ~aium
14 H~_ S~ ~ su~se ~ Sucrose
~6 0.0 0.005 0.0 .5
17 80o.83 0.0~0 64.o l.o
18 Ex~t7 ~2
- 19 ~ 0.0 o.oo8 0.0 1.0
20 80 0.59 0.013 45.0 1.6
21 As can be seen ~rom the above table, the sucrose was at
22 least 98 percent contalned over the 80 hour period in both
23 the experiments whlch ind~cates that the emu~sions re-
24 mained substantially lntact. The controlled release of
sodlum salicylate was demonstrated by releasing 64 and 45
26 percent respectively o~ the~maximum possible amounts over ;
-27 the 80 hour period~
28- -The selec.tlon of the internal phase o~ the ~j ;
39 emulsions of the lnstant invention is depe~dent on their
30 intended use. For exampleg toxlns present in the GI tract ~;~
,
31 may be removed by trapping them in ~k.~. ~n~ernal phase of-the
32 emulsion, i.e. conversion o~ a toxin which can permeate
33 the external phase of the emulsionJto an impermeable ~orm.
34 Toxins may also be converted9 ln the internal phase, to an
innocuous form, or alternatively to a ~orm which may be
36 subsequently trapped. An e~ample of this technique is the
37 converslon o~ urea9 by use o~ urease, into carbon dioxide,
~ '.,, . ' ~ :
_ " '
~ 145~76
whlch may be exh~le~, and ammonia, which may be tr~pped by
2 an encapsulated strong ~cid.
3 The varlous toxins which may be removed from the
4 GI tract by trapping in the internal phase o~ the compo~i-
tions o~ the lnstant ~nvention include
6 Table 2
7 -^~ Toxin Removal with Reagents~
8 Encapsulated in Liquid Membranes ~ -`
9 Toxineagents
Ammonia Acid - preferably hydro-
11 chloric, sulfuric -
12 : or citric
13 Phenol Base - preferably sodium
14 : hydroxide
Phosphate Calcium Salts - pre~erably~
16 a combination of
17 calcium chloride
18 and calcium
19 hydroxide
Lactic Acid Base - preferably sodium
21 . . hydroxide
~2 Iron ~ase - pre~erably sodium ~ `
23 hydroxide
24 copper : Sulfide - preferably
sodium sulfide
26 Silver Sulfide - preferably ~ :
~7 sodium sulfide
,
28 Mercury ~ Sulfide - preferably ':
29 . sodium sulfide
Examples of using the instant invention to con-
31 vert materials present in the GI tract into useful prod-
32 ucts ~nclude: '
33 ~ (1) The use of l~quid membrane encapsulated ~ .
34 amylase (an enzyme for the ~hydrolysis of starches for the ~ ~
35 digestion o~ starches), ' ~ : '
36 (2) The use of liquid membrane encapsulate~d
37 11pase (~an enzyme for:the~hydrolysis of trlglycerides : ::~
38 for the digestion of triglyc'er1des)J
.
1~4~976
( 3 ) The use o~ llquld membrane encapsulated
2 lactase to he;lp young children hydrolyze lacto~e, ; .
3 ~4) The us~ o~ liquld membrane encap~ulated
4 mixed pancreatic enzymes to promot~:,the digestlon ~nd utl-
llzation o~ protelns in childre~ with cystic fibrosis.
6 Other examples of using the compositions o~ the .
7 instant invention as reactors,-wherein mater~s-which are : :
8 not capable of being trapped by reaction or adsorption ln : ~
9 the internal phase o~ emulsions o~ the instant invention : .-
are converted into products which can be so trapped or ad-
ll sorbed, include converting glucose to gluconic acid, and,
12 l~ctose to lactic.acid.
13 The compositions o~ the ins.tant invention may be .
14 utili ed as slow release medicinals~to release, for example,
~odium Salicylate, aæ described above, Trimethaphan Camphor~
16 sulfonate, Trimethadione, Metronidazole or Penicillin 0, ~ :
17 particularly the water soluble potassium salt~
l8 In the.preparation o~ products o~ this sort, the .-.
l9 emulsion is designed so that the medicinal is only slightly . ~ ;.
20 soluble in the exter~al phase so as to provide permeation ~.
21 of the medicinal through :the external phase into the GI
:22 tract over a period~of time. In general, emulsions o~
23 this sort are designed so that the medicin~a1l ls solub}e in
24 the external phase.~rom about O.OOOl wt. % to about IO.wt.
% at 37C.
26 In carrying out the process of the instant in- .
.. ..
27 véntionJ the internal phase i9 seledted according to the
28 above crlteria to enable the ekilled artisan to carry
29 out the desired operations. For example,:when it is-desired
30 to provide a composition for the removal of ammonia in the
31 GI~ tract, an internal phase comprising a lO normal aqueous
32 hydrochloric acld solution is~emulsified in a hydrocarbon~
: :
~.: ~~ : . . :
.
1~45~76
1 solution containing a nonlonic sur~actant along w~th a
2 thickener ~or the hydrocarbon phase. ThiS thlckener, as
3 will be ~urther described below, is utilized to prov~de
4 emulslons which do not break during passage to the ~I
tract since it would be quite evident to the skilled
6 art$san that the advant~ges o~ the instant invention will
7 not be obtained wlth emulsions that are not stable durlng
8 passage through the ~I tract. The aqueous and hydrocarbon
9 mixture is emulsified under vigorous agitation to form a
stable emulsion. In this procedure, the aqueous phase is
11 added slowly to the hydrocarbon, sur~actant and thickener
12 solution over a period o~ time to form an-oil continuous
13 emulsion. This emulsion may be passed dirsctly by inges-
1~ t~on through the GI tract; hGwever, in the pre~erred em-
bodiment, thie emuleion will be mixed under conditions of
16 low agitation with water to provide a three-phase system.
17 Thi~ three-phase system may be then ingested and subse-
18 quently passed through thè GI tract. This particular
19 emulsion will pass through the stomach into the intestines
wherein ammonia present therein will permeate through the
21 external phase o~ the e~ulsion, i~e .9 the hydrocarbon con-
22 tinuous phase, into the aqueous ~.ydrochloric ac~d phase
23 wherein~ the ammonia will be converted to ammonium chloride~
24 which is impermeable and thus trapped inthe lnternal phase.
The emulsion, belng stable during passage to the GI tract,
26 then wlll be passed out o~ the human body carrying t~e am-
. .
27 monia trapped in the internal phase along with it.
28 The foLlowing are other speci~ic embodiments of ;
29 the instant invention, however, there is no intention to
be bound b~ these embodiments since variations which would
31 be obvious to the skilled artisan may be made.
: :
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': : : : ' ~. .
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; ' ' ' - . ~ . ~'
EXAMPLE 2 - Ammonia Removal
._.
2 Liquld membrane encapsulation,that ls,utillzing
3 the exterlor phase o~ fln emulsion ~s ~ membrane allows one
4 to use effective ionic reagents such as hydrochlorlc acid,
which cannot be used wlth other encapsulation methods. A
6 hydrocarbon base liquld membrane ls used. The ionic bar-
7 rier character of this membrane prevents the hydrogen and -~
8 chlorine ions of this totally ionized strong acid ~rom
9 penetrating the membrane to the bulk fluid (whlch would be - '
the gut ~luid in this application). me species to be re-
11 movedJ ammonia, always exists in equilibrium with the am--
12 monium ion (NH3 ~ H+ = NH4+). Whlch ~orm 1s dominant
13 depends on the pH. Ammonia, the molecular spe'cles NH3, "~ ' . '
.
14 which exists at gut pHIs9 can readily penetrate the liquid
membrane to contact the hydrochloric acid reagent. At the
16 very low pH o~ the encapsulated hydrochloric acid, the ' '
17 molecular ammoni~ which has moved through the li~uid mem-
18 brane is converted to ammonium (NH~). This ionic species
19 ls prevented ~rom trans~erring back out by the ion barrier
properties of this liquid membrane.
21 The liquid membrane encapsulated hydrochloric
22 acid is shown to be e~fe~cti~e experimen~ally. For any re- ;
. . , ._ . .
23 agent system to be e~fective''in the gut, i~ must remove
2l~ ammonia from the very low cjoncentrations whlch are~ound
in the gut. Tests with liquid membrane encapsulated hy-
26' drochloric acld reduced the ammonia concentration o~ a
27 solution down to less th'an 3 mg %~ i.e. 3 mg per 100 ccls.
28 In addition to removing ammonia to low levels,
29 ~mall reagent volumes are highly desirable. This could be
accomplished by using liquid membrane encapsulated con-
31 ~-centrated, lO~normal, hydrochlorlc acid.~ In this experi-
3? ment, the liquid membrane oil phase~was made ~rom~
3 -
. ' :
' " ', ', ~ . '
1~4Si~76 ` .
1 2 ~m Or Sorbitan monooleate
2 O. 5 gm of a high ~olecular weight
3 polyamlne with the structure
4 CH /CH ~ H O
5 1 3 ' \ ' ~ H O
6H-C . C ~ C - C ~
3 3 \C~ C - C ~ N-(CH2-CH2-N)4-C-CH3
H H
11 4.5 gm of a polyisobutylene with an aver-
12 ~ge molecular weight o~ about 900.
1~ 93.0 gm o~ an i~oparaffinic lubricat- ;
14 ing oil stock with a viscosity at
100F of about 600 Saybolt
16 Universal seconds
17 100 gm of oil phaseJ total
18 To the above 100 gm~. o~ oil pha~e, 50 gm of lON hydro-
19 chloric acid was added in a progression of drops~with
vigorous agitation to form an emulsion. One gram o~ this
21 emulsion was added to 100 gms of dilute ammonia solution
22 in a beaker. The combination was stirred with a propeller
23 at a very slow 50 rpm to give very mild agitation. This
24 agitatlon is probably milder than naturally occurs in the
:
gut. As too mild an agitation can produce slow removal,
,
26 it was a severe test.~ The-very encouraging rapid removal
27 o~ ammonia obtained i8 shown below in Table 3.
28 Table 3
29 Ammonia Removal by Li~uid Membranes
Ammonia Concn.
31 Contact Tlme ln Bulk Phase
32 ~(Hours
33 0 ~ ~ 26
34 ~/2 21
2 ~ 10
36 ~ 24 ~ 6
37~ Note that the ammonla level was~reduced~Prom:26 mg ~ to 10
38 mg % in the ~rst two~hours o~ this gentle contacting~
. . ' .'
97 ~
The level dropped to 6 mg ~ in 24 hQurs. The e~fectiveness
2 o~ ammonia removal was al80 ~ulte encouraging. Based on
3 the ammonia removal achieved in this experiment wlth 1 gm -~
4 o~ liquid membrane encapsulated reagent, the quan,tity re~
quired to remove all o~ the nitrogen from 12 gm/day of urea
~6 was calculated. Only 300 cc of emulsion per day is re- -
7 quired. The use of a liquid membrane suspensionJ i.e. the
8 above described emulsion suspended in an aqueous phaseJ
9 wherein 40 volume percent o~ the emulsion was concentrated
hydrochloric acid, would lower the requirements to 100
11 cc's ~or remo~al of all the urea nitrogen.
12 ~ne liquid membrane must also ~unction in gut ~;
13 fluid. To test this9 a synthetic gut fluid was prepared.
14 me synthetic gut ~luid was made with 0.5 wt. ~ NaCl to
simulate salt concentration, bu~fered with 0.4 wt. %
16 NaHCO~ to hold the proper pH and contained o.8 wt % egg
17 albumin to simulate protein content. The same type of
18 experiments described above were performed. '~he results,
19 below ln Table 4, show quite clearly that the liquid mem-
brane encapsulated hydrochloric acid removes ammonia ~rom
21 synthetic gut fluid.
22 ,T,a~le 4 ~
23 Ammonia Removal_From ,Synthetic Gut Fluid
24 Ammonia Concn. in
Contact Ti.~ ~y,nthetic ~ut F]Uid
2~ ~ ~ (mg %) ~~
27 0 38
28 1 19
29 21~ 20
3Q 48 13
31 Another quite interesting observation was made
32 when contacting the above-descr~bed emulsion with syn-
: ,
33~ thetic gut fluid. ~he stab~lity was enhanced. ThiS may
, ~ , . - ~
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,
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.
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1 be a result of protein adsorption on the suRpended e~ul-
2 sion droplets. The enhanced stabllity in gut ~luld may
3 play an important role ln the in ~ivo emulsion s~ability
4 discu~sed below. ~'
The hydrochloric ~cid reagent could be replaced
6 with citric acid, or any other acld capable of neutrallz-
7 ing ammonia, in the above example.
8 EXAMPLE 3 - Urease Encapsulation
9 The approach discussed above concerned the re- ~
10 moval o~ ammonia which had been generated ~rom urea by the ~' -
11 enzyme urease. Substantial urease activity in the gut has
12 been establlshed by the literature. However,' it'has not
13 been conclusively pro~ed that there is sufflcien~ urease
14 activity to convert all the urea that must be removed each '~
15 day. It might be necessary to in~roduce more urease '~
16 activlty'to the gut. Simple in~ection of'unencapsulated
17 urease would not be likely to be e~ective as the low pH o
18 the stomach would denature much o the enzyme. Therefore the
19 encapsulation of urease was tested in a neutral solution by
an ion excluding liquid membrane. The ion exclusion
.
21 nature of ~he liquid membrane would prevent the hydrogen
22 ions present at the low pH o the s~omach rom penetrating
.... . . . . .
23 the membrane and damaging the urease. The molecular spe-
24 cies~ ureaJ however~ co~ld readil~ trans~er through the mem-
25 brane where it would be hydrolyzed to ammonia and carbon ;
26 dioxide. The carbon dioxide, aga~n a molecular species,
27 could trans~er back out through the membrane. The 9 grams
28 per da~ o~ carbon dioxide produced from 12 gms per~day'o~ ''
29 u~rea could readily be'handled by the lungs.~ The ammonia ''~
- ~ .
produced~by the urease~encapgulated~1n the liquid membrane
31'~-could transfer out through the liquid membrane.` This~oc~
32 curs because the phase'encapsulated ln these-membranes is
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. . .
1~)4597~i
1 near neutrfll. At near neutral pH'S the main species is
2 un-ionlzed ammonia which can trans~er out o~ the ion b~r-
'
3 rier liquid membrane. The ~mmonia le~vlng the encapsul~d
4 urease may then be removed from the gut fluid by the pre- ~;
viously discussed ammoniR trapping.
6 The system described above was experimentally
7 checked for the transfer of reactant and products into and
8 out o~ the urease containing internal phase as well as the
9 activity and e~ective isolation of the urease. A liquid
membrane forming emulslon was made by dissolving o.o46 wt.
11 % urease in water and addlng it dropwise into an oil phase
12 under vigorous agitation. The oil phase consisted o~:-
13 2 wt. % Sorbitan monooleate
14 3 wt. % ~igh molecular weight
polyamlne with the structure
16 C~3 / CH3~ H 0
17 ' l ~ H 0
18 ~C~ C - C - C ~ ' "
21 ~ CH3~ C ~ N-(CH~C~l2-~)4-C C~I3
22 H H 0
23 95 wt. ~ Isoparaf~inic lubricating oil
24 ~ stock with a viscosity at 100F of
2~ about 100 Saybolt Universal Seconds~
26 In the final emulsion9 the weight ratio o~ the urease
27 solution to oil phase was 0~82. TWo ml o~ the above emul-
28 sion was added to 30 ml o~ a solution contalning 0.43
29 Molar Urea, 0 1 Molar NaCl, o~ooo8 Molar phosphate bu~fer
30 and containing O.lU 0~ Clelands reagent. Moderate stir- ~ ;
31 ring was used to disperse the e~ulsion in liquid membrane
32 form. ~he pH o~ this bulk urea containing solution was
33 held at 6.7 ~ 0.05 b~ an automatic titrator whioh neu-
34 tralized the~excess product ammonia~with lO normal HCl.
(At the 6.7 pH one-half o~ the~ammonia produced is in ex~
36 cess~over the quantity forming blcarbonate~with the carbon
- 17 - ~
.
45976
1 dioxide.) In the~e experiments, t~e quantity of HCl re-
2 quired to baL~nce the excess product ammonia wa~ recordcd
3 with t~me. The liquid membrances were removed durlng
4 the experiments and relntroduced at a later time.
Increasing the HCl was required initially,
6 indicating that the enzyme ~atalyzed reaction as well as the
7 tr~nsfer of urea lnto and carbon dioxide and ammonia out
8 of the uresse containing internal phase was occurring.
9 When the emulsions were removed, the reactio~ stopped. This
shows that the enzyme did not penetrate the liquid membrane
11 to ~he bulk phase and that the initial measured reaction
12 rate was that produced by liquid membrane encapsulated
13 urease. Reintroduction of the emulsion started the re-
14 ~ction again. The formatlon o ammonia in these experiments
wa~ also confirmed by independent specific analysis o
16 ammonia built up with time.
17 The ra~es of reaction were abo~t 1/50 of those
18 measured under similar conditions with freshly dissolved
19 urease in the unprotected bulk phase. This is a reasonable
rate and the reduction from bulk phase includes the effects
21 of ~everal factors. The denaturation o~ the enæyme during
22 encapsulation, and any limitations in transerring material
23 through the liquid membrane or inside the encapsulated
24 phase would all decrease the measured urease ac~ivity.
EXAMPLE 4- Phosphate Removal
26 Since the phosphate ion is difficult to remove
27 by hemodialysis an adj~nct method of removal would be
28 particularly useful. The reagent system selected for en
29 capsulatlon is suggested by nature. Excess phosphate in
the body can precipitate with calcium in non-physiologic
~ 18 - ~ -
:
.
: :
1~45976
1 modes. Th~ system selected encapsul~tes calclum salt~ in
2 an anion transferrlng liquid membrane. The catlon calclum
3 is retained in the llquid membrane. The flnlon phosphate
4 transfers through the liquid membrane to react with the
calcium ~orming the calcium phosphate precipitate which i8
6 trapped in the internal emulsion phase.
7 mis ~y~tem was experlmentally tested~using a--15
8 weight percent CaC12 and a 5 weight percent Ca(0~)2 reagent ~ -
9 encapsulated in an anion transporting liquid membrane.
~ne oil phase o~ this emulslon consisted o~
11 95 wt. % Isoparaf~in lubricating oil
12 sto~k with a vlscos~ty at 100F o~
13 about 100 Saybolt Uhi~ersal Seconds
14 2 wt. % Mixture o~ primary and
secondary aminas with a molecular
16 weight range o~ 353 to 393 which
17 has an ion exchange capacity of
18 about 2.7 meg/gm.g e.g. Amberlite
19 IA 2 available ~rom Rohm and Haas
2 wt. ~ Polyamine with a molecular
21 weight o~ about 2000 w~th the
22 s~ructure
23 CH3 CH H 0 ;~
24 ' , 3 ` ~ H 0
2~H-C` - C - , C - C ` ' "
27 ~ 3 ~ CH3 C - C ,- N (C~{2-CH2-~)4-C-~H3
29 ~ H H 0
30 . 1 wt. ~ Sorbitan monooleate
31 The aqueous phase was added to the oil to'~orm 33 wt. % o~
32 the tota~. aqueous plu~ oil pha~e~with vigorous agitation.
33~ m is emulsion (281 gms) was then dlspers~ed in a phosphate
34 solution t500 gm). ~he rapid phosphate removal is shown
in Table 5 below.
5~7~ - :
Table 5
2Rapid Phosphate Removal
3 ` Tlme ~i Phosphate , ,~
4 ~ ',` (wt.
0 0.273
2 0.123
~ 1~ 0 07
9 ~4 0.004
Assuming the removal o~ all the phosphate ion
11 (1/2 gms/day as phosphorous) was desiredJthe quantity of
12 liquid membrane suspension, iOe~ the emulsion suspended in
13 an aqueous phase, requlred can be calculated. Based on
14 the above reagent concentration and the reagent occupying
15 40 volume percent o~ a liquld membrane suspension~ 57 cc ;~
16 would be required per dayO
17 Exar~ e 5 - In V~yo Stability o~ Em_lsions ''
18 Emulsions that are used to treat chronic uremia ,
19 by ingestlon must be stable throughout the gastrointestlnal
tract. As'a critlcal test of stability, high doses o~ a
21 poison were encapsulated in an emulsion to see i~ the ''~ ,
22 stability of the liquid membrane barrler was su~ficient to
23 prevent killing test animals. The poison selected was
24 'sodium cyanide at 10 tlmes the lethal dose (10 X LD 50). ~`
25 The liquid membrane formulatlon was'the same ion excluding ~'
26 ~ormulation whlch was used in remo~ing ammonia ~rom solu~
: '.,
27 tion and synthetic gut ,M Uid. Wistar strain~ albino rats
28 'were used ~or this study. In addition'to the rats used to
29 determine the LD 50 of this popu~ation, three groups o~ 10 ''~
rats were used. One group recelved distilled water en-
31 capsulated in the liquld membrane. A second group re- ~ `
.
32 'ceived 10 times the lethal dose of sodlum cyanide encapsu~
33 lated in the liquid membrane~ The encapsulated aqueous
34 phase was 0.5 wt. ~ sod~um cyanide. This sodium cyanide ~'
~: ., . .................... . : ~ .
.. ~ , . . .... . .
1~45976
1 801ution w~s emul8ified a~ ~ 33 wt. % level in the 8ame
2 oil phase composition a~ u~ual in the ammonia removal
3 examples. Thi8 emulsion was then suspended in an equal
4 volume o~ water prior to administrat~on. In the third
group~ the hydrocarbon solution and the sodium cyanide
6 solution were introduced as ~epara~e liquids so there
7 we~e no liquid membranes. All the materials were admin-
8 istered by oral intubation. The result~ are summ~rized
9 below in Table 60
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1~45976
Additionally, the ra~s administered 10 times the
2 lethal dosage of NaCN in the emulsion were. observed to
3 hAve no slgns of toxiclty or pharmocologlc erfects through-
4 out the test. It was concluded that the emulslons o~ the `~
5 lnstant inventlon have good stability in vlvo. - ~
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