Note: Descriptions are shown in the official language in which they were submitted.
wo 93/11799 Pcr/uss2/os336
212~400
...
SOYBEAN PROTEIN OR HYDROLYZATES IN PHARMACEUTICAL COMPOSITIONS TO PROTECT
BIOA~TIVE PEPTIDES FROM ENZYM~TIC INACTIVATION.
This invention relates to enhancing the bioavailability ot proteolytically labile
therapeutic agents by administering the therapeutic agent in combinaUon with ~
protecting agent comprising a protein, a puritied natur~i protein, a molecular wei~ht
10 tractionated protein, or a p~tially hy~rolyzed protein.
Backaround ot the Invention
PepUde drugs and dn~gs conWning a peptidase labile bond are among the
most promising medicinal agents ot modem times, but their instability in the presence
ot proteolytic enzymes in the gastrointestinal tract and other mucosal tissues usually
15 requires that they be administered parenterally. A)though patients can be tsught to
inject parenterally, there has been a long telt need to develop a non-invasive method
for selt admlnistraUon ot peptide drugs.
Protease hhlbitors and penetration enhancers are means o1ten considered to
circumvent the enzymaffc and penetraUon barrie~ to peptide and protein absorpUon20 frorn mucosal routes ot administraUon. ~ecause of such barriers, the bioavailability ot
pepffde and protein drugs from mucos~l routes is poor.
Non-puenteral adminlstraUon of peptide drugs in parUcular ott~n results in very
low bioavailability because of hydrolysis of the peptides by proteolytic enzymes. For
leuprolide, this ranges from 0.05% followin~ or~l administration to 38% follo~nng vaginal
25 administration; for insulin, ~e oorresponding figures are 0.05% and 18%. Lee, Journai
of Controiled Release, 13, 213 (199~
Examples of proteoly~c enzym~s which inactivate proteolytically-labile
therapeuUc agents ~r~ pepsin, trypsin, chyrnotrypsin, elastase, ~nd carboxypeptidase
in the intesUnal lumen, and the ~minop~pUdasss located on the mucosal surfaoes of
30 the Gl tract, nose, and vagina.
Transport ot intact oligopepUdes aeross adult mamm~lian jejunum has been
demonstrated in vitro and in vivo ~s well as in combination with pepUdas~ inhibitors.
Friedman and Amidon, Pharm~ceutic~l Research 8, no. 1, P. 93, t1991).
Fujii et al; US 4,639,435 (198" daims the use of 1~sopropyl~l~(1,2,3,4-
35 tetrahydron~phthoyloxy)ber~oyll Piper~zine methanesunonate as an inhibitor ofchymotrypsin to be co dosed or~ly or rectally ~hth a chymotrypsin~abile drug (kallikrein
WO 93/11799 PCI`/US92/09336
12~
or caicltonin). The reference also discloses the use of benzoylpiperazine esters for this
purpose. The reference does not describe tne mechanism of these inhibitors.
Cho and Flynn; IntemaUonal Pabnt Application WO-90/03164 (1990) disclose
the use of protease inhibitors in oral forrnulaUon but do not describe the nature of such
5 inhibitors in detail; the only protease inhibitor whlch appeus in the examples is
aprotinin.
Iadron, et al; US 4,579,730 (1986) disdose the use of protease inhibHors in oral~ormulaUon o~ Insulin. Soybean 11our is disclosed as a source ot soybean trypsininhibitor (Bowman~Birk trypshJchyrnotlypsin inhibitor; molecular weight 8000 daltons).
av, d al; Biochem. Ph~rrnacol. 36, 1035 1039 (1987) disclose the use of the
protease inhibitor aprotinin to enhance the or~l absorption o1 proteins.
Losse, et al; East Gemnun Patent DD 252 539 A1 (1987) disclose the use of
epdlon-aminocaproic acid and ap-otinin as protease inhibaors in oral forrnulaUon of
pepttde~.
Lee; J.; Controlled Release 13, 213 æ3 (1990) reviews the use o~_protease
inhtbitors in brmulations o~ peptides tor oral, nasal, buccal, r~ctal, vaginal, pulmona y,
and ocu~r routes.
Certain small pepffdes containh~ up to four amino acids have b~n shown to
enhanoe the bioavailability o~ peptide dnugs.
Hussain, et al, Biochemical and Biophysical Reseai ch CommunicaUons,1 ~, no.
3, 923 (1985) ~uggested ~at nasai ~dminlstraUon of peptid~s may become an
importnnt routQ provid~d that pepffdases in the nasol mucos~ can b~ transienUy
inhtbited via ooadminis~tion ot phannacolo~ically inac~ve pep~dase substr~tes.
Faraj, et al, Joumal of Ph~nnaceutical Sciences 79, no. 8, 698 (1990~ showed
25 U at in ~e presen¢e of the small peptides, L-~yrosyl-L-tyrosine and tri-L-tyrosine methyl
ester, the hydrolysis ot leucine enkaphalin was reduced, suggsstin3 th~t competiUve
InhibiUon of nasal peptidases was caused by these small pepUdes.
Friedman and Amidon, supra, demonstrated that cope~fusion ~f the tripeptide
YGG with enkephalin ~YGGFL) r~suited in increased absorpUon o~YGGFL in a perfused
30 ratintestine.
Hori et al; J. Ph~m. Sc~. 72, 4~439 (1983), disclose the use of various amino-
protected pepWes to protect insulin from degradaUon when injected subcutaneously.
ThewWusedwerebenzyloxycarbonyi~ly-Pro-Leu~ly,benzyloxycarbonyl-Gly-Pro-
WO 93/11799 2 12 5 ~ O O PCl`/US92/09336
Leu, dinitrophenyl-Pro-Leu Gly, and benzyloxycubonyl~ly-Pro. Numerou~
public~Uons disclose enzym~Uc tre~ment of v~etable proteins. An early U.S. Patent
No. by John R. Tumer (2,4~9,208) discloses a pepsin modified whipping agent
eomponent. An alk~llne matcrial such as sodium sulfite, sodium carbonate or sodium
5 hydroxide is used to extract glycinln d ~ pH 6.4-6~8. The ~Iycinin is then precipibted
from the extract (e.g., pH 4.24.6) ad i~ isoebctric pH in which sulfur dioxide may be
uUlked as the adjusUnçl acid. The precipit~ted ~Iycinin product is then modi1ied with
pepsin under temperature and pH condiUons conducive to hydrolysis of protein. Thçllycinin is hydrolyzed with pepdn unUI its w~ter~olubility is increased to 40 S09~.
Simihrly, U.S. Patent No. 2,502,482 by Sdr et al. reports the enzymatic modi1ication of
sllycinin wTth Wsin to produce ~n isolate wherein at leas160% by weight of the pepsin
modified isolde is wder~oluble at ~ pH 5Ø
Puski reports the enzymatic modifying ot soy isolates (precipitated at pH 4.5) ~-
~Ath Aspergillus oryzae in Moditic~lion of function~l Properties of Soy Prot~ins by
Proteolylie Er~yme Tredment (Cereal Chem. 52, pa~es 655~66S (1975)).
Severd publieations dso report usin~ ~line solutions 10 extraet soy proteins.
A publieation by A. K. Smi01 et ad. ~lr. Amerk~n Ghemical Soeiety, Vol. 60, Juno t938,
page~ 1316-1320) repons ~e e3~rac00n ol ~ n meal ~h pH 6.7 water aion~ yields
more protein extraet than an aqueous extraetion in tt~ presenee of neutral s~ts.U.S. Patent No. 4,131,607 by Petit diseloses atwo-stage alkaline extraction. Theextrac~on is initially eonduct~d in U~e presence of ~odium sulphite and magnesium salt
at a pH 7.0 8.5 which is then inere~ed to a pH 10.010.510 completë the extraetion.
Th~ protein extracts are thsn precipitated or curded by adjusting the extract 10 a pH
4.~5.5. A patent issued to Martine2 et al. (U.S. Patent No. 3,579,4~6) similarly25 diseloses a multiple solvent extraetion process.
Summaly ot the Invention
We have found that proteins, pepUdes, purified natural probins, ~nd filterèd,
solvent-extracted, moleeular weight-~fractionated or partblly hydrolyzed proteins
hereina~ter referred to as protecting agents enhance the oral, nasal, recW ~nd vagin~l
30 bioavaibbility of proteolytically~bile ~4~c agents which, in the absence of the
protecting agents, would suf~er-rey~c inacUvation upon attempted oral, nasal, recbl
or vaginal ~dministration.
,~ .
WO 93J1 1799 PCI`~US92/09~36
.~1 25~00
~
Detailed Descri~tion of the Invention
This invention comprises a protectin~ agent and a pharmaceuticaily effective
amount of a proteolylically-labile therapeutic agent, with the proviso that when said
protecting agent is soy tlour said therape~ic a~ent may not be insulin.
In another aspectthis invention comprises a method of enhandn~ bioavailabllity
of a proteolytic~lly-labile the~apeutic a~ent to a mammai or other animai in need of said
therapeutic agent comprising administering ~id therapeutic a~ent in combtnaUon ~th
a bioavailability enhanchg amour~t o~ protecting agent ~hth the proviso that when said
protectin~ agent is soy nour said therapeutic agent may not be insulin.
A proteolytically labile therapeutic agent, which is an active in~redient in a
pharmaceutical composition ot the invention, includes those which possess pepUdobonds in their structure or which, upon ~posure to various proteoly~c enzymes preser~t
in the digesffve tract or nasal or vagin~l mucosa, are inactivated by decomposition,
d~on or other means. When adminlstered orally, nasally, rectally or vaginally,
15 ~rdore, ~ ~*ive U~dc agents cannot be absorbed or cannot produce their
U~c effecls to a sdisfactory extent.
Examples ot such proteolytically labihtherapeuUc ~gents are peptid~s, such ~s
calcitonin, prol~ctin, adrenoconicotropin, thyrotropin, ~rowth homnone, ~onadotropic
hormone, oxytocin, vasopressin, ~astnn, tetra~as1~in, pentagastrin, glucagon, secreffn,
20 p~nin, substance P and gonadotropin. OUlff examples of proteolyU~lly-labile
therapeutic ~gents are luteini~ing releasing honnone, leuprolide, enkephalin, follicle
stimulating hormone, chobcystokinin,~ymopentin, endoUl~lin, n~ur~tensin, inte~feron,
interbukin~, insulin, and insulinotropin. OU~er ex~nples aro U~erapeutic antibodies,
such as those used to treat septic shoek.
As the proteoly~cally~abDe therap~utic agents, U~are may ~Iso be used purNied
extr~cts of natural ori~in and their chemi~l mod fications as well ~s products obtained
by tissue cuitur~ ~nd products obtained by culUvaUng microorganisms or cells rendered
produc~ve by geneUc engineering techniques. The proteolyUcally~abile Ulerapeuticagents may also include synthetic peptides and denvateed syntheUc pep~ides such as
30 Terbkiren(lsopropyl-N-[N~(~orpholine~carbonyl)-L~henylalanin~S~ne~yl cysteino]-
2(R)-hydroxy-3(S)~nino~cyclohexylb~e) (US Patent No. 4,814,342).
Protectin~ agents ot the present invention may be chemic~lly synthesked
proteins and peptidas, natu~l proteins, purified natural protdns, chemically modHied
~ ~ .
WO 93/1 1799 PCr/USg2/09336
2125400
natur~l proteins or putidly hydrolyzed natural protein~, or proteins which have been
tr~ction~ted according to mohcular weight, polarity, or charge, or mixtures thereof.
Natur~l, food~rade proteins or putially hydrolyzed tood~rade prohins are preterred.
The mohcular weight of the protectir~ a~ent should be ~reater than 1000.
The procedures br molecular wei~ht traction~on ot the prote~n~ a~ent may
be varbd to produce any desired molecular wel~ht trac~on of a natural protdn. Solvent
~on may be used to separate proteins accordin~ to molecula wei~ht, pobriqr or
charge. Ereym~tic or chemical hydrolysis of a protein may be hllowed by ~bn
of the desired mohc:ular weight trac~on by ultrahltraffon membranes or dialy~
m~rb~es. Molecubr weight fractionaUon may also be eflected by ~el
chromato~raphy or other means.
Hydrolysi~ ot prote~ or pcptides may be carried out by hed treatm~nt, or by
b~nt with acid or base or cy~nogen bromide or by other chemical means.
Ereylr~ie tr_nl may be ccnhd out with a singh proteolytic enzyme, or with
15 var~ combination~ ot proteolytic enzym~, actin~ concurren~y or sequenUally. Avariely af proteolytic er~ may be u~d, includin~ but not limited to trypdn,
chymotlypsin, eiast~se, carbo~peptidase, uninopeptidase, p~psin, and colb~se.
Fractionation and ~etre~s to prod~lcetho protectin~ a~ents dthis
invention may be applied to a wide variety of protelnaceous materials. Ndurally
20 occurring proteins of anlmal or vegetable origin ue preferred. Such proteinaceous
~tarting materials irclude but are not limited to soy flour, soy protein, wheat gluten,
almond powder, peanut powder, casein, fish protein, ~nd the like.
W~thout intending to be limited thereby, it is believed that the protecUng agents
of this invention function as saaNbid protease inhibitors which thereby enh~noe the
25 bioavailabilit,v of pharmaeeutical agenk that are labile to certain proteases 1hat can
degrade the pharmaceutic~l agents upon oral, nasal, vaginai or rectal administrstion.
Coadministration of these protecting agents with the labile pharmaceuUc~i`agentsresults in (1) competitive occupancy of the degrading proteases by ~d protectingas~ent~, (2) inhibition of proteaso de~radation of the pharmaceutical ~ents r~ul~ in
30 enh~d ab~onption ~ ~c elf~veness, and (3) ultimate mebbolism and
~on ot 1h~ protectin~ 9~.
- In view ot the above proposed m~anism of action it is believed to be desirsble
~ 1O ~ maloh the dissolution rate of the protecting agent to the dissolution rate d the
~: :
,,::
WO 93/11799 2 ~ ~ ~ 4 0 0 PCl`/US92/09336
proteolyUcaily~abile therapeutic agent. Generally, we hsve tound that short dissolution
times of the protec~ng agent are more eftective tor low mohcular weight therapeutic
agents. PepUdes ranging In molecular weight of ~1000 to < 100,000 are preterred, with
a molecubr weight ot ~1000 to ~30,000 being especially preterred.
S The e1tective protectin~ agent fraction must be m~tched to the particubr lability
characteristicc of the proteolytic~lly-labile therapeutic agent. Thus, hr a therapeu~c
agent which possesses aminopeptidase lability, a protecting agent fraction which has
a fast dissolution rate and is etlecth~e against aminopepUdase is preferred. For the
aminopeptidase-labide therapeutic a~ent D Ala-D-Leu-enkephalin (YdAGFdL), a
prefoQred protecting agent is the <30,000 MW traction of pepsin-treated decanted coy
11our, as d monsl~ted in E~wnple 15. In general, br a proteolytically-labile therapeutic
agent ~sing lability to one or more lumenal or mucosal proteases, a prderred
proteding agent is one which improves the systemic bioavailability of the ti erapeutic
~ent when the protecting agent is dosed at a practical toW dosè, as described above.
15 ~P~d plotscbng a~ont fractions, for a particular proteolytically~abile th~c
agont, are obtained by tho decantinç~, fi~on, extraction, hydrolysis, and ske
1hc~or~tion p~s described herein. Preferred protecting agent fractions for a
p~r proteolytically~labile ~c agent are identitied u~ilizing in vit~o and in
vivo p~dures wch as those exemplified herdn.
The ph~rmaceutical composition according to the present invenUon is prefer~bly
adminktered to a marnm~l or other animal in need ot such treatment in sny torm in
which a protecting agent and proteolytically~abile therapeutic agent ar~ ~llowed to
coexist in the ir~testine, ~or exunple, in the torm of tablets, gr~ les er capsules, with
both in~redients provided with an enteric coating either separately or composltely. The
2S composition may also be administered rectally orvaginally in theform of suppositofies
pr~pared by adding both ingredients to a suppository base in ordinary use. Likewise,
the protec'dns agent and proteolytically~abiletherapeutic agent may be dosed together
in ~ nasal spray. Where desirabh, these dosa~e forms may be added with various
pharmaceu~lly acceptable additives such as excipients and emulsifiers.
The dose of the proteolylically bbile therapeutic agent is preferably 0.0001 to
1 'dmes the dose n such substance k administered orally in the prior art. The amount
; - ~ ot the~ pr~ading agent will depend upon the route of administration, the labiliq~ of the
~ ~ ~uac a~ent, and the dose ot the therapeutic agent. For oral, re~tal, and vaginal
WO 93/11799 PCI`/US92/09336
2125~00
~,
administration, the protecting agent will generaily be dosed d about 10-1500 m~. In
the case of orally dosed solutions or suspensions, the protec~n~ agent will be dosed
at 10 mg to ~bout 15 gm. For nas~l ~dministration, the dose ot protectin~ ~gent will
be generally lower, in the range about 1-100 m~.
5Several pharm~ceuffc~l compositions for intestinal absorption ~ccording to thisinvenUon were ev~luated wlth respect to their effectiveness, wilh the resuns given
below.
The tolbwing examples are intended only to hrther illustrab the invention and
~re not intended to limit the scope of the invenUon which is ddined by the ci~ims.
;.~.
EXAMPLE 1 -
Processing of Commci~l Proteins and Fiours.
Fr~c0Onation by SolubilkaUon.
15Soy Flour.
Soy Flour (from Sigma Chem. Co.), 4.5 9 h 135 ml 0.01M pH 7.5 phQsphate
Wer and 15 ml 0.05% Thimeros~l was dirred for 15 minules, sonicated for 10 minutes
and aoit~ted for 25 hours at room temper~ture. The m~tur was oJlowed to ~e, the
u~ drawn otf, oen~ilug-d, a~ tiltered. In this way th- direc~y-sdubb tractbn
20 was obWned tor reeovery and use or tulther processing.
.,
EXAMPLE 2
Processing Ot Commercial Proteins and Flours.
- 25Moleeular Weight Fraetionation by SolubilizaUon
~nd Dialysis. So~ Flour
The procedure of Example 1 was followed to prepare a solution of deeanted and
tiltered soy nour whieh was processed further as follows to aehiew molecular weight
(MW) diserimination. The solubilked fraetion was evaporated to dryness at 55C in a
30 ~ vaeuum oven. The evaporah was dissolved in water and dialyzed in 1000 Moleeular
- Weight Cut-Olf (MWC0) Spectrum dialysis tubing against water over a period of 24
hour~ wlth periodie changes ot diaîysing medium. The retentates were evaporated
s~iving 1.179 ot a ~1000 MW solubile d soy flour f raction .
-
.. . . . .. .. .. .. ..
WO 93/1179921 2 5 ~ O n PCr/USs2/09336
EXAMPLE 3
Processing o~ Commercial Proteins and Flours.
Molecular Weight Fractionaffon by
5SolubilizaUonand Ultratiltration (U~):
lK 30K and 30K-l00K Fraction.
Commercial Soy Flour, Si~ma No. ~9633 (288 ~) was added wi~h sUrrin~ to ~
solution of 8640 mi of an 0.01M pH 7.$ phosphate buffer and 1920 ml ~t 0.1%
10 Thimerosal. The suspension ~s mixed tor ~n add~ionai 15 minutes with a magnetic
sUrrer. The mixture was then sonicat~d tor 10 minutes and then stirred at room
temperature ~or 24 hours. The soluUon wa~ then centrifuged at 2500~000 rpm for 1hour. The supernatant liquTd was separated by ultrafiltraUon using ~1001C Nomlnal
Moleoulu Weight Umit Pellicon (Millipore Corp., Bedford, MA) Membr~ne. The
15 retentate (~lOOK~ was discarded, and the pemleate (~100K) was ~eparatec usin~ a
30K nomin~l moiecular weight Pellioon membran~. Ths second (30K-1001~ was s~ved,and ~e second penneate wss ~ep~rated on a 1K nominal molecul~ wdght P~licon
m~mbrane. The third r~tentat~ ~1 K~ s saved, ~nd ~e third penneate (611~ wa~s
discarded. Th~ 1K 30K~ction wss freeze drbd, ~nd th~ 30K-lOOKfr~c~on was drbd
20 in a v~uum ov~n. Th~ yield of U e 1K 30K ~r~ction (7.33 ~) was 2.5% o~ ffio s~rting
soy flour. The yield of the 30K^100Kfraction (5.25 ~) was 1.8% of ffle ~ns
~MPLE 4
Processing of Commer~ial PrGteins and Flours.
Hydrolysis by P~psin ancl Mobcubr Weight Fractionation
b Diahlsis. Soy~
Soy flour w~s hydrolys~ to lower mol~cular wei~ht (MW) *agments using
pepsin and tha hydrolysate w~s fraction~ted into 1000~500, 3500 6/8K and 6/8K-
30 12/14K MW traetions according to the tollo~nng procedure.
.
Soy flour (Sigma # ~9~633; 6.4 9) and Thim#rosal (50 ppm in fin~l ooncentra-
tion~ werc added to 180 ml of a solution containing pH 1.9 0.2N KCIJO.2 N HCI and
mixed for on~h~lf hour after which Pepsin ~Sigma #P4887; 18.0 mg) was added.
Aliquo~s of 2Q ml each were placed in each of 9 pieces of 12,00014,000 Molecular35 Weight Cut-Off (MWCO) Spec~um dialysis tubing, and were dialyzed ag~inst 55 ml of
pH 1.9 KCI buffer at 37C in ~ shaking water bath. The buffer was changed after 2
WO 93/11799 PCI`/US92/09336
2125400
hours and aner 6 hours and dialysis was continued for 24 hours. Permeates (~121141C)
from each Ume period were combined ~nd evaporated ~t 65C in a vacuum oven.
The 2, ~ and 24 hour s~nples were placed in 1000 MWCO tubing and dialyzed
a~ainst water. The resultin~ re~entates (1K-1V14K) were evaporated at 5SC In
5 vacuum oven, giving a total weight ot 449.5 mg; 418 mg of this material was dissoived
in 30 ml deionked water and placed h h~o pieces of 3500 MWCO dialysis tubing, and
dialyzed against 55 ml water at room temperature for 24 hours. The water was
changed dter 2, 6 and 24 hours dialysis, and the permeates (1 K~.5K) were combined
and evaporated at 55C in a vacuum oven.
The retentates *om each piece of tubing were each placed in 6000/~000 MWCO
tubing and treated as above. Evapor~Uon ot the permeates provided fragments of
3500 6000/8000 MW. The retentates, r~presenting the 6000/8000-12000/ 14000 MW
~rac~on, were also combined and ev~porated. Scheme I summarees the described
trac~onaffon procedure.
SCHEME I
PEPSlN-~RE~rEO
SOY ~LOUR
12~1~K nuco
LYSIS
R TEN T~t PERnEaTE
( >12~1'1K) ~<12~1~1K)
lK nuco
DI~LYSIS
RETENTRTE PERnEQTE
~lK - 12~1~K~ S<lK)
3.5K nu~o
DI~LYSIS
~
RTENT~T PERnEaTE
(3.5K - 12~1~K) (lK - 3.5K)
¦6~8K ~UCO
OlaLYS15
/ \
RETE~TaTE PERnE~TE
(6'8K - 12~1~K) (3.5K - 6~8K)
WO 93/1 1799 PCr/US92/09336
`?,1,25400 , ....
-1~ ~';'
EXAMPLE 5
Processing of Proteins and Flours. Treatment
with immobilized Pepsin.
Soy flour (Sigma Chem. Co.; S-9633) was processed as in Example 1, snd was
dried. This materbl w~s ultra1iltered with a 30K MWC0 membrane. The retentate
(~301~ was collected and dried. 13.8 5 ot this material w~s dissolved in ~00 ml water,
and ff~e pH was adjusted to pH 2.0 with 0.1 N HCI. 1.19 ~ Immobilized pepsin
~immobilized on 4% cross~inked beaded asarose Sigma Chemical Co.; P 3286; 40
units/mg) was added. This suspension, maintained at 37C, was ultrafiltered through
three 30K MWC0 membranes. The permeate (~30K) was collected in 15 minute
Int~als and was freeze dried. The retentate (~301<) was recyded through ultrahltration
processin~. The products of pepsin hydrolysis appear in
the penneate. Table I presents the volume of each permeats *action, and the mass15 of hydrolyz~d peptide in each dried fraction.
T~ble 1. ~leld of pepsin hydrolyzed pepUdes, obtained ~rough the m~thod~ d~scribed
in Example 5.
~_ . _ ~ ~
Collection Volume Mass
r~m~ (m~n) (ml~
600 361.1
_ , . ~ ~
. 300 386.0
_ _ _ _ . . . . ~ ~
300 341.5
60~ ~ 300 . _ _ 361.3 _
275 403.2
, ._ . _
. 90 . 350 39t~.1
_ _ .
105 500 557.8
. _ . ~ . _ _ _
120 600 410.5
l _
.35 550 261.6
, . _ .
WO 93/1 ~799 PC~/US92/09336
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EXAMPLE 6
Processing ot Commercial Proteins ~nd Flours.
Hydrolysis by Sequential Enzyrne Treatments.
TryDsin and Elastase. Soy Flour
Soy flour (600 mg) was dispersed in 20 ml 0.01 M potassium phosphate bulfer,
pH 7.5 with 50 ppm Thimerosal. 2 Mg of trypsin was added and the m xtur~ was
placed in 12/14K MWC0 di~ysis tubing. The mkture was dialysed against 50 ml of
buffer at 37C in a shaking water bath. The buffer w~s ch~nged aner 2 hours ~nd
tO hours ~md the dialysis continued for 24 hours. 50 ppm Thimerosal was addad to the
hour buffer. This procedure was carried out in triplicat~.
The 2, 6 and 24 hour penneate samples for each of the triplicate dialysis
sarnples were combined, absorbance at 280 nm detennined and ff~e samples
ev~porated in a ~5C vacuum oven. Thethr~ evaporation residues were r~const tut~15 wi~ 20 ml deionized water, 50 ppm in Thimerosd.
2.0 mg o~ elasta~e (0.182 ml o~ elastase solution, 11 mg protein/ml) was added
to each and the samples wer~ placed in 12/14K M~VCO dialysis tubin~ s~d di~lysedagainst buffer. The buffer was ehanged after 6 hour~ and dialysis was continued for
24 hours. ~er determining absorba~nce at 280 nm, Ule perme~tes were evaporated ~s
20 described above.
Material so~btained was designated as sequenUally-treated trypsin/elastase soy,
12t14K.
~L~
Enhancement of Terlakiren Or~l ~so~ption ~y
~=OD~O~
Renin anta~onist tripepUde tsrlakiren (200 mg of solid crystalline drug powder
in a hard gel~tin capsule formul2bon) was coadministered to tour tas~ed E~ayle dogs
wiUl an aqueous slurry o~ 1 g of the bst inhibitor in 1~0 ml water. Serum levels of
tripeptlde were measured a 6 time points post~ose: 15 min, 30 min, 1 hr, 2 hr, 3 hr
and 4 hr. Four tasted dogs were used for each study, ~ach serving ~s its own ~n~rol
on ~ preceding week. Serum w~s e~rac~ed with N~utyl chlorida ~ollowed by
incubation with an aqueous solution of chymotFypsin. The degradation product wasassayed, after derivit zation with lluorescamine. ~he fluorescencQ d~tectorwas a ~tos
36 Spectroflow 280. The column was a Waters Novapak C-18. The emission w~vebngU
WO 93/11799 PCI`/US92/Og336
2125~00
-12-
.;
was 380 nm. The mobile phase was 7~:25 water:~cetonltrile and ~ow rde 1~0 mi/min.
The detection limit was 10 n~/ml. Zero-to-four hour sreas under cuNes (AUCs) wercalculated trom the concentraUon-vs-Ume plots tor esch dog using the trapezoidal rule.
Tsble 11 demonstrates that commercially available soy protein (PP 620 trom ~ -
S Protein Technologies Inc.) and a 1-301(trsction ot processed oy llour (p~d as in
Example 3) enhance the oral bioavailabili~y d terlakerin, a chymotrypdn-labile
U~c ~ent.
_
TABLE ll
Area under the plasma level vs.
time cuNe (AUC) for dogs
,~
AUC ~-hr/ml) : .
.
, Dog #
: : .
Fomlula00n ~112 34101 L~ 04094 M~n
~PP620 _ 0~36 --0417
1t ~ S~y ~1~ 0.~20 o.æo 0.078 O.æ6 0.286
~: ~ _
~ ~ ~ ~ol 0.043 o.oe6 0.022 0.106 0.049
-
f~dio: 20.1 16.8 5.3 3.9 11.5
PP620/Control
_ .
Soy (1~ 9.8 12.3 3.~ 3.1 7.2
/Control
~: ~ ~ . ~
EXAMPLE 8
Protedtion from Chymotlypsin De~rad~on
ot Teri~kiren: In Vitro Methodologv
A standard procedure was employed to ~ssess the in vltro inhlbitory potency of
various protdns u~d~proo~ed products thereof vs the chymotrypsin degrad~tion ot
~dr~, as toUcws. ~T:est ~olutbK ot alph~?hymo~h (0.67 x 10~M), terbklren
(O.aO5~ml~ the t~? inhibitor (d conc~ns of ~bout 0.1 to/or 0.5 mg/ml) were
in~ ~pH~6.5 ci~c acW (0.10 M)/disodium phosphate ~0.20M) bulfer at a find
,, ,
" '?:: ~ .
Wo 93/11799 Pcr/US9~/09336
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buffer concentration of 300 mOsm and incubateci 37OC. Samples were tal<en at Umes
0 and then at 5 minute intervals, quenched with HCI to pH 2.0 preparatory to HPLC
analysis. HPLC andysis of terld~iren was carried out using a Wders Resolve 5u C 18
column. The mobile phase was ~ water:acetonitrile (50:50) mixture to which was added
5 1 ml of phosphoric acid per liter. Dda was expressed as % inhibition ot terl~kiren
degrad~tion based on companson with time 0 control vdue, as calculated *om the
following equaUon:
% inhibition = 100 x l1~kJ
where l~",h is the initial degrad~Uon rate of terlakiren in the presence of the protectin~
10 ~gents and ke is the initiad degradaUon rate of terlakiren without protecting agents.
% InhibiUons were detennined on decanted, ultrafiltered soy flour, 1K30K
frac~on, ~s shown in Tdble lll.
TABLE lll
15 Reduction ot chyrnotrypsin - cataiyzed degradation of
terbkiren by the 1-30K frac~on ot dec~nted, ultrahltered ~oy flour
. .~
Inhibitor Inhibdor
Concentration Concentraffon~ ~
(mg/ml) (mg protein/ml) % Inhibition
_ . _ _ . . .
0.5 0.04905 - 75.4
_ . __ _ _
0.25 Q.02453 79.8
. . . _ ~
0.1 0.00981 21.5
_ _
0.05 0.00491 29.3
. ~ _ .
0.01 0.00098 4.8
. . . - _ ~
25 -- Corrected tor proteln content.
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~ MPLE 9
a-Chymotrypsin Hydrolysis of Terlakiren
Methodoloav to D~termine 1~ Values of Inhib~ors
Ki is defined as the Inhib~ion Michaelis-Menton constant - a conventional
5 measure ~f the affinity of ~n inhibitor for the ~ctive ~ite and, henoe, its pohncy as an
inhibltor of the enzyme. ~4 determinaUons c~n be curied out from the Initial
degradation rate dat~ ~cquired ~t several concentr~tions o~ inhibitor at constant
~ubstrate and ereyme concentrations. Initial r~te~ are expressed as millimoles
terlakiren degraded per minute, as shown in Table IV.
.u ,~
TABLE IV
ReducUon of chymotrypsin-catalyzed degradation
.of Terlakirell by 1 K 30K C )ecanted
and Uitrafiltered Soy Flour
_ ~ _
Inhibitor Inhibitor _
Concentr~tion ConcentraUon~ k*
(mg/ml) (mg protein/ml) (m mol/min)
_ . _ __
0.5 0.04905 1.93 X 10~
_ ~ .~ _ _ . .
0.25 0.02453 1.59 x 1~
, _ _ _ _
0.1 0.00981 ~.17 x 10~
_ , _. _ ___ . . _ _ .
0.05 0.~0491 5.56 x ~0~
~ _~ ~
~.01 0.00098 7.48 x 10~
, ,, ~ . - . -
PooleJ control 7.86 x 104
.~
Initial rate loss o~ Terlakiren .
~ Corrected for protein content.
~ ~
AJtema~vely, determination of Ki for a single inhibitor concentration ~single-point
Ki~) can be carried out using the s~andard Michaelis-Menton equation for competitive
inhibi~on. Determination of Ki for multiple concentrations (~multipl~point Ki-) can be
WO 93/1 1799 P~/US92/09336
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.~
carried out using the same relationship, Mting the data to the equation using nonlinear
regression analysis.
EXAMPLE 10
S The dissolution time is an Important factor for the performanee of the protectin~
agents of this invenUon. For the purpose of this disdosure, the reported dissolution
Ume is the time required tor a 0.5 m~/ml slurry of the test ~olid to dissolve completeiy
in a 0.1 M eitrie aeid/0.2M disodium phosphate pH 7 bulfer At room ~emperature rotating
end over end at 8 rpm. ~Isual inspection was used to determine the endpoTnt for
10 eompleb dissolution.
EXAMPLE 11
The 96 protein was d~termined forvarious materials which are protecting ager~ts.The eoneentrations ot earbon, hydrogen, and nitrogen in the sample were determined
15 using a Perlci~Elmer 2400 C, H, and N Elemental Anaîyz~r. Appro)dmately two m~ of
sample was aeeurately weighed ~nd placed into the analyzer. The % nitrogen in 1h~
sample was muttiplied by 6.25 to give the estimate of 96 protein.
EXAMP~E 12 ~;
The ability ot a variety o~ commQr~al snd processed protein fractions to reduce
the degradation ot terlakiren by chymotrypsin was d~termined. Soy nour was trom
Sigma Chem. Co.; almond ~our and peanut ~our were from Pert L~bj; whe~t ~luten
w~s ~rom Total ~ods Corp.
Soy flour ~rom Sigma Chemical Co. is unroasted, and thus oontains ac~va
Bowman-Birk t~ypsin/chymotrypsin inhibitor, which h~s an 8000 MW.
Soy protein (#PP620) trom Protein Technoiogi~s, Inc. is a heat-treated
prep~ration, in which the Bowrnan-Birk trypsin/chymotrypsin inhibitor has b~n
inacffvated. Percent inhibition was determined as described in Example 8. T~ble V
demonstrates 1hat the tested prote~ting agern fractions reduce th~ chymotrypsi~
30 catal~zed degrada~on of terlalclren, A chymotrypsin-sensitive renin inhibitor.
WO 93/11799 PCI`/US92/09336
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,~ _ ~ ~ ... ..
TABLE V
In vitro Inhibition ot Degradation o~ Tel1akiren by
Commercid and Processad Protein~ (0.5 mg/ml)
, _ _
# Inhibition (according to ~ :
Matorial Source/Description Example 8)
1. Soy 1bur, pepsin-treated and dialy~ed (>1K) 87
2. Soy 1bur, pep~in-treated and frac~onated
(Exunpb 4)
1000 3500 MW 24.0
3500 6/8K MW 46.6
6/8K-12/14K MW _ 85.1 _
3. Soy Flour 69.9 _ _
4. Soy nOur, decanted and ultrafiltered
1K~OK MW
75.4 and 93.3 (two prepuations)
30K-100K MW 75.~ and 86.8 (~vo prepuation~)
5. Soy Flour, ~1000 MW dblysi~ 97.6
6. Wh at ~luten, decanted and ultrafinered
tK-3OK MW 95.8
7. Wh-d Giuten 76.8 _
8. Peanut 1bur, dec~nted ~nd ultrafiltered
1K-30K MW ~
~8.6
__ . _ _
9. Il.lnK~nd llour, decanted and ultr~littered
1K-30K MW 37.6
, , _ _ _ _, ~
10. Soy Protein (Protein Tech., Inc.) 86.0
.. ~ ~
EXAMPLE 13
For a variety o~ commercially available protein materials and for processed
protein tr~ctions, the % protein, dissolution Ume, and 1~ we~e deterlTlined (as described
in Examples 11, 1 0, and 9, respec~ively). These da~ are pr~sent~d in Table Vl. These
d~t& demonstrate that degradation-redudng tractions cam be prepared which exhibit
both a low l<i and a short dissolution time.
Soy flour trom Sigma Chemical Co. is unroast~d, and thus contains active
Bowrnan-Birk tlypsin/chyrnotrypsin inhibitor, which has an 8000 MW.
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~ . . ,, . . ~ ~ .
..
TABLE Vl
Dissol~nion Rate and Inhibition Constants of Representative
Commercial ~nd Processed Protdn
_ . . .,_ . , . .'
Dissolution Determined ~:
M~teri~l Source/Description % Protsin rlme IG__ :-
A. Commefci~l Proteins/Flours
1. Casein (Sigma C 5890) 87.94 _ ~ 4 d ys 1 0.240
2 Soy Flour 51.69 ~ 4 days 0.1~
3. Wheat Gluten _ 78.81 4 days_ 0.091 * _
B. Ultraffltered (1 K 30K) Substances
__ . __
1. Soy Flour 9.81 _ 0.1 min 0.00765
2. Almond Flour 10.37 2.3 min 0.0618*
3 Cssein 71.31 1.8 min _
. , . ~ . , ~
4. Peanut Flour 13.19 3.1 min 0.12û~
. _~ ~
5. Whe~t ~;lulen 37.81 3.8 min 0.0060*
, _ _ _ , _ , _ , , .
C. Ultrafilter~d (30K-100!~ Substances ~
, , _ _ _ _ _ .
201. Soy Flour 44.44 2.6 min 0 0029
D. Dialysis, ~1K
_ . . _ __ ~
1 Soy FloLIr 76.5 ~ 4 day~ ~ - U.010
_ _
E U tr~fitbred, Pepsin-Treated (c30K)
~ ~................... ~_
1. Soy FloLIr (Ex. 16) ¦ 8û.1 0.1 min 0.0094
~ . _ .
25 * singl~point Ki estimation (concentration of inhib~or 0.5 mg/ml)
.- - ,, ~ = _ -- -- . . =
EXAMPLE 14
Rabbit Intestinal Brush Border Membrane Vesicle
(BBMV) Enzymatic Dcgraeiation of Cholecystokinin~ (CCK~).
Inhib~ion by Sov Protein Fr~ctions.
R~S 3ejunal brush border membrane vesicles (BBMY) were prepared according
to the method of Kessbr et aJ (Biochem. Biophys. Acta 506 (1978) 136). BBM\~ (25
WO 93/11799 PCI/US92/09336
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micro~m protein) were incubated with CCK-8 (10 miaomolar) in the presence and
absense of protecting agents (0.1S m~/ml) in a totd volume ot 1 ml, at 37C. Samples
were withdrawn at 1 and 3 minutes, were quenched by acetonitrile in an ice bath, and
were assayed for undegraded CCK-8 using a high pertormance liquid chroma~o~raphy5 assay. Dda presented in Tabh Vil demonstrate thd two dfflerent molecular wei~ht
tractions ot processed ~oy protein were active as inhibitors of the degraddion ot CCK-8
by BBMV proteases, presumably aminopepUdases.
TableVil. Degradation of CCK 8 by BBMV protease. Effect ottrac'donated soy protein.
%CCK-8 Remaining
Incubation nme lK-30K MW 30K-10ûK MW
(min) No inhibitor Inhibitor Inhibitor
1 min 429~ 5996 6796 -
~ 3 min 8% 10% 16%-
- : ~ _
:: . ~ ~ _
EXAMPLE 15
~rotection aadnst dearadation by aminoDeDtidase in vivo.
Thepentapeptidénkeph~lin~logu C.~l~lln(YdAGFdL)(1.0
m~g)~ was dlrec~y ileally dosed to chronic~lly ile~lly fishJated rats. Radb~
YdAGFdL (1.12 microaram) was dosed wffll e~ch of tour protccting agents: (1)
20 decanted soy flour, ~1K MW; (2) decanted ~nd ultrahltered soy tlour, 1K-30K MW; (3)
pepsi~treated (FMG ~CTI-MOD) ~oy ~lour, ~30K MW; (4) the potent aminopeptidase
inhibitor amastaUn (posiUve control). Blood was collected from a ~ugular vein cannula
a~ vuious times post~osing, and intact YdAGFdL was quantilied by a radiometric 1hin
layer chromatography (TLC) method ufflizing reverse phase KC-18 TLC pl~tes
25 ~Wh~man Co.). TheTLC plates were developed wiUl 30:701~ropanol/0.1 M phosphate
buff (pH 4.1).
Tabb Vlll presents absolute bioavailabUiUes from various treatments. The
pepcin~t~ (FMC ACTI~MOD) ~oy flour (MW <30K) was particularly effecBv as a
nt againd intestinal aminopeptidases, as evidenced by an almost 11-fold
30 ~ i~ ln % ~bsorbed. The potent aminopeptidase inhibitor amastaUn was dso
lbdiw, d~ng that the bioavailabUity of YdAGFdL is partially limited by
, . , . ~ ,
WO93/117g9 PCl`/US92/09336
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degr~tion by intestin~ aminopeptid~ses. Unro~sted soy tlour offered no protection
to deg~d~tion by intestinal aminopeptidases.
T~ble Vlll .:
l r i , ., ~ ~ ~ , , _
Protectin~ ~gent n % YdAGFdL
Bbavdl~bility
_
S None (control) 16 1.78 :t: 0.4S
Decanted soy tlour;. 4 1.74 ~ 0.6B ..
c1K MW (100 mg) _ . .
Decanted, ultr~tiltered 4 2.76 ~ 1.43
soy 11our (FMC ACTI- . .
MOD); 1K-3OK MW
1~) . ~,.'
Pepdn-treated 4 19.54 :t 13.75
oy1hur, <30K
~W (100 m~) '
b~ ) 6 8.76 :~ 4.47_ ...
~ oor~ol, Si~ma .
Ch~ Co.) .
_
Soy Flour, Unroasted 2 . 1.88 ~ 0.67 .
(Sbm-) 50 m~ . ,
EXAMPLE 16
PreparaUon ot Pepsin-Treated Soy Flour,
Usina Immobileed P~sin
Soy flour (Si~ma Chem. Co.) (36 gm) w~s suspended in 1080 ml w~er and 120
ml of a 0.1% (wh) soluUon of Thimerosol. The suspension was mixed at room
temperature for 24 hr, and cer~trifuged tor 1 hr at 3600 rpm. The supernatant was
ultratinered using a 30K MWCO membrane. The >30K MW *action was collec~ed,
concentr~ted, and~adjusted to pH 2 with 0.5 N HCI. This >30K MW fraction was fed30 into an ACTI-MOO spiral reactor module loaded with 8 gm immobileed pepsin (FMC
Corp., Pinebrook, NJ). The outllow of the ~yme reactor was ultratiltered using a 30K
MWGO m-mb~. The <30K MW *action was saved, and tho ~SOK MW *action was
~*cubted ~h the uynN reactor. Afler 2 hr total enzyme tre~nt and
d~bn, th- toW ~30K MW fraction was saved, and was designded 'Ultr~ltered,
;~ 3S P~Tr~d (c301~ Soy Flour~.
.
~, .
WO 93/1 1799 PCr/US92/Og336
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EXAMPLE 17
InhibiUon of Trypsin - Cd~îyzed De~r~dation --
of Benzovl-Arainin~par~-Nitroanilide In Vitro
The following procedure was employed to ~ssess the in vitro inhibitory potency
5 of a protecting agent of this Inven~on vs U-e ~ypsin catalyzed degradaffon of benzoyl-
ar~inine~ua-ni~roanilide (BAPNA). TesS solutions ot trypsin (1.25 u~/ml, 103 BAEE
units/ml), BAPNA 0.5 mg/ml, and test Inhibitor (O.S m~/ml) were prepued in a pH 8
TRIS (0.048 M) / CaC12 (0.019 M) buffer containins 3.75 uglml of bovine senum albumin
and incubated at 37C. Samples were taken at 5 min~nes and then at 5 minute
10 intemals up to 40 mln~nes and quenched with an equaî volume ot 30% vh acetic acid
preparatory to analysis. Analysis of the decay product of BAPNA hydroîysis,
nitroaniline, was carried out using ~ Perlcin-Elmer Lambda 3B w/~rls
Spectrophotometer. Absorbance of the quenched samples was measured at 410 nm.
Data were expressed as % inhibition of BAPNA degradation based upon comparison
15 with a control, which contained no Inhibitor, as calculated from the follo~nng e~uation:
% inhibition = 10096 x ~1 - (S~,h,Sl,
where, Sj"h is the rate of ch~ngs of absorbance ~ Ume in the presence of inhibnor
and S0 is the rate o~ change of absorbance wiUl Ume in the ab8ence ~f inhibitor.Percent inhibition was determined using filtered soy flou-, 30K - 100K fraction.20 This lot of processed soy flour was p~par~d accordin~ to ~e soh6blleation andultrafiltraffon me~hod described in Example 3. The results arQ giv~ in Table IX ~nd
dQmonstra~e that the tested process~d pro~in fraction reduced the t~ypsin catalyzed
dcgr~daUon of 8APNA.
Tabb IX. Reduction uf trypsin -c~talyzed degradation of ~PNAbythe 30K- 100K
25 traction of processed soy flour.
Table IX
, ~ ~ --. -~ =c:=~ ~5
Inhibitor Inhibitor Protsin
Concentraffon ConcentraUon
, . . _ _ __ . I
(mg/ml) (mb protein/ml) % Inhibition
_ . . I
0.5 0.41 73
. _ ~ -
WO 93/11799 2 1 2 5 ~ O O PCr/US92/Og336 :-
EXAMPL~ 8
Additional *actionation ~nd enzyme treatment F?roc~dures
The procedures for molecular weight tractionaUon of proteins ~nd peptides
S described in Examples 1~ are vuied to produce any desired molecular weight *actton
by appropriate choice of ultr~hltration membranes or di~lysis membranes. Molecular
weight tractionation is dso effected by gel chromatography.
Enzymatic treatment, as exernplitted in Exarnples 4-7, is carried out with asingle
proteoly~ic enzyrne, or with various combinations of proteolytic cnzyrnes, acttng
10 concurrently or sequentially. A variety of proteolytic enzymes are used, including but
not limited to trypsin, chymo~ypsin, elastase, carboxypeptidase, aminopepUdase,
pepsin, and collagenase.
The fracUonaUon and enzymatic treatments of Examples 1~ UQ appOied to a
~dde variety of protein~ceous materials of animsl or vegotable origin. Such
15 proteinaceous startin~ materials include but are not limited to ~oy flour, soy protein,
wheat gluten, almond powder, peanut powder, casein, and fish protein. -~
EXAMPLE 19
Th~ ~bility of a protecting agent to dissolve quickly and to begin acting
20 immedhtely upon being rele~sed in vivo is an imporbnt factor for the performance of
the de~ratior~reducing agents of this invention. For this pUrpOSB, the ability of soy tlour
and o~ SOK-100K ~oy ~our, d~canted and ultrafiitered, to reduce the degrada'don ot
T~rlakir~n in a dynami¢ en~ironment were compued in vit~o. The in ~itro me~hodology
ot Exunple 8 was tollowed ~xcept the test ssluUon contalned only enzyme and
25 Terl~kir~n 3n buffer. Tes~ inhibitor (soy flour or 3~K-100K soy flour, d~anted and
ultrafil~red) was ~dded ~t a concentraffon of 0.01 mg/mi wiUlout additional mWng,
whih ~olution was shaking ~t speed 5 in a water bath (American ScienUfic, model #
YB~i31) ~t 37C. Sampling was at 19 seconds, 1 minute, than 2 minute inteNals unUI
11 minut~s had elapsed, then at 5 minut~ intcrvals 1Or ~ total o146 minutes.
30 Quenehing, HPLC analy~is and data analysis were as in Example 8.
WO g3/1 17g9 PCI'/US92/09336
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. . """"_,.. , .,~"",._ ~ ,
Test Inhibitor k~ %
On1 mg/ml (m mol/min) Inhibition ~:
Soy flour 2.96 x 104 27.1
Soy 11our decanted 80.86 x 10~ 78.8
unrafinered
30K-100K
l l l __
Although the invention has been described with regard to its preferred
embodiments, whi¢h ¢onsUtub ~e best mode presenUy known to the inventors, it
~d bo understood that varbw ¢han~e~ and modifications ~ would bo obvious to
one having ordinary skill in this art may bo m~de without depalting from the scopo !
the invenffon which is defined in the claims.
16
