CA2125400A1 - Soybean protein or hydrolyzates in pharmaceutical compositions to protect bioactive peptides from enzymatic inactivation - Google Patents

Abstract

Proteins or peptidic substances, which may be prepared from naturally occurring proteins, enhance the bioavailability of proteolytically-labile therapeutic agents which, in the absence of the protein or peptidic substance would suffer enzymatic inactivation upon administration.

Description

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-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 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.
;.~.

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.
., 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 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 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~ ~';' 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 212~00 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 .;
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 ~: ~ ~ . ~

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 212~400 -1~ .

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.

WO 93/l 1799 PCI'/US92/09336 ,~125~00 ~ 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 2125~00 .~

carried out using the same relationship, Mting the data to the equation using nonlinear regression analysis.

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.

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 2l25400 -16- ' ,~ _ ~ ~ ... ..
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 .. ~ ~

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.

W~ 93/1 1799 Pcr/~ss2/o9336 2125~0~

~ . . ,, . . ~ ~ .
..
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) .- - ,, ~ = _ -- -- . . =

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 2125~00 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%-- : ~ _ :: . ~ ~ _ ~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 212S~OO

-19- , 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~ . , 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 25~00 -2~

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. -~

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 .1.2540 -æ-. . """"_,.. , .,~"",._ ~ , 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 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.

Claims (20)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A pharmaceutical composition comprising a protecting agent and a biologically effective amount of a proteolytically-labile therapeutic agent, with the proviso that when said protecting agent is soy flour, said therapeutic agent may not be insulin.

2. The pharmaceutical composition of claim 1 wherein said protecting agent is selected from a protein, a peptide, a purified natural protein, a molecular weight fractionated protein, a solvent-extracted protein, or a partially hydrolyzed protein.

3. The pharmaceutical composition of claim 1 wherein said protecting agent is a partially hydrolyzed protein which has been hydrolyzed by enzymatic means.

4. The pharmaceutical composition of claim 3 wherein said enzymatic means is trypsin, chymotrypsin, elastase, carboxypeptidase, aminopeptidase, pepsin or collagenase.

5. The pharmaceutical composition of claim 1 wherein said protecting agent is a partially hydrolyzed, molecular weight-fractionated protein which has a molecular weight greater than 1000.

6. The pharmaceutical composition of claim 1 wherein said protecting agent is a partially hydrolyzed, molecular weight-fractionated protein which has a molecular weight range of 1000 to 30000.

7. The pharmaceutical composition of claim 1 wherein aid protecting agent is soy flour, soy protein, wheat gluten, almond flour, peanut flour, casein or fish protein.

8. The pharmaceutical composition of claim 1 wherein said protecting agent is a protein or molecular weight-fractionated, partially hydrolyzed protein which is derived from naturally occurring protein.

9. The pharmaceutical composition of claim 1 wherein said proteolytically-labile therapeutic agent is calcitonin, prolactin, adrenocorticotropin, thyrotropin, growth hormone, gonadotropic hormone, oxytocin, vasopressin, gastrin, tetragastrin, pentagastrin, glucagon, secretin, pancreozymin, substance P, gonadotropin, immunoglobulin, leuprolide, luteinizing hormone releasing hormone, enkephalin, cholecystokinin, follicle stimulating hormone, interferon, interleukin, thymopentin, endothelin, neurotensin, insulin, insulinotropin, or teriakiren.

10. A process for producing a pharmaceutical composition in which bioavailability of a proteolytically-labile tharapeutic agent is enhanced, which comprises admixing the therapeutic agent with a protecting agent in an amount sufficient to enhance the bioavailability of the therapeutic agent, with the proviso that when the protecting agent is soy flour, the therapeutic agent is other than insulin.

11. The process of claim 10, wherein the protecting agent is selected from the group consisting of a protein, a peptide, a purified natural protein, a molecular weight-fractionated protein, a solvent-extracted protein, or a partially hydrolyzed protein.

12. The process of claim 10, wherein the protecting agent is a partially hydrolyzed protein which has been hydrolyzed by enzymatic means.

13. The process of claim 12, wherein the enzymatic means is trypsin, chymotrypsin, elastase, carboxypeptidase, aminopeptidase, pepsin or collagenase.

14. The process of claim 10, wherein the protecting agent is a partially hydrolyzed protein which has a molecular weight greater than 1000.

15. The process of claim 10, wherein the protecting agent is a partially hydrolyzed protein which has a molecular weight range of 1000 to 30000.

16. The process of claim 10, wherein the protecting agent is protein or a partially hydrolyzed protein from soy flour, soy protein, wheat gluten, almond flour, peanut flour, casein or fish protein.

17. The process of claim 10, wherein the protecting agent is protein or a partially hydrolyzed protein which is naturally occurring protein.

18. The process of any one of claims 10 through 17, wherein the proteolytically-labile therapeutic agent is calcitonin, prolactin, adrenocorticotropin, thyrotropin, growth hormone, gonadotropic hormone, oxytocin, vasopressin, gastrin, tetragastrin, pentagastrin, glucagon, secretin, pancreozymin, substance P, gonadotropin, immunoglobulin, fibrinogen, leuprolide, luteinizing hormone releasing hormone, enkephalin, cholecystokinin, follicle stimulating hormone, interferon, interleukin, thymopentin, endothelin, neurotensin, insulin, insulinotropin, or teriakiren.

19. The process of any one of claims 10 through 17, wherein the proteolytically-labile therapeutic agent is teriakiren.

20. The pharmaceutical composition of any one of claims 1 through 8, wherein the proteolytically-labile therapeutic agent is teriakiren.