CA2138146A1 - Proteinoid carriers and methods for preparation and use thereof - Google Patents

Abstract

Improved proteinoid carriers and methods for their preparation and use as oral delivery systems for pharmaceutical agents are described. The proteinoid carriers are soluble within selected pH ranges within the gastrointestinal tract and display enhanced stability towards at least one of photolysis or decomposition over time. The proteinoid carriers are prepared from proteinoids having between 2 and 20 amino acids and having a molecular weight of between about 250 and 2400 daltons.

Description

- WO 93/25583 PCr/llS93/0572 PRCJ~ ID t~l2~ T~12 C: AND Z~ .OL~S
FOR PREPAR~TION AND crsE T~EREOF

This applica~ion is a continuation-in-part of U.S.
application serial no. 07~920,346, filed July 27, 1992, which in 15 turn is a con~inua~ion-in-part of U.S. applica~ion seriai no.
07/898,909, filed June 15, 19~2.

Field of the Invention This invention relates to proteinoids and proteinoid 20 carriers made from them. The proteinoid carriers releasably encapsulate active agents and have extended longer shelf life andjor photostability. Methods for the preparation of such proteinoid carriers are also disclosed.

25 Baclc~round of the Inven~ion The available modes of delivery of pharmaceutical and therapeutic agents often are severely limited by chemical or physical barriers or both, which are imposed by the body. For example, oral dellvery of many such agents would be the route of 30 choice if not for the presence of chemical and physicochemical barriers such as extreme pH in the gut, exposure to powerful digestive enzymes, and impermeability of gastrointestinal membranes ~o the active ingredien~. Among ~he numerous ph~rm~co-logical agents which are Known to be unsuitable for oral 35 ~clmi ni stration are biologically active pep~ides and proteins, such as insulin. These agents are rapidly destroyed in the gut W093/25583 PCT/US93/0~72

2 ~3 8 l 46 2 ~ by acld hydroiysis and/cr by pro~eoly~ic enzymes.
A great deal of research has been devoted to developing effec~ive oral drug dellvery meshods and systems for these vulnerable pharmacological agen~s. The proposed solutions have included :
(a) co-adminisrration of adjuvants (such as resorcl-nols and non-ionic surfactants polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether to lncrease the permeability of the intestinal walls; and (b) co-~mlnlstration of enzymatic inhibitors, such as pancreatic ~rypsin inhibl~or, diisopropylfluorophosphate (DFF) and trasylol to avoid enzymatic degradation.
The use of such substances, in drug delivery systems, is limiced however ei~her because of:
(a) their inherent toxici~y when employed at effec-tive amoun~s;
(b) their failure to protect the active ingredient or promote its absorp~ion;
(c) their adverse interaction with the drug.
Liposomes as drug delivery systems have also beer.
described. They provide a layer of lipid around the encapsulated pharmacological agent. The use of liposomes containing heparin is disclosed in U.S. Patent No. 4,239,754 and several studies have been directed to the use of liposomes contalning insulin;
e.g., Patel et al. (1976j FEBS Letters Vol. 62, page 60 and Hashimoto et al. (1979) Endocrinol. Japan, Vol. 26, page 337.
The use of liposomes, however, is still in the development stage and t~ere are continuing problems, including:
(ai poor stabiiity;
(b) inadequate shelf life;
(c) limited tG low MW (c 30,00Gj cargoes;
(d) difficulty in manufacturing;
(e) adverse interactions wi~h cargoes.
More recently synthetic amino acid polymers or proteinoids, forming microspheres, have been described for 2138146 `
W093/2~583 PCT/US93/0572~

encapsulating pharmaceuticals. For example, U.S. Patent No 4,925,673 (the '673 patentj, the disclosure which is hereby incorporated Dy reference in itS entirety, describes suc~
microsphere constructs as well as methods for their preparatior.
and use. The '673 paten~ also describes microspheres whlcn encapsulate pharmaceutical agen~s for delivery into the gastroin-testinal tract or into the blood.
While the proteinoid microspheres described in the '673 patent are useful for their intended purposes, the physicochemi-cal properties of the proteinoid microspheres, such as lightsensitivity, shelf life and the selectivity of their solubility in various portions of the gastrointestinal tract, could be improved. Additionally, there is a need in the art for micro-spheres that can encapsulate a broader range of active agents such as polar drugs.
The method employed in the '673 patent to prepare proteinoids produces a complex mixture of high molecular weight (MW) (> 1000 daltons) and low MW (c 1000 daltons) peptide-like polymers which are difficult to separate. Moreover, the method produces a small amount of the low MW proteinoids which is the microsphere-forming fraction. Hence, an improved method of preparing of the proteinoids is also desired.
Accordingly, there is a need in the art for improved proteinoid carriers as well as improved methods for their preparation.

Obiects of the Invention It is an object of ~his invention to provide proteino-ids wnich forms proteinoid carriers as a dellvery system wltn enhanced stability towards a. least one of photodegradation and decomposition over time.
It is another object of the invention to provide a proteinoid that forms proteinoid car_iers with more selective solubility under various conditions such as pH.
It is yet another object of the invention to provide W O 93/25583 - PC~r/US93/0572 2 ~ 3 8 1 4 6 4 - pro~einoid carriers encapsulatlng blologlcally active agents which are selectlvely releasable within particular portions of the gastrointestinai tract.
It is a further object of the invention to provide proteinoid carriers which promotes the bioavailability of pharmaceutical agents which otherwise display poor absorption in the gastrointestinal tract.
It is yet a further object of the~invention to provide an improved method for produclng proteinoid carriers having particular characteristics and for improving yield of the desired proteinoid carriers.
It has been found that these objects and other advantages~ which will be apparent from this specification, are achieved by the invention described below.
SummarY of the Invention The present invention relates to improved proteinoid carriers and methods of making and use thereof.
Proteinoids of a MW ranging between about 250 and about 2400 daltons and of defined amino acids are useful in preparing proteinoid carriers with improved stability against photodegrada-tion and/or decomposition. The proteinoids comprise a peptide polymer selected from the group consisting of:
(i) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid;
(ii) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamlne, and aspartic acid; and from at least one third monomer selected from the group consisting of lysine, arginine and ornithine, the proteinoid being a microsphere- and/or microcap-.

- W093/25583 ~ t 3 8 1 ~ 6 PCT/US93/0572~

sule-forming protelnold and belng soluble wl~hln a selected pH
range.
The pro~einoid molecules of the invention contain be~ween about 2 and abou~ 20 amino acid residues, preferably between about 2 and about 8 amino acid residues, and has a molecular weight which ranges be~ween about 250 and about 2400 dal~ons, preferably between about 250 and about 600, and most preferably between about 250 and 400 daltons.
The proteinoid carriers are useful as delivery systems to releasably encapsulate and carry a broad range of cargoes including pharmaceutical agents, dye reagen~s and cosmetic ingredients. In particular, the proteinoid carriers are useful as oral delivery systems of sensitive pharmaceutical agents, whlch normally would not De adminlstrable via the oral route, for selec~ive release at targeted regions of the gastrointestinai tract.

Descri~tion of the Drawin~s Figure 1 illustrates the molecular weight distribution as a function of monomer concentration of poly (Asp.Bz-co-Phe) polymer prepared by the NCA method as described in Example 3.
Figure 2 illustrates the molecular weight distribution of a function of monomer concentration of poly (Asp.Bz) polymer prepared by tne DPPA method as described in Example 5.
Figure 3 illus~rates the effecr of reaction time duration on yields of poly (Asp.Bz) polymer prepared by the DPPA
method as described in Example 5.
Figure 4 illustrates the effect of ~emperature of the molecular weight of poly ~Asp.Bz) polymer prepared by the DPPA
method as described in Example 5.
Figure 5 illustrates the effect of changing the molar ratios of [DPPA]/[M] on the molecular weight of poly (Asp.Bz) polymer by the DPPA method as described in Example 5.
Figure 6 is a photograph of an x-ray film of the - 35 western ;mmlln~hlot analysis, as described in Example 9, of W O 93/25583 PC~r/US93/0572~
21381~6 puriflea murlne mA~ 9BG5 ~2~5, lane l; lmg, lane 2; and 0.25 ~g, lane 3); emp~y proteinoid carrier supernatant after encapsulating process (no mAb~ (lane 4~ emp~y protelnold carrier pellet (lan~
5); proteinoid carrier encapsulated mAb supernatant after encapsulating process (lane 6j; and protelnoid carrier encapsu-lated mA~ pellet. Lane MW contalned standard molecular weight markers. ! '' Figure 7 is a photograph of an~x-ray film of a western immunoblot analysis of samples descri~ed in Example 10.
Flgures 8 (a-c) llustrate the levels of serum proteins which bound to lmmobillzed reovirus type 3 and VLSH under ELISA
conditions as described in Example 11. ~Empty spheres~ refers to ~n1m~1 S orally administered empty proteinoid carriers (no mAb 9BG5!; "mAb spheres~ refers to ~nlmAls orally administered mAb 9BG5 encapsulated proteinoia carriers; "IV" refers to ~nim~l S
intravenously administered unencapsulated mAb 9BG5; and "oral"
refers to ~nim~l S orally administered unencapsulated mAb 9BG5 Flgure 9 show mAb binding under conventional ELISA
procedures using immobilized reovirus type 3 and VLSH proteins with serial dilutions of purified mAb in 0.85 N citrate-0.5~ gum (Figure 9(a)) or phosphate buffered saline (Figure 9 (b)) as described in Example 11.
Figure 10 illustrates levels of erythropoietin (EPO) detected in rat serum taken from rats administered proteinoid carrier encapsulated EPO (15~g EPO/kg body weight) and encapsu-lated EPO (15~g EPO/kg body weight) as described in Example 15.
Figure 11 illustrates EPO serum levels in rats that were administered either erythropoietin (50~g/kg) or encapsulated erythropoietln (50~g/kg) directly into the proximal duodenum as descri~ed in Example 15. Serum erythropoietin levels were determined over time with a erythropoietin enzyme immunoassay kit.
Figure 12 iilustrates EPO serum ievels in rats wno wer~
orally gavaged with eitner encapsulated or unencapsulated erythropoietin (lOO~g/kg) or received a subcutaneous injection of - W O 93/25583 PC~r/US93/0572~

ei~her 2~g/kg or lO~g/kg as described in ExampIe 15. Serum erythropoietin levels were de~ermlned over time with an erythro-pole~ln enzyme lmmunoassay kit.
Figure 13 illustra~es serum calcium changes after oral administration of salmon calci~onin proteinoid carriers (0.25 mg calci~onin/kg body weight) ln cynomolgus monkeys as described in Example 17. The results are expressed as absolute change in serum calcium from baseline values. The data represents means +/- SEM.
** Serum calcium levels significally different from baseline values.
Figure 14 illustrates serum calclum changes following oral administration of salmon calcitonin proteinoid carriers (0.60 mg/kg body weight~ in rats as described in Example 18~ The resul~s are expressed as absolure change in serum calclum from baseline values. The data represents means +/- SEM. **Serum calcium levels significantly different compared to the control group at the corresponding time point.
Figure 5 illustrates serum calcium changes after intraduodenal ~ml nlstration of salmon calcitonin or calcitonin proteinoid carriers (3 ug/kg body weight) in rats as described in Example 18. The results are expressed as absolute change in serum calcium from baseline values. The data represents means +j- SEM.
** Significantly different from the unencapsulated control group at the indicated time points.
Figure 16 illustrates clotting tlmes af~er oral administration of proteinoid carrier encapsulated Factor IX (FIX
sph PO) and IV ~m;n;stration of FIX solution (FIX IV) as described in Example 20.
Figure 17 illustrates clotting times after oral administration of proteinoid carrier encapsulated Factor IX ~FIX
sph PO) and FIX solution (FIX unencap PO) or IV adminis~ration of FIX solution (FIX IV) as described in Example 21.
Figure 18 ~llustrates the percentage of intact alpha-interferon (IFN! rem~lnlng after incubating IFN and IFN prote-inoid carriers in simulated gastric fluid (SGF).

W093/25583 PCT/~IS93/0572 2 ~ 3 8 1 46 8 Figure 15 lllus~a~es ~he percentage of lntac. IFNremaining after lncubatlng IFN and IF~- pro~einoid carriers ln 5.08N HCl.
Figure 20 iiiustrates the percentage of intac~ IFN
remaining after incubatlng IFN and IFN proteinoid carriers in simulated intestinal fluid (SIFj Figure 21 illustrates the clotting times in rats dosed with heparin or pro~elnoid/heparin, both in water. The data represents an average of 6 rats. The data represents means +/-lC SEM.
Figure 22 iliustrates clotting times in rats dosed IDwlth USP heparin or heparin proteinoid carriers, both in citric acid. Each time point is an average of 12 rats. The data represents means +~- SEM.
Figure 23 illustrates clo'ting times in rats dosed orally with heparin-spiked empty proteinoid carriers or heparin protelnoid carriers. Each time polnt is an average of 12 rats.
The data represents means +/- SEM.
Figure 24 illustrates the average titers of rats lmmllnlzed orally with M1 proteinoid carriers versus unencapsulat-ed M1. Only responders in each group were averaged.
Figure 25 illustrates HA-NA titers of rats lmmllnlzed orally with HA-NA micropspheres versus unencapsulated HA-NA.

2~ Detailed DescriPtion of the Invention All patents and literature references cited in this specification are hereby incorpora~ed by reference in ~heir entirety. In case of inconslstencies, the present description, lncluding tne definitlons and interpre~ations, will prevail.
The instant invention arose from ~ne discovery that proteinoids of a MW of between about 250 and about 2400 daltons and of defined aminG acid composition can be obtained by modifying known reactions and selecting starting materials.
These proteinoids form proteinoid carriers with surprisingly enhanced stability against at least one of photodegradation and - W093/2~58~ 2 1 3 8 1 9 6 PCT/US93/0572~

decomposltior over time, In additlon, proteinoi~ carriers prepared from such proceinoids carry a broader range of pharma-ceu~lcal agents, including labile polypeptides such as insulln, alpna-interferon, calcitonin, antigens, e.g. influenza virus M1-protein, and Factor IX and display a selective releasabilitywithin various portions of che gastrointestinal tract, relative co prior art proteinoid m1crospheres The proteinoids of the invention comprise a pepcide polymer selected from the group consisting of:
10(i) peptide polymers made from at least one firsc monomer selected from the group consisting of tyrosine and phenylalanine; and from at least one second monomer selected from the group cons1sting of glutamic acid, pyroglutamic acia, glutamine, and aspartic acid;
15(iii peptide polymers made from ac least one first monomer selected from the group consisting of tyrosine and phenylalanine; at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and from at least one third monomer selected from the group consisting of lysine, arginine and ornithine, the proteinoid being a microsphere- or microcapsule-forming protein-oid and being soluble within a selected pH range.
The proteinoid molecules of the lnvention concain between about 2 and about 20 amino acid residues, preferably between about 2 and about 8 amino acid residues, and have a molecular weight which ranges between 250 and about 2400 daltons, preferably between about 250 and about 600~ and most preferably between about 250 and 400 daltons.
Proteinoid carrlers prepared from the proteinoid molecules, in accordance with the present invention, display a selective solubility at specifi- acidic or basic pH ranges, depending on the choice and amount of the second and third monomers in the proteinoid.
Proteinoid carriers which are selectively soluble under alkaline pH environments, such as those found in the distal W093/25583 PCT/US93/0~72~
21381~6 lQ
portion of the lntestine, are prepared from base-soluble protelnoids. These protelnolds contain, as starting monomers ln the reac~ion mixture, a~ leas~ one second monomer selected from the group consisting of glutamic acid, glutamine, pyrogiutamic acid, and aspartic acid. At a pH ranging between about 7.2 and about 11.0, the base-soiuble proteinoid exists largeiy as the anion and is soluble. At a pH below about 7.0, the proteinoid is largely protonated and insoluble in water.
Similarly, proteinold carriers which are selectively soluble under acidic pH environments, such as the stomach, are prepared from acid-soluble proteinoids. In this case, the proteinoid contain, as startin~ monomers in the proteinoid reaction mixture, at least one second monomer selected from the group consisting of glutamic acia, pyroglutamic acid, glutamine, and aspartic acid and at least one third monomer selected from the group consisting of lysine, arginine, and ornithine. At a pH
ranging between about 1 and about 7, the base-soluble proteinoid exists largely as the cation and is soluble. At a pH above about 7.2, the proteinoid is largely unprotonated and insoluble in water.
The pH and the solubility characteristics of the acid-soluble proteinoid depends largely, but not exclusively, upon the pH and solubilty of the last amino acid added du~ing the syntnesis of the proteinoid. For instance, the incorporation of a basic amino acid, e.g., a third monomer, selected from the group consisting of lysine, arginine and ornithine in the acid-soluble proteinoid will result in the elevation of the pI (pH at the isoelectric point) of the proteinoid.
The proteinoids of the present invention are preparable by a thermal condensation reaction by heating mixtures of the appropriate amino acids under conditions described in the '673 patent. In contrast with the '67~ patent procedures which use as many as eighteen amino acids, mixtures of two to five specific amino acids with at least one selected from each of the aforemen-tioned groups yield proteinoids which form proteinoid carriers ` W O 93/25583 2 1 3 8 1 4 6 PC~r/US93/0572~

wi~h selec~ive solubili~y at part,cular pH ranges and at nlgh ylelds .
In carrying out the thermai condensation reaction, ithas now been discovered that lnclusion of tetramethylene sulfone, an lnert, high boiling, polar solvent, maximizes the yield (>
80~) of low MW proteinoids. Omission of solvent does not produce high yields of low MW proteinoids. Presumably this is due to the poor solubility of the amino acid monomers in these solvents andjor unavoidable side reactions between the monomers and the lG solvent under the reaction conditlons.
In general, indivldual amino acids are added to a reaction flask containing tetramethylene sulfone (sulfolane) whicn has been heated to a temperature ranging between about 130C and about 200C, preferably about 175C to 195C, under an inert atmosphere of argon or nitrogen gas. After each additlon, the solution is stirred for a period of time ranging between about 10 minutes and about 5 hours, depending on the amino acid type and the order of addition.
Upon heating mixtures of amino acids to temperatures of about 190C as described above, a reaction takes place and water, oPla and carbon dioxide are produced as side-products. Water is removed from the reaction as formed and the reaction is terminated when water formation ceases. Thereafter, the proteinoid are precipitated out of the reaction sclution by quenching with excess water, under vigorous stirring. After stirring for a period of about 1 hour, the protelnoids are collected by filtration, washed with water and dried under vacuum.
Chemical condensatlon methods which utilize derivatized 3u amlnG acids are also useful for maklng the proteinoids of the presen~ invention as they permit greater control of molecular weight. Such reactions are general~y conducted at lower reaction temperature and with initiators. In particular! low MW protein-oids produced by the alpha-amino acid N-carboxyanhydride (NCA~
method and the diphenylpnosphoryl azide (DPPA) method (N. Nishi W09 ~ ~ ~ 1 4 6 ~ PCT/US93tO572~
i~
er al ~1991l Makromol. Che~ ~-ol.192, pages 1789-1798j were ~ound t~ form prote~noi~ _ar-lers havlng selected solubillty Wl t~lln a parcicular pH range~
The NCA metnod involves the preparation of N-carboxyan-hydrides of alpha-amino acid esters and their subsequent polymerization, uslns low MW amines as initiators. Tt has been ~iscovered that non-NCA de-lved amino esters, e.g l ~-methyi tyrosine ester, are effectlve inltiators which are stable ana soluble in many organic solvents such as tetranydrofuran (THF).
The use of amino acids as initiators, presumably due to their poor solubility in organlc solvents and their low stability, are not knowr.. The NCA reaction produces a high yield of proteinoids with high purity.
The DPPA method lnvolves the dlrect condensation of '5 benzyl esters of alpha-amino acids in the presence of DPPA and a low MW amine, followed by removal of the protectlve benzyl groups, contained in the proteinoid product, by aikaline hydrolysis. If catalytic hydrogenation is used in place of alkaline hydrolysis, low MW proteinoids of unexpected high purities and yields are obtalned.
Proteinoids prepared by any of the above methods can be used immediately to microencapsulate an active pharmacological agent or the proteinoid can be concentrated or dried by conven-tional means and stored for future use.
The proteinoids o, the invention are purified as follows: crude proteinoids are slurrled with water at room temperature, e.g. 25C. Whiie at this temperature, the pH or the slurry is adjusted to about pH 8 using an aqueous alkaline solution, e.g. 40% sodium hydroxlde and 10~ sodium bicarbonate solutions for an acid-soluble protelnoid. For a base-soluble proteinoid, the slurry is adjusted to an acidic pH with an aqueous acidlc solutlon, e.g. 10% acetic acid solution. The mlxture is then filtered and the fiiter cake washed with a volume of water. The washes and f ~trate are .hen combined and evaporated to dryness in vacuo to afford proteinoids. If 21381~6 W093/25~83 ~ PCT/US93/0572 necessary; th~s process can be repeated untiL proteinoids of a desired puri~y level are oD_ained.
~ f desired, the proteinoid may be further purified by fractionating on a column conta~ning solid supports which include silica gel or alumina, using methanol or propanol as mobile phase; ion exchange resin using water as the mobile phase;
reverse phase column supports using trifluoroacetic acid/acetoni-trile mixtures as mobile phase. The proteinoids may also be purified by extrac~ion with a lower alcohol such as propanol or bu~anol tO remove low molecular weight con~aminants.
Proteinoid carriers are made from purified proteinoids as follows: proteinoids are dissolved in delonized water at a concentration ranging between about 75 and about 200 mg/ml, preferably about 100 mg/mi, at a temperature between about 25 C
and about 60 C, preferably a~out 40 C. Particulates remaining ln the solution may be filtered out by conventional means such as gravity filtration over filter paper.
Thereafter, the proteinoid solution, maintained at a temperature of about 40aC, is mlxed with an aqueous acid solution (also at abou~ 40~C) having an acid concentrat on ranging between about 1 N and about 2 N, preferably about 1.7 N. The resulting mixture is further incubated at 40 C for a period of time effective for microsphere and microcapsule formation as observed by light microscopy. In practicing this invention, the preferred order of addition is addlng the proteinoid solution to the aqueous acid solution.
Suitable acids include any acid which does not (a) adversely effect ~he pro~einoid, e.g., chemical decomposi~lon;
(b) interfere wi~h microsphere or microcapsule formation; (c) interfere with microsphere or microcapsule encapsulation of cargo; and (dj adversely in~eract with the cargo. Preferred acids for use in this invention include acetic acid, citric acid, hydrochloric acid, phospho_ic acid, malic acid and maleic acid.
In practicing the inven~ion, a proteinoid carrier stabilizing additives are preferably incoipora~ed into the W O 93t25583 PC~r/~lS93/05723 2i3a~46 1~
- aquesus acid sclution ~r intc the protelnoid soiutlon, prior ~v ~ne m crosphere or microcapsule formatlon process. The presence of such addltives promotes the s~ability and dispersibility of the proteinoid carriers in solution.
The additives ma~ be employed at a concentratlon ranging between about 0 ~ and 5 ~ (W/V), preferably about Q.5 ~
(W/V). Suitable, but non-limlting,~ examples of stabilizlng additives include gum acacia, gelatin, polyethylene glycol, and polylysine.
Thereafter, the proteinoid carriers may be used immediately or may be stored at 4 C or lyophilized and stored under desiccant at room temperature or below.
Under the aforementioned conditions, the proteinoid molecules forn spherical proteinoid carriers comprising protein-oid microcapsules and protelnoid mlcrospheres of less than 10 micron diameter. As defined hereln, a "microsphere" is spherica homogeneous mesh work structure having no discrete inner chamber.
A "microcapsule~ refers to a spherical structure having a proteinoid wall which forms a hollow or chamber. If the proteinoid carriers are formed in the presence of a soluble material, e.g., a ph~rm~ceutical agent in the aforementioned aqueous acid solution, this materiai is believed to be encapsu-lated within the hollows of cAe microcapsules and confined within the proteinoid wall defined by the spherical structure or entrapped within the matr~x of protelnoid molecules in the microsphere structure. In this way, one can encapsulate or entrap pharmacologically active materials such as peptides, proteins, and polysaccharides as well as charged organic molecules, e.g., quinolones or antimicrobial agents, having poor bioavailability by the oral route. The amount of pharmaceutical ager.t which may be encapsulated or entrapped by the proteinoid Carrler lS dependent on a number of factors which include the concentration of agent in ~he encapsulating solution.
The proteinoid carriers of the invention are pharmaco-l-ogical~y harmless and do not alter the physiological and W O 93/25583 2 1 3 8 1 4 6 PC~r/US93/0572~

biologlcal properties of the actlve agent. Furthermore, the encapsulatlon process does no~ alter the pharmacological properties of the active agent. Whlle any suitable pharmacologi-cal agent can be encapsulated within proteinoid carriers, it is particularly valuable for delivering agents wh ch otherwise would be destroyed or rendered less effective by conditions encountered in the animal body before it reaches its target zone and which are poorly absorbed in the gastrointestinal tract.
The proteinoid carriers of the invention are particu-larly useful for the oral administration of certain pharmacologi-cal agents, e.g., small peptide hormones, which, by themselves, pass slowly or not at all through the gastro-intestinal mucosa and/or are susceptible to chemical cleavage by acids and enzymes in the gastrointestinal tract. Non-limiting examples of such agents include human or bovine growth hormone, interferon and interleukin-II, calcitonin, atrial naturetic factor, antigens, monoclonal antibodies, and Factor IX, a vitamin K-dependent blood coagulation proenzyme.
The choice of a particular proteinoid for use in encapsulating or entrapping a pharmacological agent depends on a number of factors which include:
(1) the acidity or basicity of the agent;
(2) the targeted area for release in the gastrointes-tinal ~ract;

(3) the solubility of the drug at certain pH ranges;

(4) efficiency of encapsulation;

(5) interaction of drug with proteinoid.
For example, proteinoids made from glutam1c acid, aspartic acid, tyrosine, and phenylalanine are especially suitable for encapsulating polysaccharides like heparin.
In addition to selective pH solubility, the particle size of the proteinoid carrier plays an important role ir.
determining release of the active agent in the targeted area of the gastrointestinal tract. Proteinoid carriers having diameters between about ~ 0.1 microns and about 10 m crons, preferably W093/2~58~ PCT/US93/05723 2~381~6 1~
be~ween about 5.0 microns anc abou~ 0. microns, and con~ainlng encapsulated or entrapped active agents are sufficiently small to effec~ively release the a_tlve agent at the targeted area within the gastrointestinal tract. Large proteinoid carriers (>10 microns) tend to be less effective as oral delivery systems.
The size of the prote;noid carriers formed by con~act-ing proteinoids with water or aqueous solution containing ac~ive agents can be controlled by manipuLating a variety of physical or chemical parameters, such as the~pH, osmolarity or salt content ~0 of the encapsulating solution, and the choice of acid used in the encapsulating process.
By tailoring both the solubility characteristics of a proteinoid and the particle sl~e of the proteinoid carriers, active agent bearing proteinoid carriers can be produced from base-soluble proteinoids which are stable in the highly acldlc stomach (normal pH of from about 2 to about 6), but which dissolve in the distal portion of the intestines. Such systems are suitable for oral ~m~n;stration of peptlde hormones, e.g., insulir., and polysaccharides, e.g., heparin, which otherwise would be quickly destroyed in the ~I tract. They also are suitable for protecting the stomach from gastric irritants, such as aspirin. When such aspirin-containing proteinoid carriers are orally ~m; n; stered, they pass through the gastrointestinal mucosa and release the aspirin far more rapidly than conventional enterlcally coated aspirin, whicrn first must traverse ~he stomach and then must enter the bloodstream from the intestine after the enteric coating has dissolved.
It also is possible to produce systems from acid-soluble proteinoids which are stable under weakly basic condi-tions (pH of about 8j, but which release active agent under acidic conditions (pH ~f about 2 to 5). Such systems are suitable for the intravenous administration of pharmacological agents such as caicium regulators and redox carrier systems for dopamine or gamma-aminobutyric acid.
The proteinoid carriers of the invention may be orally W O 93/25583 ~ PC~r/US93/0572~

adminis~ered alone as scl1ds ~n the form of ~ablets, pellets, capsules, and granulates sul~able for suspension ln liquids such as edible oils. Similarly, the proteinoid carriers can be formulated into an orally administrable composition containlng one or more physiologically compatible carriers or excipients.
These compositions may contain conventional ingredients such as gelatin, polyvinylpyrrolidone and fillers such as starch and methyl cellulose.
The proteinoid carriers of the invention may also be administered by injection.
The following examples are illustrative of the invention but are not intended to limit the scope of the invention.
5 Example 1: Preparation of a Base-soluble Proteinoid by a Thermal cQn~n~Ation Reaction 750 ml of tetramethylene sulfone was heated to 190C in an inert nitrogen atmosphere in a 4 liter flask with stirring.
294 g of glutamic acid was added and the mixture was heated for one-half hour. 266 g of aspartic acid was added and the mixture heated as rapidly as possible ~o 190C and held there for 15 minutes. 362 g of tyrosine was added and the mixture heated at 190~ for 3 hours. 330 g of phenylalanine was added and the m1xture heated at 190C for 1.5 hours. The hot melt was then poured into 5 liters of water with vigorous stirring. After stirring for about 1 hour, the mixture was filtered and the filtrate discarded. The cake was reslurried in 5 liters of water, filtered and the cake was again reslurried in 5 liters of water. The pH of the slurry (at 25C) was adjusted to 8 using 40~ sodium hydroxide solution. The mixture was filtered and the cake washea with a small amoun~ of water. The washes and filtrate are combined and evaporated tG dryness ln vacuo to give Glu/Asp/Tyr~Phe proteinoid.
Appendices A, B, and C describe examples of other proteinoids prepared by the thermocondensation method.

W093~2~3 8 1 ~ 6 PCT/US93/0572 Example 2: Preparation of an Acid-soluble Proteinoid by a Thermal Con~ncation Reaction 750 ml of tetramethylene sulfone is heated to 190C in an inert nitrogen atmosphere ln a 4 llter ~lask with st~rring.
294 g of glutamic acid is added and the mixture is heated for one-hal hour. 362 g of tyrosine is added and the mlxture i~
heated at 190C for 3 hours. 330 g of phenylalanine is added and the mixture is heated at 190C for 1.5 hours. 266 g of arginine is added and the mixture is heated for an additional 1.5 hours.
The hot melt is then poured into 5 liters of water with vigorous stirring. After stirring for about 1 hour, the mixture lS
filtered and the filtrate is discarded. The cake is reslurried in 5 liters of water, filtered and the cake is again reslurried in 5 liter~ of water. The pH of the slurry (at 25C) was adjusted tO 5 using 10~ acetic acld solution. The mixture is filtered and the cake is washed with a small amount of water.
The washes and filtrate are combinea and evaporated to dryness ln vacuo to give proteinoid.
Appendices A, B~ and C describe examples of other proteinoids prepared by the thermocondensation method.

Example 3: Preparation of Proteinoids by the NCA Method ~sin~ Amine Initiator This example illustrates the NCA method for preparing copolypeptides consisting of ~sp.Bz, Glu.Bz, Phe, and Tyr components. The NCA monomers of these amino acids were prepared according to the reported method.
The reactions were carried out in tetrahydrofuran (THF) or in dichloromethane using benzylamine ~BzNH2) or 4-methylbenzyl amine (MeBzNH2) as initiator a- room temperature ([M] = 10~). The characterization of the resul~ing copolymers was performed by iH
NMR and GPC. The results ob~alned are listed in Tab'e 1.
As shown in Table 1, proteinoids having Asp and/or Glu as the second monomers and Phe and/or Tyr as the first monomers were obtained in high yield from the polymerization initiated with BzNH2 at the ratio of [M]j[I~ = 5 (No. 2-1 to 2-7).

W O 93/25583 PC~r/~lS93/0572 , 9 The GPC curve (Figure 1~ for poly(Asp.Bz-co-Phe), ~rom which a polydispersity of 1.91 was determined. Similar molecular weight distributions were observed for other copolymers.
Polydispersity lS def1ned hereln as the molecular weight distribution of a sample. The dlstribution is assigned a numerical value derived from ~Ae molecular weight (MW) divided by the molecular number (Mn)A The polydispersity value for a homopolymer is 1 because the molecular weight is equal to the molecular number. Any polymer with a polydispersity value of 1 ls considered to have a very narrow dlstribution. A polymer with polydispersity value of 1.6 to 1.7 is considered to have medium distribution. A polymer with a polydispersity value of 2.0-2.' is considered to have a broad distribution.
The homopolymerization of NCA of Asp.Bz and the copolymerizations of NCAs of Asp.Bz, Glu.Bz, Phe, and Tyr were also carried out using MeBzNH2 as initiator (No. 2-11, 2-15, and 2-16). Similar results were obtalned for reactions initiated by BzNH2 .

W O 93/25583 PC~r/US93/0572 2 ~ 3 8 1 ~ TliELE 1 COPOLYMERIZATION OF NCAs INITIATED WITH ~MT~-~
5STORED AT ROOM TEMPERAT~RE FOR 4 DAYS

POLYM. COMONOMER INITIATOR SOLVENT YIELD Mw NO. COMPOSITION (IM11111) (%) 2-1 Asp-GluPheTyr BzNH2 (5:1) THF 84.1 830 11:1:1:1) 1 O 2-2Asp Phe (1 :1) BzNH2 15:1) THF 70.9 730 2 3Asp Tyr 11 :1) BzNH2 15:11 THF 88.6 1000 2-4 Asp-Tyr 12:1) BzNH2 15:1) THF 89.3 1050 2-5 Glu-Tyr 11:1) BzNH2 15:1) THF 84.9 870 2-6 GluPheTyr BzNH2 15:1) CH2CI2 68.8 790 12:1:1) 2-7 Glu-Phe-Tyr BzNH2 15:1) CH2CI2 53.7 1000 (1:1:1) -2-11 Asp MeBzNH2 15:1) THF 88.3 870 2-15AspGlu-Phe-Tyr MeBzNH2 15:1) THF 76.4 11:1:1:1) 2 16Asp-Glu-Phe-Tyr MeBzNH2 15:1) THF 76.4 630 11:1:1:1) Example 4: Preparation of Proteinoids by the NCA Method ~sinq ~-MethYl TYTosine Ester as Initiator This example illustrates the method of conducting NCA
polymerizatior.s, using ~-methyl tyrosine ester (Tyr.Me) as the initiator. The reaction condi~ions are essentially the same as described in Example 4 except tetrahydrofuan (THF) solvent was used. The results are listed ir Table 2.

W093/2~583 21 3 81 9 B PCT/US93/05723 PROTEINOID ~YNl~SIS BY NCA INITIATED WITH AMINO
ACIDS STORED AT ROOM TEMPERAT~RE FOR 4 DAYS

POLYM. COMONOMER INITIATOR SOLVENT YIELD Mw NO. COMPOSITION (IMIIIII) (%~
28 Asp-Glu-Phe Tyr.Me (1:1) CH2CI2 100 450 11:1:1~
2 9 Asp-Glu-Phe Tyr.Me ~3:1~ CH2CI2 71.4 450 (1:1:1) 2 10 Asp Glu Phe Tyr.Me (5 1~ CH2CI2 68.0 730 ~1:1:1) 2-12 Asp Tyr.Me (1:1) THF 100 460 2-13 GluTyr 11:1) ,~-Ala 12:1) THF 67.4 480 Suc.An (2:1) (reflux) 2-14 Asp Tyr.Me (6:1) THF 91.8 890 2-17 Phe Tyr.Me ~1:1) THF 73.0 ND
2 18 Tyr Tyr.Me ~1:1) THF 65.7 ND
2-19 Phe Tyr.Me ~5:1) THF 78.3 ND
2-20 Tyr Tyr.Me ~5:1) THF 63.3 ND

It was found that the in tiation by Tyr.Me is very fast (No. 2-;7 to 2-2G) and all the NCA has been converted after 2 hours. From GPC data, it was observed that the molecular weight of the polymer increased with increasing ratio of [M]/[Tyr.Me]
and rhe polydispersity is quite narrow. The existence of a Tyr.Me residue in the polymers was conflrmed by 'H NMR spectra.
In conclusion, Tyr.Me is a novel and effective initiator for the polymerization of amino acid NCA's.
Sample No. 2-13 represents a polymerizarion initlatea with ~-alanine and terminaced with succinic anhydride. As ~-alanine is lnsoluble in most organic solvents, the reaction was carried out in refluxing THF. As a result, the polydispersity of W O 93J25583 PC~r/US93/05722 1 3 ~ ~ ~ 6 22 - che polymer obtalned was broaaer ~han that of the polymers ini~ia~ed by Tyr.Me.

Example 5: Preparation of Proteinoids bY the DPPA Method (#l) This is an example of a direct polycondensation of Asp.Bz in the presence of DPPA and triethylamine (TEA) as a base under various polymerization conditions ((a), (b), (c), and (d)).
The molecular weight of the polymers, as well as polydisperslty, was evaluated in each case by GPC. The polymers were character-ized by IR and NMR spec~roscopy.
Asp.Bz was prepared by the esterification of L-aspartic acid as follows: L-aspart;c acid (26.6 g, 0.2 mole? was suspended in 300 ml of freshly distilled benzyl alcohol in a 50C
i5 ml round bot~om flask, followed by addition of 45 ml o concen-trated hydrochloric acid (12N). The mixture was heated up to 60 C under vigorous stirrlng for 30 minutes. Thereafter, the reaction solution cooled to room temperature. Triethyl amlne (about 56 mlj was added ~o neutralize (to a pH of about 7) the solution. The crude product was collec~ed by filtration, washed with ethanol and acetone, dried in vacuo, and crystallized twice from ho~ water. 18 g of product was obtained (~ yield = 44~).
M.pt = 2170C.
Commercial DPPA was used without further purificatlon.
TEA was distillated before use. Solvents for polymerization were purified by conventional methods. The direct polycondensation of Asp.Bz was carried out by stlrring a dimethyl formamide (DMF) solution of the monomer ir. the presence of DPPA and TEA. The mixture was stirred for l h at 0-10C followed by stirring at room temperature for two days. The resulting polymer was precipitated in a large amount of water, collected by filtration, and then dried ln vacuo.

2. Effect of Monomer Concentration Listed ln Tab7e 3 are the results for the pc'ymerlza-tion of Asp.Bz in DMF at room ~emperature for two days.

21381~6 W093/2558~ PCT/US93/05723 Poly(Asp.Bz~s were obtaine~ from these direc~ polycondensations in hish yleld.
The molecular weig~t of the polymers was found to be dependent on the concentration of the monomer [M]. Low molecular weight polymers with broad distribution were obtained from a low [M] (Figure 2, curve A). On the other hand, when [M] was greater than 0.2 g/mL, a polymer with a bimodal molecular weight distribution was obtained (Figure 2, curve B). The lower molecular weight oligomers (-1000) may be due to an intramolecu-lar termination between the tenminal amino and the ~-carboxylic groups. After several reprecipitations from THF/methanol, a polymer with a higher molecular weight (~ = 22,000) and narrow polydispersity (M~/~ = 1.68) was successfully isolated from the polymer mixture prepared at [Mj = lg/mL. The separation was also performed using GPC column with Bio-Beads.

~-L OF T~E MONOMER CO~ KATION ON POLYMERIZATION
20OF Asp.Bz BY DPPA IN DMF AT ROOM TEMPERAT~RE:
[DPPA~/tM~ = 1.3; [TEA~tM~ = 2.3 IMI (glml) YIELD ~%) MnX103'C' MwlMn 0.025 71.5bl 1.4 4.15 0.033 74.7'~; 1.0 3.50 0.05 67.2b' 1.1 5.11 0.10 63.2~' 0.91 3.70 0.20 85.4i~1 16.3 (60.7), 1.84, l.l3 1.0 139.3) 0.50 86.5~' 11.0 159.4), 2.22, 1.08 0.92 (40.6) 1.0 97.6~i 15.1 ~71.4, 1.81, 1.05 0.88 ~28.6~

a) The polymer was collected by centrifugation after polymerization for 2 days;

W093/25~83 PCT/US93/0572~
?~38~4~ 24 - ~ The polymer was coliected by filtratlon after poiymer-izatlor ror 2~5 daysO
c) The values in paren~neses are molar ~ercentages.
5~ .
b. Effect of Reaction Time and Temperature The yield of the resulting polymer increased with the reaction ~ime: 75~ conversion .n 5 h and 95~ in 4 days (Figure 3, curve A,. The molecular weight of the resulting polymer also increased with time in ~he inirial phase (up ro 4 h) and then became almost constant (Figure 4j. The polymerization decreased with increasing temperature (Figure 3, curve B). Polymers obtalnea at 60 and 80C were of yellow color and insoluble in THF
but soluble in DMF and DMSG. This may be due to the formation of an imide ring which has been reported to occur durlng thermal polycondensations of aspartic acid.

c. Effect of Molar Ratios [DPPA]/[M] and [TEA]/[M]
The dependence of the yield and the molecular weight of the polymer on the molar ratios of [DPPA]/[M], as well as [TEA]/[M], was investigated (Table 4). The highest yield was obtained at a [DPPA]/[M] of 1.3 and a [TEA]/[M] of 2.3 (Figure 5). These observations are in agreement with the results reported by Nishi et al. Higher molecular weiynt products were obtained in the range of [DPPA;/~M] = ;.3-2.C and ~TEA3/~M] =
2.0-3.0, respectively~

W093/25~83 2 I 3 814 6 PCT/~S93/0572~

EFFECT OF T~E ~OT~ RATIOS OF DPPA AND TEA ON POLYMERIZATION
OF Asp.Bz IN DMF AT ROOM TEMPERATURE: [M~ = O.50 g/ml IMIIDPPA~MIIITEA] YIELD MnX103'A' MwlMn (%) 0.5 2.3 16.3 0.81 4.09 1.0 2.3 69.63.1 (45.4), 0.39 154.6) 2.58, 1.48 1.3 2.3 86.511.0 (59.4), 0.92 ~40.6)2.22, 1.08 10 1.5 2.3 69.415.9 134.2), 0.83 165.8)1.77, 1.21 2.0 2.3 64.313.1 158.3), 0.89 (41.7)1.87, 1.09 1.3 1.5 58.46.0 (39.3), 0.63 (60.7) 2.43, 1.37 1.3 2.0 78.313.3 (64.3), 0.92 (35.7)1.87, 1.19 1.3 3.0 74.613.6 (64.8), 0.83 (35.2)1.98, 1.18 15 1.3 3.5 65.08.3 (60.0), 0.80 (40.0) 2.70, 1.10 a) The value in parentheses are molar percentage.

d. Effect of Solvent A comparison of the polymerizations in different solvents is shown in Table 5. It can be seen from this table that the yield and the molecular weight of the polymer are infiuenced by the solvents used. Higher yieids were obtained in DMF while higher molecular weights were obtained in THF and in bulk. On the other hand, the polymerization in dioxane gave a lower molecular weight product, and cherefore is preferred.

W O 93/25583 PC~r/US93/05723 2~3~ 4 TliBLE 5 EFFECT OF T~E SOLVENTS ON POLYMERIZATION OF
Asp.Bz AT ROOM TEMPERATURE FOR 2 DAYS
5[M]/[DPPA] = 1.3, [M]/[TEA] = 2.3, [M] = O.50 g/ml SOLVENT YIELD 1%) MnXto3lb~ MwlM~
DMF 86.511.0 (59.4), 0.92 140.6) 2.22, 1.08 DMSO 70.611.5 (78.9~, 1.05 (21.1~ 1.87, 1.13 THF 49.929.6 (74.6, 1.14 (25.4) 1.31, 1.13 ACETONITRILE 71.120.3 (79.3), 1.05 (20.7) 1.65, 1.14 DIOXANE 70.54.7 168.5), 0.82 131.5) 3.80, 1.13 NONE'i 61.229.8 (82.8), 0.86 (17.2) 1.32, 1.16 a, Bulk polymerizatlon.

b) The value in parentheses are molar percentage.

Example 6: Preparation of Proteinoids by the DPPA Method (#2) Copolymerizations of Asp.Bz with other amino acid monomers such as ~-benzyl glutamate (Glu.Bz), ~-alanine (Ala), Phenylalanine (Phe), and O-benzyl tyrosine !Tyr.OBz) in the presence of DPPA were carried ou. using the same procedure as that for the homopolymerization of Asp.Bz (Example 5~. Random copoly(amino acids) were obtained in high yield ~> 77~) as shown in Table 6. This indicates that the copolymerization of amino acids using DPPA is a useful approach to copolypeptide synthesis.
~_modal molecular weight distributions were also observed ir, ihese cases similarly to the homopolymerization of Asp.Bz.

W093/25583 2 1 3 8 1 4 6 PCT/US93/0572~

COPOLYMERIZATION OF ~-AMINO ACIDS IN THE PRESENCE OF
DPPA AS CONDENSING AGENT IN DMF AT ROOM TEMPERAT~RE FOR 2 DAYS

POLYM. COMONOMER YIELD Mw MwlMn NO. COMPOSITION (%~
Co.lDPPAAsp.Bz-Glu.Bz 97.415900, 1080 1.76, 1.13 10Co.2DPPAAsp.Bz-~ Ala 91.2 1590 1.18 (1:1) Co.3DPPA Asp.Bz-Phe 89.713700, 800 1.89 1.25 (1:1) Co.4DPPAAsp.Bz-Tyr.OB~ 87.39000, 1000 1.78, 1.17 (1:1) Co.5DPPAAsp.Bz-Glu.Bz-Phe-Tyr.OBz 92.516800, 960 1.66, 1.14 (1:1:1:1) Example 7: Reductive Debenzylation of Proteinoids Produced bY the DPPA Method The example illustrates a preferred method for the removal of benzyl protective groups in poly(Asp.Bz) and poly(Glu.Bz) by catalytic hydrogenation.
The hydrogenation of the polymers was carried out according to the following procedure: To a solution of the polymer in THF/methanol (1:1, v/v`, Pd on active carbon (10~) was added in the amount of 1/10 of the polymer weight. After the replacement of air by nitrogen, hydrogen gas was introduced into the system and maintained with ~ balloon. The reaction mixture was stirred at room temperature overnight. After removing the catalyst by filtration and concentrating the solution, the mixture was poured into a large amount of petroleum ether to precipitate the polymer. The polymer obtained was then dried ln - 30 vacuo.
The completion of the hydrogenation was confirmed by 'H
NMR of the polymer. In most cases, useful water-soluble polymers W O 93t25583 P~r/US93/05723 2&
~3 ~ere produced. The hydrogenation ls an effective and clean ~ procedure for benzyl group removal.

Example 8: Preparation of Empty Proteinoid carriers with Glu, As~, TYr, Phe Proteinoid This Example illustrates a method for the preparation and cleaning of empty proteinoid carriers.

PRG~u~E
1. Reagents:
a. Proteinoid powder prepared as described in Example 1 b. Anhydrous citric acid (USP) c Gum acacia NF
d. Deionized water e. Glatial acetic acid 2. Equipment:
a. Ph meter b. Water bath, 40C
3. Preparation of Solutions:
a. Proteinoid solution - Dissolve lOOmg proteinoid in lml deionlzed water (or multiples thereof). Filter through a Whatman #' filter paper (if necessary) and keep at 40C in a water bath. This is solution A.

b. 1.7 N citric acid with 0.5~ acacia - Dissolve 5g of acacia and lO9g of ci.ric acid in 1 liter delonized water. Incubate at 40C. This is solution B.
4. Preparation of Proteinoid carriers:
a. Add all of solution A to solutlon B rapidly in one step while swirling solution B by hand, in a 40C water batr,.

W093/25583 2 1 3 8 1 1 6 PCT/~S93/0~72~

Example 9: Preparation of ~urine IgG Mo~oclonal Antibody-.. cont~; n ~ n~ Proteinoid Carrier This experiment describes encapsulati~jon of anti-reovirus monoclonal antibody ~mAb) 9BG5, an mAb directed against the slgma-1 gene product ~Hemaglutinin, HA3) of the Reovirus ffl e 3. HA3 binds to the cell surface receptor for Reovirus type 3, - and mAb 9GB5 interferes with viral binding to the receptor.
Mouse IgG monoclonal ancibody 9BG5 was prepared and puriried as described W.V. Williams et al. (1991) J. Biol. Chem., Vol. 266(8), pages 5182-5190, as well as references cited therein, using a purified Reovirus type 3 preparation (W.V.
Williams et al. (1988) Proc. Natl. Acad. Sci. U.S.A, Vol. 85, pages 6488-6492~. The purified 9BG5 used in this Example had a prote1n concentration of 1O5 mg/ml in phosphate buffered saline ~pH 7.2).
Proteinoid carriers encapsulatin~ mAb 9BG5 were prepared having final concentrations of Glu/Asp/Tyr/Phe protein-oid ~ 1:1 mole ratio of Glu, Asp,Tyr, and Phe in the reaction mixture) 50 mg/ml, mAb 0.7 mg/ml and gum arabic 0.5~ in 0.85 N
citric acid. Empty proteinoid carriers were prepared to contain the same final concentrations, except mAb was omitted. Aliquots (0.5 ml), in duplicate, of both mAb and empty proteinoid carriers preparations were centrifuged at 5000 RPM. Pellets and superna-tants were frozen prior to anaIysis by Western blotting to determine antibody encapsulation efficlency.
Figure 6 is an x-ray film of a western blot analysis of purified mAb 9BG5, empty proteinoid carriers (no mAb added), and proceinoid carriers containing 9BG5. The analysis was done by immunoblotting with anti-mouse IgG which specifically reacted wl~h mAb 9BG5. The lanes correspond co the following:
Lane Sample 1 2 ~g 9BG5 mAb 2 1 ~g 9BG5 3 0.25 ~g 9BG5 3i MW molecular weig;~ markers 4 Empty procelnoia carrier supernatant after encapsula-tion Empty proteinoid carrier pellet W093/255~3 PCT/US93/0572~
?,~3~46 3~

6 mAb contalning supernaranr after encapsulatlor

7 mAb containlng protein carrier peller The data indica~es tha~ the 9BG5 proceinoid carrlers contained abou~ 40~ of tne mAb ln the pellet and the remaining 60~ did not incorporate ln the proteinoid carriers and was left in ~he supernatant. The empty proteinoid carriers did not contain antibody in the supernatanc or the pellet as was expected.
The relative mobility (molecular weight) of the pure mAb is slightly differenc than the mAb in the proteinoid ca.riers. Thls is most likely due to different salt concentra-- tions ln the samples, because the encapsulation process employed 0.8 M salt solution.

Example 10: Effect of Additives on Stability of Proteinoid Carriers with Encapsulated Murine mAb 9BG5 Various proteinoid carrier formulations were screened, with or without additi~es, to determine optimal carrier-forming conditions and concentratlons of mAb required for carrer formation.
The mAb 9BG5 preparations used to prepare the encap-sulated proteinoid carriers had a protein concentration of approximately 2 mg/ml in phosphate buffered saline.
Final proteinoid concentration was 50 mg/ml and 5~
(w/w) gum acacia ("gu~.") or gelatin ("gel"). All protelnoid carriers were prepared in 0.85 N citric acid. Empty carriers were included for use as controls, and they were prepared in the same manner with the omission of mAb. Duplicate (0.5 ml) aliquots of proteinoid ca~rler suspension were centrlfuged at 5000 RPM. Pellets and supernatants were frozen in dry ice prior to analysis.
Table 7 lists samples that were prepared. Numbers in parenthesis indicate amount of mAb added.

21381~6 W 093/25583 PC~r/US93/05723 TliBLE 7 SAMPLE PROTEINOID ADDITIVE FINAL PROTEIN
(MG/ME) 326 Gum 0 3 326 Gel 0 334 Gum 0 7 334 Gel o 9, 10 326 Gum 0.5 11,12 326 Gum 0. 25 13 326 Gel 0. 25 1015, 16 334 Gum 0. 25 17, 18 334 Gel 0. 25 In order to test resistance to freeze and thawing on the integrity of the proteinoid carriers containing mAb, one of each pair of duplicate pellets were washed by gentle resuspension in 0.25 rnl of 0.85 N citric acid. The pellets were then analyzed next to the unwashed pellets to test whether any mAb was lost in the washing.
The samples were analyzed by conventional Western blotting as described in Example 9. Pellets were dissolved in sodium dodecyl sulfate with 0. 05 N NaOH and analyzed under reducing conditions (breaks up the mAb into 50 kDa and 25 kDa bands)~ Aliquots (50~1) of supernatants were analyzed under non-reducing conditions (expected intact 150 kDa mAb). This was done to determine differentially whether the mAb left behind is denatured or intact.
As can be seen from the X-ray film from the Western Blots (Figure 7), pellets of samples 9 and 10, and 11 and 12 contain between 5 and 10 ~g of mAb. The washed samples did not lose any significant amount of mAb, suggesting that the prote-inoid carriers r~m~; n~ intact after freeze-thawing.
The supernatants of samples 9 and 11 had no significant amount of mAb, indicating that unincorporated material was lost W093/25583 PCT/US93/0~72 during preparation.
Sample 17 had some mAb encapsulated which was losc after washing (see number 18). This sphere preparation was not resistant to freeze-thawing. Additionally, a band at a MW of 150 kDa for sample 17 supernatants indicates that a significant amount of mAb is left behina after proteinoid carrier formation.
Based on these results, it appears that the mAb r~mAlns intact and therefore the encapsulating procedure does not degrade it. The empty proteinoid carrier controls did not produce any bands, as expected because they have no mAb.

Example 11: Efficacy of Encapsulated Murine I~G Monoclonal AntibodY
In this Experiment, a mAb 9BG5 proteinoid carrier preparation and unencapsulated mAb 9BG5 were evaluated in rats.
The mAb 9BG5 (1 mg/ml), prepared as described in Example 9, was encapsulated in Glu/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Glu, Asp,Tyr, and Phe in the reaction mixture) protein carrier formulation with gum arabic. The mAb proteinoid carriers suspension contained 0.25 mg/ml mAb and 50 mg/ml proteinoid in 0.85 N citric acid-0.5~ gum. Empty proteinoid carriers were prepared similarly, but did not contain mAb. Since 30~ of the mAb was found to be encapsulated, the mAb proteinoid carriers were estimated to contain G.075 mg/ml mAb and this value was used to determine dosages. The mAb proteinoid carriers were examined microscopically and appear to be a fairly homogeneous prepara-tion.
For AnlmAl dosing, appropriate aliquots of proteinoid carriers were centrifuged at 5000 RPM for 15 minutes, and pellets 3C were resuspended in 1.0 ml of 0.85 N citric acid-0.5~ gum.
A purified mAb solution (0.95 mg/ml mAb ln 0.85 N
citric acid-0.5~ gum) was used for oral gavage. This solution was prewarmed to 40 C prior to A~m~ nl stration. For IV adminis-~ration, a purified mAb solution (1 mg/ml mAb n phosphate buffer saline) was used.
The amounts and administration routes employed in the W O 93/25583 ;~ 1 3 8 1 4 6 PC~r/US93/05723 expe-lmen~ are as follows-1. Empty proteinoid carrlers (no mAb): 1 ml aliquot containing 50 mg empty proteinoid carriers by oral gavage (rats # 2312 and 2313).
2. mAb 9BG5 proteinoid carriers: 3.7 mg mAb/ kg body weight of rat by oral gavage (rat # 2287, 2288, 2290, and 2291).
3. unencapsulated mAb 9GB5 0O73 mg/ kg body weight of rat by intravenous administration ~rats #2292, 2293, and 2311).
4. unencapsulated mAb 9BG5: 3.7 mg/ kg body weight of rat by oral gavage (rats #2314 and 2315).
Baseline blood samples (1 ml aliquots) were withdrawn from each rat just prior to dosing ~''0'l time~. After dosing;
blood samples were drawn at 1 h, 6 h and 24 h. The blood samples were processed immediately and sera were stored frozen at -20C.
Thawed serum taken from the experimental ~n;m~l S were analyzed by conventional ELISA techniques, in triplicate, using purified reovirus type 3 and VLSH dimeric peptides im.mobilized in multi-well plates (W.V. Willia-m-s et al (1991) J. Biol. Chem., Vol. 266(8), pages 5182-5190). Control plates included wells having no immobilized reovirus and VLSH peptides to which mAb (lmg/ml) was added. VLSH peptide (W.V. Williams et al. ibid, Table 1) is a synthetic variant of VL peptide, the latter which corresponds to a portion of the light chain variable CDR II
region of 87.92.6 antibody. The 87.92.6 antibody displays idiotypic and anti-idiotypic behavior towards reovirus type 3 receptor and mAb 9BG5, respectively (W.V. Williams et al. ibid)~
The bound protein content of each well were measured by standard protein methods, e.g., Lowry method, and the results for each multi-well plate are shown in Figures 8(a-c), respectively.
Figures 8 (a-ci illustrate the levels of serum proteins which bound to immobilized reovirus type 3 and V~SH as detected by measurement of protein concentration. These Figures show that the serum levels of bound proteins, after 24 hours post-dosing, were highest for ~n;m~ls orally ~m;n;stered mAb proteinoid carrlers and ~nim~ls ~m;n;s~ered unencapsulated mAb by the IV

W093~25583 PCT/US93/057t3 2 ~3 8 1 46 34 - rou~e. ~ower levels of bound serum proteins were found in ~nimAIs orally adminstered uncapsulated mAb. Serum taken from the ~nim~7s receiving empty proteinoid carriers (no mAb) showed non-specific serum IgG protein binding, as expected, under the assay conditions.
Figure 9 show mAb binding under conventional ELISA
procedures using im.mobilized reovirus type 3 and VLSH proteins.
Serial dilutions of mAb treated with 0.85 N citrate-0.5~ gum (Figure 9(a) or phosphate buffered saline (Figure 9 (b) were employed. The Figures show that the bound protein levels were higher for mAb in citrate buffer than for mAb in phosphate.
Without being bound by any theory of operation for this inven-tion, it iS belleved that the binding enhancement may be due ~o changes in the chree ~lmpn~ional conformation resultlng from citrate-protein binding.
In su-mmaryl serum levels of mAb, as reflected by the absorbance of bound proteins, were greater in ~nim~ls receiving encapsulated mAb by the oral route or unencapsulated m~Ab by the IV route, than an ~n;m~l receiving orally ~ministered unencap-sulated mAb.

Example 12: Preparation of Proteinoid carrier cont~i n ~ n~Heparin This Example describes a metnod for the preparation and cleaning of heparin proteinoid carriers.

PROCED~RE
i. Reaqents:
a. Proteinoid powder prepared as described in Example b. Heparin c. Anhydrous citric acid (USP) d. Gum acacia NF
e. Deionized water f. Desiccant g. Liquid nitrogen W O 93/25583 2 1 3 8 1 ~ 6 PC~r/US93/0572~

2. Equipment:
a. Magnetic stirrer b. Buret c. Microscope d. Clinical centrifuge e. Dialysis membrane tubing (Spectrum 6, 10 mm, 50,000 M.W. Cutoff) f. pH meter g. Lyophilizer (Labconco #75035) h. Lyophilizing flasks (150-300 mL) i. Rotating shell freezer j. Isopropanol/dry ice bath or liquid N2 k. Mortar and pestle l. Storage containers (500 mL) m. Eppendorf pipet (0-100 uL) n. Plastic closures for dialysis tubing (Speccrum) o. 2 mL syringe with 0.45 um Acrodisk 20 3. Preparation of Solutions:
a. Proteinoid Solution A (80 mq/ml):
Dissolve 160 mg proteinoid in 1 ml of deionized water.
Using a 2 ml syringe fitted with a 0.45 um Acrodisk, the proteinoid solution was filtered into a 10 ml test tube and kept at 40 C.

b. Solution B (1.7 N citric acid with 1~ qum):
Dissolve 10 g of gum acacia and 109 g of citric acid in 1 liter of deionized water.
c. Solution C (Heparin solution):
Dissolve heparin in Solution B at 150 mg~mL and keep at or multiples thereof.

W09~/2~5338 1 46 36 PCT/US93/05723 . Preparation of Prote~noid carriers~
a. Add all of solution A to solutlon C quickly while swirling solution C slowly, by hand, in a 40C water bath.

5. Dialysis of Heparin Pro~elnoid carriers:
It has been found the presence of citric acid in the encapsulated proteinoid carriers interferes with a subsequent lyophilization process. Hence, proteinoid carrier encapsulates prepared with citric acid solutions are preferably dialyzed against 5~ acetic acid solution for at least two hours with at least four changes of the dialysis solution to remove citric acid by an exchange process. Thus, a. Transfer the suspension with a syringe (no needle) to dialysis tubing and seal with plastic closures. Tubing should be no more than 70~ full.

b. Discard any amorphous material sedimented and/or aggregated on the surface.
c. Dialyze the proteinoid carrier suspension against acetic acid solution (using 20 mL of acetic acid solution per ml of proteinoid carrier suspension) while stirring the acetic acid solution with a magnetic stirrer.

d. Replace the acetic acid solution every hour. Continue dialyzing for a total of 3 hours.

6. Lyophilization:
a. Add one part of 50~ trehalose (Sigma Chemical CG., St.
Louis, M0, USA) into nine parts of dialyzed proteinoid carrier solution. Flash freeze proteinoid carriers in a freeze-drying flask using the shell freezer adjusted to rotate at ca. 190 rpm and immersed in a liquid 2138I46 O 93/25583 PC~r/US93/05723 nitrogen bath.

b. Freeze dry for 24 hours or until dry as evidenced by lack of self-cooling.

c. Record weight of dry proteinoid carriers.

d. Grind to a fine powder with mortar and pestle.

e. Transfer proteinoid into an amber container, seal with desiccant, and store at room temperature.

7. Resuspension:
a. Weigh the lyophilized powder and calculate the amount of proteinoid in the powder.

b. Add aqueous 0.85 N citric acid into the lyophilized powder at 40C. The final concentration of proteinoid in solution is 80 mg/ml.
Example 13: Preparation of Insuiin-contAi n; ng Proteinoid Carrier This Example illustrates a method for the preparation of insulin proteinoid carriers.

PROCED~RE
1. Reagents:
a. Proteinoid powder b. Anhydrous citric acid (USP) c. Gelatin (USP) d. Porcine insulin (Novo Nordisk) e. Deionized water (USP) 2. Equipment:
a. Water bath W O 93/25583 P(~r/US93/05723 3 a ~ 4 6 38 b. 0.2 micron Acrodis~ fil~er c. Sterile syringe (lOcc) d. Glass or plastic vessel of appropriate volume for desired amount of proteinoid carrier solution.

3. Preparation of Solutlons:
a. 1.7 N citric acid with 5.0~ gelatin:
Dissolve 109 mg anhydrous~citric acid and 50 mg gelatin per 1 ml of deionized water at desired volume and incubate in water bath at 40C until gelatin is completely dissolved. This may be prepared and stored at 40C for later use.

b. Insulin solution^
Dlssolve 12 mg insulin per 1 ml of 1.7 N ci~rlc acid with 5~ gelatin at 40C at desired volume.

c. Proteinoid solution:
Dissolve 100 mg proteinoid per 1 ml deionized water at room temperature and desired volume. Using syringe and 0.2 micron Acrodisk, filter the solution to ensure a clear liquid and incubate in a water bath at 40C. See Section Sb.

4. Preparation of Proteinoid carriers:
a. Proteinoid solution and insulin solution are combined at equal volumes sufficient to produce the final desired volume of proteinoid carriers.

b. Rapidly add the filtered protelnoid solutlon to ~he insulin solution at 40C while simultaneously and constantly swirling the insulin solution to ensure a ' Proteinoid and Insulin solutions should each be prepared at one-half the total volume of the final microsphere solution desired.

W093/2~583 2 1 3 8 1 4 6 PcT/us93/o572~
thorough mixing.

Example 14: Procedure for Preparation of Erythropoietin - Containinq Proteinoid carriers Encapsulation of human erythropoietin tEPO) in proteinoid carriers was performed in the same manner described in Example 13. EPO was obtained from Genetic Institute (Cambridge, MA, USA, now available from Amgen Corp., Thousand Oaks, CA, USA).
A solution of Gln/Asp/Tyr/Phe (1:1:1:1 mole ratio of Gln, Asp, Tyr, and Phe in the proteinoid reaction mixture) proteinoid and a 150 ug/mL EPO solution in 1.7 N citric acid with 1~ gum was used in preparing the EPO-containing proteinoid carrier.

Example 15: Evaluation of Erythropoietin-cont~i n; n~
Proteinoid Carrier In this Example, an EPO-containing protein carrier, prepared as described in Example 14, was evaluated in rats. An EPO experimental synopsis is given below.
Rats weighing 150-200 grams are anesthetized with ketamine (8.5mg/kg) and thorazine 3.75mg/kg) with intramuscular injection. The rat is then ~m; n; stered either unencapsulated erythropoietin or encapsulated erythropoietin by oral gavage. In brief, an 8 french nelaton catheter is inserted down the esophagus of the rat until the 10cm mark on the catheter is even with the incisors. The test or control solution is drawn up into a syringe and attached to the catheter. Holding the ~nlm~l upright, the solution is expressed into the stomach of the rat.
The experimental results are summarized ln Figures 10-12.

W O 93~25583 PC~r/US93/05723 2 ¦ 3 8 1 ~ 6 ~K~l~OPOIETIN EXP~TNn~Nr~L SYnNOPSIS

Batcn Dose Ra~s Respondinq Comments Control 15~g/kg 0/4 Fasted 15 hours.
251<3K 15~g/kg 0/4 Access to bedding.
254~3K 15~g/kg 2/4 Gavaged Control 15~g/kg 0/2 251~3K 15~g/kg 0/2 Fasted 36 hours.
254~3K 15~g/kg 1/4 5~ sucrose.
270K 15~g/kg 1/3 No bedding.
270G 15~g/kg 3/3 Gavaged.
Control 15~g/kg 1/5 Fasted 24 hours.
264CP 15~g/kg 1/4 Access to bedding.
270G 15~g/kg 1/6 Gavaged.
Controi lO~g/kg 0/5 Fasted 24 hours.
270G lO~g/kg 3*/6 No bedding.
Control 30~g/kg 0/3 Fasted 24 hours.
Control 60~g/kg 1/4 No bedding.
270G 30~g/kg 1/3 Direct injection 270G 60~g/kg 1/4 into the stomach.
Control 50~g/kg 0/3 Control+
Pepsin SO~g/kg 0/4 Direct injection 270G 50~g/kg 2/4 into the intestine.
270G +
Pepsin 50~g/kg 0/4 Control lOO~g/kg 1/5 Multiple Dosing 270G lOO~g/kg 1/5 (5 dosing intervals I.V. 50~g/kg 2/2 at t 1/2) S.C. 50~g/kg 2/2 Gavage by stomach tube.

~Rats were foaming at nostrils.

Figure 10 illus~ra~es levels of erythropoietin (EPO) detected in rat serum taken from rats ~m;nlstered Gln/Asp/Tyr/-Phe proteinoid carrier encapsulated EPO (15~g EPO/kg body weight) and encapsulated EPO (15~g EPO/kg body weight) at t = 0.5, 1, and 2 hours. Serum erythropoietin levels were determined over time with an erythropoietin enzyme ;mml~noassay kit (Amgen, Thousand W O 93/25583 2 1 3 8 1 ~ 6 PC~r/US~3/05723 Oaks, CA, USA~. The resuits show that EPO serum ievels in rats administered erythropoietin proteinoid carriers were relatively higher at all time poin~s compared to rats (control! which received unencapsulated material. At t = 2 hours, the EPO levels remained at approximately 300 pg/mL serum in rats ~ml n; stered erythropoietin proteinoid carriers while the control rats had undetectable EPO levels.
Figure 11 illustrates EPO serum levels in rats tha-were ~mln;stered either erythropoietin (50~g/kg) or Gln/Asp/Tyr-/Phe proteinoid (1:1:1:1 mole ratio of Gln, Asp,Tyr, and Phe inthe reaction mixture) proteinoid carrier encapsulated erythro-poietin (50~g/kg) directly into the proximal duodenum. Serum erythropoietin levels were determined over time with the aforementioned erythropoietin enzyme immunoassay kit. The results show that EPO serum levels in rats administered eryth-ropoietin proteinoid carriers steadily increased at a rate of approximately 50 pg/mL per hour over a range of two hours. In contrast, rats (control) which received unencapsulated EPO had EPO levels peaked at 100 pg/mL at 1 hour following ~mlnistration and steadily decreased to about 50 pg/mL at the end of 2 hours.
Figure 12 illustrates EPO serum levels in rats who were orally gavaged with either Gln/Asp/~yr/Phe proteinoid (1:1:1:1 mole ratio of Gln, Asp,Tyr, and Phe in the reaction mixture) p-roteinoid carrier encapsulated or unencapsul~ted erythropoietin (100~g/kg); or received a subcutaneous injection of either 2~g/kg or 10~g/kg. Serum erythropoietin levels were determlned over time with the aforementioned erythropoietin enzyme immunoassay kit. The results show that EPO serum levels in rats (#640-64Sj orally ~mi nl stered erythropoietin proteinoid carriers were relatively higher up to t = 2 hours, compared to rats (EPO) which received unencapsulated material.
The results obtained in this Example provide evidence that proteinoid encapsulation markedly improved the oral bioavailability of EPO.

3~6 42 ~xample 16: Preparation of Calcitonin-contAi n i ng Proteinoid carrier Encapsulation of salmon calcitonin in pro~einoid proteinoid carriers was performed ln the same manner described in Example 13. Calcitonin, a peptlde hormone which acts predomi-nantly on bone to lower serum caicium concentration, was obtained from Sandoz (Basil, Switzerland). Calcitonin proteinoid carriers were prepared by mixing a 1:1 volume ratio of a lOOmg/ml aqueous solution of Gln/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Gln, Asp, Tyr, and Phe used in the proteinoid reaction mixture) and a 150 ug/mL calcitonin solution in 1.7 N citric acid solution with 1~ gum acacia, as described in Example 13. The efficiency of calcitonin encapsulation was approximately 40~. Calcitonin concentration was determined directly by HPLC after dissolving the calcitonin proteinoid carriers ln 60~ aqueous acetonitrile.

Example 17: E~aluation of Calcitonin-contA;ning Proteinoid carriers in Monkey~
In this Example, the calcitonin proteinoid carriers, prepared as described in Example 16, were evaluated in cynomolgus monkeys. Male cynomolgus monkeys weighing 4-5 kg were fasted overnight, anesthetized (approximately lOmg/kg ketamine HCl) and placed into a primate restraint chair for dosing and blood sampling. A single oral dose of calcitonin proteinoid carriers (0.25 mg/kg body weight) was ~m;n;stered to each of four monkeys by nasogastric gavage. The dosage was based on the body weight taken on the morning of dosing. Blood samples were collected from saphenous vein catheters at hourly intervals, starting at t = 0 prior to administration of the proteinoid carriers, and hourly, from 1 to 7 hours post-dose for serum calcium determina-tion. The hypocalcemic response following oral calcitonin nistration was used as an index of ph~rm~cological response.
Serum calcium concentrations were quantitated by a conventional O-cresolphthalein complexone method.
35Figure 13 ~mon~trates the response obtained in cynomolgus monkeys following naso-gastric gavage of microencap-21381g6 W093/2~583 PCT/~IS93/05723 43 ~
sulated calci~onin. Signiflcan- cnanges from basellne serum calcium ccncentra~lon were observed. Slx hours followlng doslng, seru~. calcium concen~ra-ions de-reased by 13 ~g/ml. A signifi-cant pharmacological response was still apparent seven hours after the administration of calcitonin proteinoid carriers.

Example 18: Evaluation of Calcitonin-contA;ninq Proteinoid Carriers in Rats In this Example, the calcitonin proteinoid carriers prepared in accordance wi~h Example 16 are evaluated in fasted male Spraque Dawley rats weighing 100-150g. Calcitonin proteinoid carriers and calcitonin were administered by either oral gavage or lntraduodenal in]ection. The rats are divided into the following groups:
1. calcitonln proteinoid carriers: 60 ug calcitonin/kg body weight by oral gavage (3 rats);
2. calcl~onin proteinoid carriers: 3 ug calcitonin/kg body weight by intraduodenal gavage (3 rats);
3. calci~onin: 60 ug calcitonin/kg body weight by oral gavage (3 rats) (Control).
4. calcitonin: 3 ug calcitonin/kg body weight by intraduodenal gavage (3 rats) (Control).
Oral gavage dosing of rats is performed. Calcitonin proteinoid carriers are prepared lmmediately prior to dosing and Groups 1 and 2 each receive an appropriate dosage of the proteinoid carrier suspension. Groups 3 and 4 receive the unencapsulated calcitonir ~no proteinold carriers). Approximate-ly 0.5 ml of blood is serially withdrawn from tne tail artery of each rat just prior to dosing ("0" timej and 1 h, 2 h and 3 h post-dosing. Serum from the blood samples are stored a~ -2CC
for serum calcium concentration determination.
Figure '4 is the serum concentration-time curve for oraliy ~mi ni stered microencapsula~ed calcitonin and unencapsu-lated calcitonin in rats. Experimental results in rats demon-strate a significant increase in pharmacological response (i.e., W093/25583 PCT/US93/0572~

decreasing serum calcium levels) when proteinoid encapsulated calcitonin is compared to the unencapsulated vehicle control group. One hour after dosing serum calcium concentrations decreased 23 ~g/ml in the ~ats receiving encapsulated calcitonin compared to a decrease of only 6 5 ~g/ml ln the con~roi group.
Fur~hermore, the responses were dose-dependen. (data not shown).
The results of intraduodenal injection of encapsulated or unencapsulated calcitonin ln rats is shown in Figure 15. The results demonstrate a time-dependent decrease in serum calcium levels for the encapsulated preparation. The control group showed no response. One hour after intraduodenal administration, serum calcium levels in the calcitonin proteinoid carrier group decreased by 18~g/ml, whereas unencapsulated calcitonin was unchanged. These results indlca~e that tr~n~m~mhrane transport of calcitonin is enhanced by protelnoid encapsulation.
The results obtained in this Example and in Example 17 provide evidence that proteinoid encapsulation markedly improves the oral bioavailability of calcitonin. The data also indicate that the oral drug delivery system is not species-dependent.
Example 19: Preparation and Evaluation of Factor IX-contain-inq Proteinoid Carrier Factor IX is a vitamin K-dependent blood coagulation proenzyme, MW 56 kD. Factor IX deficiencyi known as hemophilia B, occurs in approximately ' out of every 25,000 males. To date, treatment of this disoraer is accomplished by intravenous administration of Factor IX, although a recent report details efforts to supplement by subcutaneous injection (Thompson (1986) B~ood, Vol. 67(3), pages 565-572).
Encapsulation of Factor IX (FIX) in proteinoid carriers was performed, following the procedure described in Example 13, by mixing (1:1 v/v) 100 mg/mL of Glu/Asp/Tyr/Phe proteinoid ~ mole ratio of Glu, Asp, Tyr, and Phe used ln the proteinoid reaction mixture) in deionized water and an aqueous solution of FIX. Two proteinoid carrier suspensions were prepared and evaluated in vivo separately as described in -W O 93~25583 2 1 3 8 1 4 6 PC~r/US93/05723 Examples 20 and 21.
FIX protelnoid carrler suspension A _ontained 50 ~g/ml of proteinoid and 500 U/ml FIX (FIX is available from the American Red Cross, Roc~vil~e, Maryland, USA) solution containing 4~ acetic acid, 2~ gum acacia, 002~ PEG 14 (available from Union Carbide, Danbury, CN, USAi, 14 mM CaCl" final pH 3.81.
The second suspension, FIX proteinoid carrier suspen-sion B, contained 50 mg/ml proteinoid and 116 U/ml FIX solution containing 3.8~ acetic acid, 1.5~ gum acacia, 0.15~ PEG 14, 11 mM
lG CaCl2, final pH 4.58.
The stability of FIX proteinoid carrier preparations was assessed over a short time course in vitro. The protein carriers encapsulating FIX were ~X~mi ned by optical microscopy and laser light scatterlng~ Aliquots of pro~einoid carrier suspension were withdrawn every 30 minutes for 1.5 hours~ FIX
proteinoid carriers were lsolated by centrifugation at 4500Xg and dissolved in activated partial thromboplastin time (APTT) assay buffer (0.05M histidine-O.OlM NaCl-O.l~ bovine serum albumin-0.01~ TWEEN-40, pH 7.47) to release soluble FIX and proteinoid.
Quantitation of FIX activi~y by APTT employed both FIX st~n~Ards (0.025, 0.05, and 0.1 U/ml) and "empty" proteinoid carrier suspension as control. APTT assay kits are commercially available, e.g. Sigma Diagnostics (St. Louis, MO, USA).
Based on the above analysis, it was determined that FIX
protelnoid carriers of grea~er stability are obtained by encapsu-lating FIX at a higher pH, e.g., pH 4.9. Furthermore, the efficiency of encapsulation is approximately 20~ of available FIX
units and activity levels remain constant for at least 1.5 hourc wher FIX proteinoid carr~er pellets are stored at about 4 C.
Example 20: Evaluation of FIX-cont~; n i nq Proteinoid carriers (A) in Rats In this Example, FIX proteinoid carrier suspenslon A, prepared as described i. Example 15, were evaluated in male Sprague Dawley rats (ave. weight 300g)~ Appropriate aliquots of suspension were centrifuged at 4500Xg to pellet the FIX protein .

W093/2~583 PCT/~IS93/05723 i3 ~ar~lers, -~hi~h we~e subsequently resuspended in the same buffer for anlmai doslng~ The rats are dlvided into tWO groups as fol-lows:
1. Oral FIX proteinoid carrlers (FIX sph PO): 2709 U FIX/kg body weight by intragastric g~avage (4 rats);
2. Intravenous FIX (no pro~elnold carriers) (FIX IV!: 200 U/kg body weight by intravenous ~njection. 32 rats received 0.7 ml FIX in 0.11 NaCl-0.02M sodium citrate, pH 6.85 by tail vein injection.
The FIX proteinoid carrier suspension and solution are prepared immedlately prior to dosing. One ml of blood was witndrawn rom each rat just prior ~o dosing (~lol~ timej and 1 hj 2 h and 4 h (post-dosingi, a cl.rate anticoagulant was added to the blood, and plasma from the blood samples were stored at -70C.
Plasma samples were zssayed by a modified APTT assay uslng FIX coagulated deficient plasma (assay kit is available from Ortho Diagnosis (Raritan, New Jersey, USA). ~hanges in clotting times were calculated by subtracting individual baseline (0 hr) values from subsequent clotting time values. The data shown in Figure 16 are the mean values for a given group. Values below baseline indicate the presence of exogenous FIX.
As shown in Figure 16, significant amounts of FIX was delivered to blood via oral adm~nisrration of FIX proteinoid carrlers. The relative plasma level is lower in the FIX
proreinoid carriers group, however the ~im~lnltion in clotting -ime at 0.5, 1.0 and 2.0 hours is notable. This is achleved by oral doslng with approximately 14 times tne IV dose. Moreover tnese results are particularly lnteresting since Factor IX is an acid labile protein whose hal -life is approximately less than one hour at 37-C at pH 5.G. The FIX protelnoid carriers in this experiment were at pH 3.81 and encapsulated 14.8~ of the available FIX units during prepararion. The results support that FIX proteinoid carriers remain viable in the GI tract to facilitate delivery.

W093/2~83 2 1 3 8 1 9 6 P~T/US93/0572~
4~
xample 21: Evaluation of FIX-containinq Proteinoid Carriers (B) in Rats In this Example, FIX pro~einoid carrier suspension B, prepared as described ~n Example 15, were evaluated in male Sprague Dawley ra~s (ave. weigh~ 300g)~ Resuspended FIX
proteinoid carriers were prepared as described in Example 20.
The rats are divided intc two groups as follows:
1. Oral FIX proteinoid carriers (FIX sph Po!: 1006U FIX/kg body weight by intragastric gavage (5 rats).
2. Intravenous FIX (no proteinoid carriers) (FIX IV): 185 U/kg body weight by intravenous injection. 3 rats received 0.3 - ml FIX in 0.11 NaCl-0.02M sodium citrate, pH 6 . 85 by tail vein injection.
'. Oral FIX ~no pro~einoia carrlers) (FIX unencap PO): 2760~T
FIXtkg body weight by lntragastric gavage. 4 rats receivea 1.0 ml of FIX in sallne solution containing 3.8~ acetlc acid, pH 6.85.
The FIX proteinoid carrier suspension and solutions were prepared immediately prior to dosing. Plasma samples were obtained and assayed as described in Example 20. Changes in clotting times were calcula~ed by subtracting individual baseline (0 hr) values from subsequent clotting time values. The data shown in Figure 17 are the mean values for a given group. Values below baseline indicate the presence of exogenous FIX. The FIX
proteinoid carriers, prepared ar pH 4.56, encapsulated 23.1~ of the FIX units.
As shown in Figure 17, at oral dose levels of only 5 times that of ~he IV dose, significant orai delivery was observed. In addi~ion, na~ive FIX ~pH 6.85) dosed at 15 times the IV dose level resulted in no detectable levels of exogenous FIX in the plasma.
- Thus, the results sAown in this Example and in Example 20 support that oral delivery of FIX can be accomplished via the use of FIX proteinoid carriers. These protelnoid carriers appear to adequately protect FIX during transit through the GI tract and deliver FIX to the blood stream.

W093/25~83 PCT/~S93/0572 x ~ ~3 a ~ 46 4&
E le 22: Preparat~on of alpha-Interferon (IFN)-containing Proteinoid carrier ~r. this ~xample, a study was undertaken tO
evaluate the protective capabillty of proteinoid carriers on enzymatic degradation under slmulated gastrointestinal condi-tions. The in vitro stability of IFN in proteinoid carriers was examlned in simulated gastric fluid (SGF) containing pepsin in 0.G8 N HCl and simulated lntestlnal fluid (SIF) containing pancreatin in pAosphate buffer. The reagents and stability assay procedure are described in the "United States Pharmacopocia"
(Vol. XXII, 1990, pages 1788 and 1789).

Preparation of IFN-containinq proteinoid carriers Encapsulation of IFN ln proteinoid carriers was performed in the same manner descri~ed in Example 13. Alpha-IFN
is available from a number of commercial sources. One commercial IFN product includes Roferon-A (Hoffman LaRoche). IFN proteinoid carriers were prepared with an aqueous solution of Glu/Asp/Tyr/-Phe proteinoid (1:1:1:1 mole ratio of Glu, Asp, Tyr and Phe used in the proteinoid reaction mixture), and an IFN solution containing 1.7 N citric acid solution with 5~ gelatin. The IFN
proteinoid carrier suspension contained 80 mg/ml proteinoid, 600 ug/ml IFN, 0.6N citric acid, and 2.5~ gelatin, pH 3Ø

Stability of IFN proteinoid carriers in SGF
SGF (2 ml) was added into 1 ml of IFN proteinoid proteinoid carrier suspension. The solution was incubated at 40-C wi~h shaking, and aliquo~s were taken serially after SGF
addition as described in the "U.S. Pharmacocopia" (ibid). An equal volume of stopper solution (pepstatin A in phosphate buffer, was added to each aliquot immediately after sampling to stop the enzymatic degradation and to open the proteinoid carriers. The IFN concentration in all samples was then determined by HPLC. As a comparison, the stability of IFN alone in SGF was evaluated. The experiment were performed as described above, without the proteinoid carriers. As another control, the W093/25583 2 1 3 8 1 ~ 6 PCT/US93/0572~
4c stability of IFN proteincla ca~riers was evaluatea ln 0.08 N HCl.

Stability of IFN-containin~ Proteinoid carriers in SIF
SIF (2 ml) was added into ~ ml IFN proteinoid carriers.
The solution was incubated at 40 C with shaking and samples were taken serially as described in tne "United States Pharmacocopia"
(ibid). An equal volume of stopper solution (aprotinin and trypsin/chymotrypsin inhlbitor in phosphate buffer) was added to each aliquot immediately after sampling to stop the enzymatic degradatior. The IFN concentration was analyzed by HPLC.
To study the study the stability of IFN alone in SIF, 600 ug of IFN was dissolved in 0.85 N citric acid or 0.01 M
phosphate buffer. SIF (2 ml) was added to 1 ml IFN solution.
The solutlon was sampled and analyzed as described above.
Results and Discussion (a) Protective Effects of Proteinoid carriers in SGF
As shown in Figure 18, after 1 hour of SGF incubation, approximately 50% of IFN remalned intact. After incubation in SGF for 6 hours, approximately 20~ of IFN was not degraded. As expected, IFN alone (in the absence of proteinoid carriers), was found to be completely destroyed by pepsin in SGF within 20 minutes.
Another control was performed using IFN alone in 0.08 N HCi. IFN alone was stable ln SGF without peps1n (0.08 HCl~.
There was only a slight decrease after 2 hou~ incubation. This sugges~s that IFN was rather stable in HCl at pH 1.2 up to six hours (Flgure 19).
The results suggest tthat proteinoid carriers can retard 3G IFN from pepsin digestion, while TF~- alone cannot survlve ln tne stomach for more than 20 minutes. These observations demonstrate the protective ability of proteinoid carriers on enzymatic digestion of protein drugs in the stomach.

W093/2~583 PCT/~IS93/0572~
~ 3~ 6 50 ~b~ Protective Effects of Proteinoid carriers in SIF
As snown in Flgure 2G t IFN proteinoid carriers were much more stable than IFN alone lin ~he absence of protelnoid) ln SIF. IFN alone at pH 7.4 was completely degraded withln 10 minutes when ncubated with SIF~ However, approximately 70~ of the IFN/protelnoid carriers survlved after 6 hours ln SIF, indicating that considerable s~abili~y lS provided by the proteinoid proteinoid carrier.
IFN alone was slightly more stable in SIF a~ pH 3 than at pH 7.4. After 6 hr incubation in SIF at pH 3, there was approximately 10~ of the IFN remaining. The stability of IFN in SIF at pH 3 is attributed ~G rhe low pH, which appears tc suppress enzymatlc activity of the intestlnal proteases.
5 Example 23: Evaluation of HeParin-containinq Proteinoid carriers in Rats In this Example, a study was undertaken to ascertain whether proteinoid carriers are required for protective capabili-ty or whether (1) proteinoids (soluble proteinoids--not in carrier form) may be used and whether (2) alternative methods of carrier loading, such as incubating the therapeutic compound with preformed proteinoid carriers, are useful.

Preparation of Heparin-containinq proteinoid carriers Encapsulation of neparln in proteinoid carriers was performed in the same manner described in Example 12. Heparin (USP grade) was used and this material is available from a variety of commercial sources lnciudlng Eil Lilly (Indlanapolls, USAi. Heparin proteinoid carriers were prepared, foilowing the procedure Or Example 12, uslng a 1:1 volume rat1o of 150 mg/ml of Glu/Asp/Tyr/Phe/OrnOs (i~ mole ra~io of Glu, Asp, Tyr, Phe, and Orn used in the proteinoid reaction mixturei pro~einoid ln deionized water, and an 20mg/mL aqueous heparin solution con.alnlng 1.7 N cltric aci~ soiution and 0.5~ gum acacla. The heparin proteinoid carrier suspension was dialyzed in acetic acia solu~ion as described in Example 1~ Heparin proteinoid car_iers WO ~3/25583 2 1 3 8 1 4 6 PCr/l 'C93/05723 were ~hen centrifuged at 4800Xg (15 minutes~ and totai heparin was measured by assaying tne pellet and the supernatant with a modification of the Azure A method (Gundry et al. Amer. J. of Surgery (1984; Vol. 148, pages 191-194). Proteinoid was assayed by dissolvlng the protelnoid carriers with 0.1 N NaOH and measuring absorbance at 294 nm.

Pre~aration of heDarin-spiked ~mptY ~roteinoid carriers Empty protelnoid carriers were prepared following the same procedure described above for the heparin proteinoid carriers, witn the modification being that no heparin was present. The lyophilized empty protelnoid carriers were resuspended in O.85N citric acid and 0.5~ gum containing heparin at a concentration of 20 mg/ml. The amoun~ of heparin co-isolated with the proteinoid carriers was measured as describedabove.

ExPerimental Procedure Male Spaque Dawley rats weighing approximately 350g were dosed by oral gavage or lntraduodenal (ID) injection (just anterior to the pyloric sphincter and into the duodenum). Rats were dosed orally or ID with one of the following: lyophilized heparin proteinoid carriers, heparin-spiked empty proteinoid carriers, proteinoid/heparin in water, heparin in G.85N citric acid and 0.5~ gum and heparin alone in water. In both oral and ID injection experiments, weignt ratios of neparin:proteinoid were constant. The total heparin dose in the oral studies was 100 mg/kg body weight; in ID injections studies, it was 50 mg/kg.
The proteinoid dose was 40 mg/kg for oral gavages and 20 mg/kg for ID injections. The dosing volume was approximately 0.3 to 0.5 ml. Approximately 0.5 ml of blood is serially withdrawn from the taii artery of eacA rat just prior to dosing ("0" time) and 1 h, 2 h and 4 h post-dosing. Serum from the blood samples are stored at -20C for heparin activity determination.

W O 93~5583 PC~r/US93tO5723 ~1 38~ ~6 5G
.e~L~g and DiScussion ' The resulcs ob~alned suggest that heparin alone as well as soluble pro~e;noia anG neparin (both in water, dosed orally or by ID injection) did not appear to be absorbed from the GI tract in amounts sufficient to increase APTT values (Figure 21).
Heparin in citric acid elicited some increase in APTT values, but only when dosed directly into the duodenum.
Heparin proteinoid ca~rriers gave the highest APTT
value~, indicated increased absorption of heparin when dosed orally, as well as when directly injected into the duodenum ~Figures 22 and 23). While tne observed activity was lower than observed with heparin prote1noid carriers (Figure 23), heparin-spiked empty proteinoid carriers showed increased APTTs over baselines. Both types of proteinoid carriers showed a much greater increase in APTT values ~han that observed with citric acid/heparin.
The results obtained in this Example suggest that, in the proteinoid system, proteinoid carriers are necessary for the observed increase in heparin absorption, as soluble proteinoid did not show detectable activity within the experimental limits.

Example 24: Preparation and Evaluation of M1-contA;n;ng Proteinoid carrier In ~his Example, influenza virus antigen-contalning proteinoid carriers were prepared and evaluated in rats.

Preparation of M1 Proteinoid carriers ~ncapsulation of M1 in proteinoid carriers was performed in the same manner described in Example 13. M1 protein, a major internai component of influenza virus, was obtained by purification of a swine influenza vaccine aonated by Drug Directorate, Health Protection Branch, Bureau of Biologics, Ottawa, Ontrario C~nn~. The vaccine was prepared with the high-yielding recombinant strain X-53Aa, which derives its HA and NA from the parent strain A/N~/11/76(HlNl) and its internal W093/25~83 ~ 1 3 81 ~ 6 PCT/US~3/05723 pro~elns, includlng Mlj tro~ th~ paren~ straln A/PR/8/34 ~R.B.
Couc et al. (1983) Ann. Rev~ Microbiol., Vol. 37, pages 529-~49 and B.R. Murphy ~1982) Infec. Immun., Vol. 36, pages 1102-L108).
Ml was purified as described by Khan et al ( (1982) J.Clin.Micro-biol., Vol. 16, pages 813-820io Ml proteinoid carriers were prepared, by mixing (at 40 C), equivolumes of an aqueous solution of 100mg/ml of Glu/Asp/Tyr/Phe proteinoid in deionized water and a 10mg/mL solution of Ml protein in 1.7N citric acid and 5~ gum arabic (pH 2.0). The final Ml concentration in the suspension ;0 was l.Omg/ml.

Pre~aration of HA-NA-containinq Proteinoid carriers and unenca~-sulated antiqens HA-NA antigen was isolated accordlng to the procedure of Gallagher et al. ((1984i J.Clln.Mlcrobioi., Vol. 20~ pages 80-93). Influenza virus (A/PR8/34) was centrifuged at 90,000 ~ for 60 min. The viral pellec was solubilized with 0.05M acetate buffer (pH 7.0) containing 7.5~ octylglucoside and re-centrifuged under the same conditions. The resulting supernatant contained approximately 90~ HA and 10~ NA as determined by SDS-PAGE.
HA-NA proteinoid carriers were prepared following the same protocol as for the Ml proteinoid carriers but substituted Ml for HA-NA. The final concentration of HA-NA in the suspension was also 1~0 mg/ml.
"Empty" proteinoid carriers were prepared foilowing the sampe procedure described for the Ml proteinoid carriers, with tne only modification being that a 1.7 N citric acld/gum solutlon was usea in place of the Ml/citric acid/gu~ solution.
Unencapsulated antigens, Ml and HA-NA, was diluted ir 1.7 N citric acid, 10 mg/ml gum arablc to the same final lmg/ml concentration.

Experimental procedure Male Spraque Dawley rats (about 350g weight) were usea in this experiment. Oral dosage was by gavage. Four groups of wo93/23 ~3~ 46 54 PCT/~'S93/05723 five rats each (the subcu~aneous control group had 4) were dosed as foiiows Group l wa~ dosed orally with lmg of M1 proteinoid carriers per ra~ (1 ml1, C-roup 2 was dosed oraily with 1 mg per rat cf "empty" proteinold carrier. Group 3 was dosed with 1 mg of unencapsulated M1 per rat of '~empty" carrler, Group 3 was dosed with 1 mg of unencapsulated M1 per rat in 1 ml and Group 4 was dosed subcutaneously (SC) wlth 25 ug per rat of M1 in 0.3 ml.
Blood samples (300 ul) were taker. from each rat by tail bieeeding before dosing and at 1, 2, 3 and 4 hours post-dose (to assay for an~igen) and at 14, 28, and 42 days post-dose (for antibody assay). Solutions for subcutaneous control-M1 in TRIS (no SDS) was diluted to a concentration cf 167 ug/mL. An equal amount of Freunds Complete Adjuvant (FCA, Slgma) was added and the mixture was -horoughly homogenizea. Tne rlnal concentration of M1 in the mixtue was 83.3 ug/ml. HA-NA solutions for subcutaneous administration were prepared in the same manner except that phosphate buffered saline replaced TRIS-SDS buffer.
The same lmmllnization and bleeding schedule was follwed when dosing with HA-NA prote~noid carrier, with the following modifications: all rats received an oral hooster with HA-NA
proteinoid carrier (250 ug/rat) 42 days after the first oral dose and blood samples were again taken 14 days after the booster dose. Serum derived from the samples were stored at -20 C until assayed.
Serum an~i-M1 and anti-HA-NA specific IgGs were assayed by an ELISA method as described Khan et al. ((1982) J.
Clir. Microbiol., vol. 1~, pages 813-820).

Results and Discussion Attempts to measure antigen in plasma samples were unsuccessful. M1 antigen could not be detected in rat plasma samples taken 1-4 hours post-dosing in all groups, including the subcutaneous control.
Plasma sample~ _rom rat~ dosed orally wi'h "empty"
proteinoid carriers showed no significant antibody titer against either Ml or HA-NA antigens wher assayed by ELISA (Table 8). As expected, rats dosed with 25 ug of either M1 or HA-NA antigen (with FCA) subcutanouesly developed a vigorous antibody response wi~h tlters that ranged from 54,000-330,000 in the case of M1 and 176,750-909,000 in the case of HA-NA (Table 8).
Plasma samples from three of the f1ve rats dosed wltn M1 prote1noid carriers showed a signficant primary response to M1 antigen. All three rats had titers ranging from 760 to 2150 as early as 14 days post-dosing, compared to c30 in all rats that received the amount of unencapsulated M1 (Table 8~. Titers in the group that received proteinoid carriers increased to 1150-5200 by 42 days (Figure 24).
Four out of six rats immlln;zed with unencapsulated HA-NA did show a moderate anti-HA-NA IgG response, with titers of 3400-17,675, while two of six rats dosed with HA-NA proteinoid carrier showed a significant response (Figure 25). The rats that did respond, however, reached titers at least eight times higher than those obtained in the controls. Although several rats showed higher titers after the oral booster with HA-NA proteinoid carriers given 42 days post-dose, most did not show a significant increase in titers.
The results support that a single dose of Ml proteinoid carriers was capable of inducing a significant IgG response to M1 as ear ! y as two weeks post-dosing, while rats dosed with same M1 (no proteinoid carriers) total dose showed no aetectable antibody response. Similarly, a sing'e dose of HA-NA proteinoid carriers induced a response in 33~ of the rats used in the study. This response was up to eight times greater than rats dosed with unencapsulated HA-NA.

2~3~46 56 ANTI M PROTEIN ANTIBODV TITERS IN SERUM FROM RATS
DOSED WITH M PROTEINOID CARRIERS VS CONTROLS

14 day 28 day 42 day Dosing rat ~titer titer titer oral M protein 197 c30 ~30 ~30 unencapsulated 198 <30 ~30 c30 1 mg/rat 199 <30 ~30 ~30 200 ~30 ~30 35 201 ~30 ~30 56 empty carrier 203 ~30 ~30 82 204 ~30 ~30 70 205 ~30 ~30 60 206 ~30 ~30 86 207 ~30 ~30 45 M proteinoid 209 ~30 ~30 64 carriers 210 2,150 820 5,200 1 mg/rat total 211 860 430 1,150 212 760 1,850 3,000 213 ~30 ~30 62 subcut. control 21540,000 62,000 330,000 0.025 mg/rat 217 34 8,000 54,000 in FCA 218 430 8,000 125,000 219 270 6,600 78,000 wo 93/2~s8~ 2 1 3 81 ~ 6 PCr/~1~9~/0~72~

App nd8~ A
P-u- 1 o- 8 Prot-inold B-tch--T-mp n", Siph-r- B teh 61--Bt No #AA C ' Addhiw C Ihrl ~ Mol-- Op r tor D te 086 S 13LU ASPW ILEU --- 170 3.0 INSii MT1 0,0 08d 3 GLU ASP2 VAL --- 170 S.O INS4 MTO HEPO 0.0 087 3 QLU ASP LEU --- 170 3.0 INS6 MT3 HEP6 0.0 088 2 GLU2 ASP2 DQU 6EE HEHO 0.0 0.0 OB8 2 GLU2 ASP2 DQU -- 170 3.0 INS6 HTO 0.0 080 3 OLU2 ASP2 VAL -- 170 3.0 INS3 HTO HEP1 0.0 081 3 ~ILU A6P LEU -- 170 3.0 INS2 HT1 0.0 082 3 QLU ASP THR -- 170 3.0 INS2 HTO 0.0 081 4 GLU2 ASP2 VAL PRO -- 170 3.0 INS2 HT2 0.0 084 3 GLU ASP CY6-H -- 170 3.0 INS1 HT1 0.0 086 4 PRO 8ER THR CYS -- 170 3.0 0.0 086 3 QLU ASP VAL2 --~ 170 3.0 INS3 HTO HEP4 0.0 087 3 tlLU ASP VAL -- 170 3.0 INS2 HT1 0.0 088 3 GLU ASP CYC~H --- 170 3.0 INS4 HT1 0.0 088 2 GLU2 ASP2 DQU --- 170 3.0 INS4 0.0 188-CP 4 PYGLU ASP TYR PHE PA 17e 4.0 IN60 HT4 HEP6 0.3 188-CP 4 GLU ASP TYR PHE H20 100 88.0 HTO INSO HEPO 0.0 202A-CP 4 OLU2.4 A6P2 VAL2 OLY -- 170 4.0 INS3 HTO O.ô
202B-CP 4 OLU2.4 ASP2 VAL2 QLY -- 170 4.0 HTO INS3 O.ô
20dA-CP 4 OLU ASP-TYR PHE 8ULFA 176 4.6 IN64 HT4 HEP3 O.IS
2011B-CP 4 GLU ASP-TYR PHE SULFA 176 4.6 O.ô
2011C>3K 4 GLU ASP-TYR PHE SULFA 176 4.6 O.ô
207A-CP 4 GLU ASP-TYR PHE 6ULFA 176 10.0 IN66 HT4 HEP4 2.0207E-CP 4 OLU A6P-TYR PHE SULFA 176 10.0 HTS INS4 HEP4 2.021 lA-CP 4 OLU A6P-VAL LYSFB SULFA 180 4.3 IN66 HT6 HEP6 W 0.3 211B-CP 4 GLU A6P-VAL LYSFB SULFA 180 4.6 0.3212A-CP 3 GLU-TYR PHE SULFA 186 6.0 INS4 HT3 HEP4 0.3212B-CP 3 OLU2-TYR PHE 6ULFA 186 li.O 0.3 214-CP 3 GLU LYSF8-ARG SULFA 180 7.0 IN60 HTO HEPO 0.0 223-CP 4 LY6F82 ARG2 LEU PaLU SULFA 1W8.0 INSO HTO HEP2 0.3 227A-CP 2 VAL2 GLY2 6ULF 180 1.6 INSO HTO HEPO 0.1 2278-CP 2 VAL2 QLY2 ~:ULFA 180 1.6 HTO INSO HEPO 0.1 228A-CP 3 VAL2 ~lLY2 PGLU SULF 1S0 2.6 INSO HTO HEPO 0.1 228B-CP 3 VAL2 QLY2 PI~LU 6ULFA 180 2.6 HTO INSO HEPO 0.1 248 -CP 3 OLU ASP LEU -- 180 4.0 INS3 HTO HEP- 0.0281iA-CP 4 ~ILU ASP-TYR PHE 6UL 166 4.0 INS4 HT4 HEP6 1.0 201;8 4 QLU ASP-TYR PHE 6ULPOLANE 166 4.0 1.0 2811A-CP 4 GLU LYSH PHE ASP SUL-H 180 3.0 INS4 HT2 HEPO 0.6 283B-CP 4 GLU LYSH PHE ASP 6UL-H 1W 3.0 0.11 288 -CP 4 GLU A6P-TYR PHE l;UL-H 180 1.6 INS1- HT3 HEP4 0.6 301 -CP 4 aLU AW-TYR PHE 6UL 176 8.0 INS4 HT2 HEP3 2.0302 -CP 4 GLU ASP-TYR PHE BH-PO 180 1.6 INS4 HT2 HEP3 0.3 308 -CP 4 GLU ASP TYR PHE BHP 170 1.0 INS4 HT4 HEP4 0.3 308 -CP 4 -t~LU1.3 ASP1.3 TYR PH31.3 SULPOLANE 180 1.6 INSR MT30-HEP4 0.3 310 -CP 4 QLU A6P TYR PHE SULPOLANE 1804.0 INS4 HT2 HEP6 1.0 Glo---ry: = rnorphou- o = oll ~ = wr~ino ~ I = eook tim ch nge SUBSTITUTE SHEET

WO 93/2~i~i8:~ PCr/~'S9?~/0572~

?,~3a~46 App~ndi~l A
68 P-g- 2 ot Prot-lnold B teh--T-mp nm- *h~r B tch a.-llt ho ~AA '`~ Addhlv- C ~hrl ~9 Ml-! OD r tOr D t-038 2 GLU A6P2 EQU 1S0 1.6 o.O
03~ 3 ASP2 ARO ILEU -- 170 0.0 HTO o.O
040 2 OLU2 A8P2 EQU -- 176 3.0 0.0 041 2 OLU2 Al;P2 EQU PA 170 3.0 QO
042 2 OLU A6P2 EQU OLYC 170 S.O HTO 0.0 043 2 OLU2 A~iP2 EQU OLYC 170 S.O II~S4 HT4 0.0 044 2 OLU2 ASP2 EQU OLYC 170 3.0 HTO o.o 046 2 OLU2 ASP2 EQU PA 170 S.O HTI o.o 04B 2 OLU2 A6P2 EQU OLYC 1110 0~0 HTO 0.0 047 2 OLU2 ASP2 EQU PA 180 0.0 HTO 0.0 048 2 OLU2 ASP2 EtlU --- 1110 0.0 HTO 0.0 04~ 2 OLU2 ASP2 EQU -- 1110 J.O HTO 0.0 060 2 OLU2 A8P2 EQU -- 170 3.0 HTO 0.0 061 2 OLU2 ASP2 EQU -- 170 0.0 0.0 062 2 OLU2 A6P2 EQU -- 170 0.0 HTO O.o 063 2 OLU2 ASP2 EtlU -- 170 4.0 IN80 HTO 0.0 064 2 ~LU2 A8P2 EQU -- 200 3.6 IN64 HTO 0.0 066 2 OLUZ ASP2 Eau -- 160 3.6 HT~VERY 8H 0.006B 2 OLU2 ASP2 EQU -- 110 4.3 HTO 0.0 067 2 OLU2 A6P2 EQU -- 160 3.6 HTO 0.0 068 2 OLU2 ASP2 EIIU -- 180 6.0 o.o 069 2 OLU2 ASP2 EQU -- 160 3.0 IN60 HTO 0.0 OBO 2 OLU2 ASP2 EQU --- lBO 3.0 HT3 o.O
Oôl 2 OLU2 ASP2 EQU -- 1B6 3.0 HT NO AM 0.0 062 2 OLU2 LEU --- 170 3.0 HTO 0.0 003 2 OLU2 A6P2 EQU --- 170 3.0 O.o oe4 2 OLU2 LEU --- 170 3.0 . IH82 HTO 0.0 066 3 OLU2 AliP2 LEU -- 170 3.0 INS6 HEPO H 0.0OOô 2 OLU2 OLY -- 170 3.0 HTO 0.0 007 2 ASP2 LEU -- 1~16 3.0 , 0.0 008 2 A8P2 LEU -- 0.0 0.0 003 2 OLU2 Al;P2 DQU -- 170 3.0 INNS6 ~ A 0.0 070 3 OLU2 A8P2 LEU -- 170 0.0 HE 0.0 071 3 OLU A8P3 LEU -- 170 2.11 0.0 072 2 OLU A8P2 DQU -- 170 3.0 U~O HTO 0.0 073 3 OLU A8P PRO -- 170 4.0 INltO MTO HEPO 0.3074 2 OLU2 ASP2 DQU -- 170 3.0 IN80 0.0 078 2 OLU2 AliP2 DQU -- 170 3.0 HT3 NO AMO 0.0077 2 OLU2 ASP2 DQU -- 170 4.6 INI;6 o.o 078 2 OLU2 ASP2 DQU -- 170 4.0 INC6 0.0 073 4 OLU A8P PRO LY63 -- 170 4.6 LOST BATCH 0.0OSO 3 OLU2 ASP2 ILEU -- 170 4.0 INS4 HTO HEPO 0.0OS1 2 ARO LYS DQU -- 170 3.0 0.0 082 2 OLU A8P2 DQU --~- 170 4.0 INI;4 HT3 0.0 083 3 OLU ASP2 ILEU -- 170 B.O INS4 HT1 HEP4 o.o084 3 OLU2 ASP2 ILEU --- 170 3.0 INS4 HT3 0.0 ~o~ = morphou~ o -- oll ~ = v r~lng , I = eool~tlm- ch r~g~
SUBSTITUTE SHEET

,~138146 WO 93/25~8?~ PCrt~'S9?~/0572~

App ndh A
6i3 P-v- 3 ' P~ot-inoid 8-~ch-e T-mpnn~e 6ph-~ Ibteh Si2-

8~ No 8AA O Additiv- C Ihr~ R~ino ~ OP rD_ 311-CP 4 -iiLU2LY6H2PHE2ASP 6ULFA 180 1.7 IND40iMT30HEP3 1.7 312-CP 4 ~i3LU2 LYiSH2 PHE2 A6P 6ULFA 1i30 0.7 IN6~ MT2 HEP4 17.8 313-CP 4 -OEiLU2 LY6H2PHE2 A8P 6ULFA 180 3.0 IN63 MT3 HEP3 o O.i3 314CP 4 -A6P TYR PHE POEiLU 6ULF0 130 2.6 tiN62 ~ . 0.8 316-CP 4 OEiLU AiSP-VAL LY8HB 6ULF0 130 '.0 IN64 MT4 HEP3 0.3 310-CP 4 OEiLU AiSPrTYR PHE 6ULF0 1i30 21.0 IN6~ MT3 HEP 0.3 317-CP 4 QLN-A6P TYR PHE 8ULFA 176 '.. 0 IH86 MT6 HEPlj 0.3 318-CP b GLU2 A6P2 TYR2 PHE2 ORN6ULFA 180+ .0 MT1 INS4 HEPJ~ 1.0 313-CP 4 -TYR PHE A6P POLU 6ULFA 180 2.6 iNi1~HT4 HEP~ 0.3 320-CP 4 ~TYR PHE POELU A6P 6ULF0 180 1.6 i~HT4 HEI 4 0.3 3Z1-CP 6 ~3LU2 A6P2 TYR2 PHE2 ORN6ULF0 180 + 3.0 iN63 HT2 HE~ 1.0 322-CP 4 GLU2 LY8H2 PHE2 AfiP- 6ULF0 182 1.2 11~i62 MT2 HEP2 O.d 323-CP 4 OLU ASP TYR PHE- 6ULF0 130 .0 A8iORT 1i~i.0 324-CP 4 -OLU A8P TYR PHE iSULFO 130 3.0 INS4 MT4 HEPi~ 2.0 326-CP 6 OEiLU2 ASP2 TYR2 PHE2 ORN 6ULF0 1i30 + 3.0 liW;li~ MT2 HEP 1.0 326-CP 4 -OEiLU A6P TYR PHE 6ULF0 130 8.6 iN63 HTO HEP3 18.0 327-CP 4 -OLU A6P TYR PHE 6ULF0 180 '.0 IN6~ HT6 HEP3 17.0 328-CP 4 -OEiLU A6P TYR PHE 6ULF0 180 3.0 IN66 - 1 L. 17.0 328-7- 4 -13LU A6P TYR PHE 6ULFO .0 IN63 HTO- HEP4 .0 328-CP 4 -QLU A6P TYR PHE 6ULF0 176 t.6 IN66 HT3- HEP6 1.0 330-CP 2 ASP PHE 6ULF0 180 3.0 IN60- HT1- HEPO 0.6 331-CP 2 A6P2 PHE 6ULF0 180 S.O IN60- HTO- HEPO 0.6 332-332 2 ASP3 PHE 6ULF0 180 3.0 IN6~HT1-HEPOc 0.6333-7A 4 -OEiLU ASP TYR PHE 6ULF0 180 6.0 IN62 HT~ HEP6- 17.0 33470V 4 -OELU ASP TYR PHE SULFO 180 6.0 INIU~HT6 HEP4 17.0 336-CP 2 -A6P PHE2 6ULF0 180 3.0 IN61-HT2 HEP1- 0.6 33i3-11 6 -OELU2 A6P2 TYR2 PHE2 ORN6ULF0 180 .0 IN63- HT3- HEP4 2.0 337-337 2 -A6P2 TYR 6ULF0 180 6.6 IN62 HTOeHEPOc 0.6338-CP 2 -A8P TYR 6ULFO t80 3.0 IN60 HTO HEPO 1.0338-CP 2 -A6P3 TYR 6ULF0 180 3.0 IN60- HTO HEPO 0.6 340 4 -i3LU A6P TYR PHE 6ULFO .0 1.6 341 4 -13LU A6P TYR PHE 6ULFO .0 17.0 342 342 2 -ASP TYR2 6ULFO .0 IN60HTOHEP0 0.6 342~CP 2 -A6P TYR2 6ULFO .0 U~60 HT2 HEPO 0.6 343 4 -OLU A6P TYR PHE 6ULFO .0 17.0 344 4 4LU A6P TYR PHE 6ULFO .0 Z.O
346-CP 2 -A6P2 PHE 6ULFO .0 IN60- HTO HEPO .0 344CP 4 ~LN A6P TYR PHE 6ULFO .0 INI;O-HT1-HEP2 .0 347 4 ~LU2 A6P2TYR6 PHE6 6ULFO .0 .0 348 2 -A6P2PHE 6ULFO .0 .0 348 2 -PHE A6P2 6ULFO .0 .0 360 2 -ASP2 PHE 8ULFO .0 .0 361 361 3 ~LU2 TYR PHE 6ULFO .0 IN63 HTZ HEP3- .0 362 6 -13LU2 A6P2 TYR2 PHE2 ORN 6ULFO .0 .0 363 6 -GLU2 ASP2 TYR2 PHE2 ORN 6ULFO .0 .0 Gb-~ = morp~ou~ o = oil = v r~inV I ~ = cool~ tin~- ch npe - SUBSTlTUtE SHEET

wo 93/2558~ Pcr/~s9~/0572~

App ndh A
~0, P~g- 4 of Prot-lnoid i~tch--T-rnp n", 6ph-ro ~t-h 6~-8t No ~AA C~ ~1Addhlv C Ihr) ~9 ~ OP rO_ Z3b-CP 1 ASP 6UL~H 180 1.6 INS2 HT2 HEP30- 0.3 2117-CP 4 OLU A6P-TYR PHE CUL-H 180 l.b INS2 HT4- HEP3 O.b Z~10-CP 4 aLU LY8 PHE ASP SUL-H 100 1.7 IN6b HT4 HEPZ 0.8 300-CP 6 GLU ORN ASP LY6 PHE ---- 180 S.0 IN63 HT3 HEP3 0.3 303-CP 4 GLU A8P-TYR PHE SUL-H 17b 8.0 IN84 HT2 HEP3- 2.0 304-CP 6 OLU A6P TYR PHE ORNO.bSUL-H 180 S.0 IN6~ HT2 HEPS 2.0 306 4 -PGLU AW.i3TYR PHE 6UL 0.0 IN S HT2 HEP3 0,3 300-CP 4 -I~LU ASP.bTYR PHE 8UL 0.0 INSS HT2 HEP2 0.3 307-CP 4 GLN ASP TYR PHE 6ULF0 17b 4.0 INfi40 HT4 HEP40 0.3 - .0 .0 000 .0 .0 001 Z OLUZ AW2 EQU -- 1704.0 0.0 002 Z GLU A6P EQU -- 14~0.0 0.0 003 2 ~LU A6P EQU -- 1030.0 0.0 Of~4 0 - 2040.0 0.0 OOb 2 OLU AU EQU -- 1703.0 0.0 006 2 GLU A8P EQU -- lb4S.O 0.0 007 2 GLU A6P EQU --- 1116 Z O O.o 008 2 GLU A6P EQU -- lb4S.O 0.0 008 2 OLU2 A6P2 EQU --- 1023.0 0.0 010 2 OLU2 AW2 EQU -- 1034.0 0.0 011 2 OLU2 A8P2 EQU -- 1006.0 0.0 012 2 1~LU2 A6P2 EQU --- lb44.0 0.0 013 2 IILU2 AW2 EQU -- 1704.0 0.0 014 2 OLU2 AW2 EQU -- 174S.b 0.0 010 2 . GLU2 A6P2 EQU -- 170S.b 0.0 017 2 t~LU2 A8P2 EQU -- 170S.b 0.0 018 2 OLU2 A8n EQU -- 1703.b 0.0 010 2 QLU ~P EQU -- 1803.b 0.0 020 2 QLU2 A6P2 EQU -- 1804.b HT 0.0 021 2 OLU2 A6P2 EQU _- 1803.b 0.0 022 2 GLU2 A8P2 EQU -- 1803.b 0.0 023 2 OLU2 A6P2 EQU --- 180S.3 0.0 024 2 OLU2 AW2 EQU --~- 17b3.3 0.0 02b 2 OLU2 ASP2 EQU ---- 17b3.0 0.0 026 3 GLU2 A6P2 ASPG ~-- 17b3.0 0.0 027 3 OLU2 ASP2 6ER -- 13b6.0 0.0 028 2 ~LU2 ASP2 EQU ---- 17b3.b 0.0 025 2 GLU2 A6P2 EQU --- 17b3.b 0.0 031 2 OLU2 A6P2 EQU -- 1703.3 0.0 032 2 OLU2 ASP2 EQU --- 170S.b HTO 0.0 033 2 OLU2 ASP2 EQU -- 17bS.O ITO 0.0 034 2 GLU2 A6P2 EQU -- 1800.0 HTO 0.0 03b 2 OLU2 A6P2 EQU --- 0.0 HTO 0.0 030 2 OLU2 A6P2 OQU -- 17bS.O HTO 0.0 037 2 GLU2 ASP2 EQU --- 17b21.0 0.0 Glo~ = morphou- o = oil = v-r~inU I ~ = cooi~ orn chonpe SUBSTITUTE SHE~T

21381~fi wo 93/2558~ Pcr/us93/0572~

App ndla A
P-g- 6 o~

Prot-lnol~ B tch--T-mp nm 8ph-~ ~tch 8~-Bt No JAA C Addi~v C Ihrl ~ ~r O~r~
243 <3K 4 m 1171 Fll,l Y8H2PGLU ~--- 180 3.0 INSO HTO HEPO 0.1 Z60 <3K 6 P'31" .P :-~Y62LEUA8P2 ----- 1W 3.0 IN80HTOHEPO 0.1 261 <3K 4 mll.~ n P.lEi SUL-H 180 3.0 INI;4HT4HEP2 0.1 262-CP 4 ICLU I ASPIVAL LY8 --- 170 3.0 IN81 HT2 HEP1 0.0 263-CP 4 GLU A8P TYR PHE 8UL-H 1S0 '.6 INS1 HTO HEPO 2.0 263 4 GLU ASP-TYR PHE 8UL-H 1S0 10.0 lNS4 HT4 HEP4 1.0 264CP 6 GLU2A8P2-TYR2PHE20RN 8UL~H 180 8.6 lNa HT4 HEP~4 0.1 266-CP 6 GLU A8PTYR-PHE ORN 8UL-H 180 3.0 INS2 HT4 HEP4 0.3 268-CP 4 GLU2LY8H2PHE2POLU --- 180 3.0 IN80 HTO HEPO 0.1 267-CP 4 GLU A8P AROH ORNH -- 180 3.0 INSO HTO HEPO 0.0 26B <3K 3 GLU A8P ARGH 180 3.0 INS1 HT1 HEPO 0.0 2611 3K 4 13LU A8P-TYR PHE 8UL-H 180 3.0 INS3 HT3 HEP3 0.3 2110 3K 4 OLU A6P~TYR PHE 6UL-H 1S0 2.6 IN82 HT3 HEP2 0.3 231-CP 4 ~3LU A6P-TYR PHE 8UL-H 180 J.O INSO HTO HEPO 0.3 21S2-CP 4 GLU ORNH A8P LY8FB 8UL-H 180 3.6 IN80 HT1 HEP4 0.3 233-CP 4 GLU LY8H2 PHE2 A8P - 180 :1.0 IN83 HT3 HEPO 0.3 Z~CP 4 13LU LY8H2 PHE2 A8P -- 180 3.2 IN86 HT3 HEP~4 0.4 ZIIII-CP 4 CLU2 LY8H2 PHE2 A8P --- 180 S.O IN84 HT4 HEP~4 0.3 207-CP 3 OLU LY8FB A8P LY8FB 8UL-H 180 + 3.0 IN& HTo HEPe 0.3 238 4 GLU A8P-TYR PHE 8UL-H 1110 2.6 IN80 HTO HEPO 0.3 21111-CP 4 OLU ORNH A8P-LY8FB 8UL-H 180 '.0 IN& HTo HEPc 0.3 270~CP 4 13LU A8P-TYR PHE 8UL-H 180 1.6 IN86 HT4 HEPO 1.6 271 3 GLU L~ IFB . .IC 8UL-H 180 1.6 IN83 HT'oHEP40 0.0 272-CP 4 13LU2 LEU2 LY8H2 TYR1 --- 180 3.0 IN& HT1 HEP4 0.1 273-CP 4 ~LU2LEU2LY8H2PHE1 -- 180 3.0 IN112 HT2HEP2- 0.1 274~CP 4 GLU LEU AR13 TYR -- 180 J.O IN& HTe HEPc 0.1 276-CP 4 13LU AROH-TYR SUL 180 1.6 ~18e HTe HEPc 0.3 27~CP 4 aW2 LEU2 AR1~2 PHE -- 180 3.0 IN83 HTJ HEP4 0.1 277-CP 3 OLU LYS TYR 8UL-H 130 1.6 IN8e HTe HE~o 0.3 278~CP 3 13LU LY8 PHE 8UL H 190 1.6 IN& HTe HEP4 0.3 2711-CP 3 13LU LY8 ALA --- 160 1.6 IN& HTe HEPe 0.3280-CP 4 m U~ I UF .3_UTYRGLPHE 8UL-H 1BO 1.6 IN84 HT3 HEP4 0.4281-CP 4 OLU1 A8P1 TYR2.6~PHE2.6 8UL-H 180 3.0 IN84 HT- HEP2 1.0 282-CP 3 OLU2 LY86 HEP2 --- 180 1.6 i~80 HTO HEP2 0.3 283-CP 4 GLU2 LY86 PHE6 TYR2 -- 1110 1.6 IN80 HTO HEP3 0.1 284 6 (tl~- ~ 2-TYR2PHE20RN 8UL-H 180 3.0 1N8R HT'oHEP2 1.0 286-CP 2 13LUI2XI A8PI2XI --- 180 3.0 IN& HTc HEPc 0.3 2818CP 2 GLU A8P12XI -- 180 2.6 lN& HTc HEPc 0.3 287-CP 2 OLU PHE -- 180 3.6 IN83 HT2 HEP3 0.3 288-CP 3 GLU ORN PHE -- 180 3.0 IN& HTc HEPc 0.3 28H 2 13LU ARG -- 180 1.0 0.3 290-CP 3 OLU ARG PHE -- 180 3.0 iN82 HT2 HEP2 0.3 2111-CP 3 13LU LY8 PHE 8UL-H 11~0 1.6 IN84 HT30 HEP40 0.3 2112-CP 6 GLU A8P ARO ORN PHE 8UL~H 180 3.0 IN80 HTO HEPO 0.3 283-CP 4 GLU A8P ARG ORN PHE 8UL-H 180 3 0 IN83 HT3 HEP3 0.3 21~CP 4 OLU2 LY8H2 PHE2 A8P --- 180 3.0 IN83 HT4 HEP4 0.3 Glo---r~: = mo~ihou- o = oil = v-r~ing . l = cooi~tim- ch-nge SUBSTITUTE SHEET

wo 93/2558~ Pcr/~sg~/0572~
~3~6 App ndbl A
o2 P-o- ~' of P~ot-lnold 8Jteh-e T-mo nm- Sph-r Ibtch 8i~e Bt No AA C AddWv C Ihrl R tino 1~ OD rD_ 182-CP 3 13LU LY6FB A6P -- 1il6 3.0 IN64 HTO 0.3 183 >IIK 4 ~aLU~A6P~TYRPHE PA 176 '.0 HTOHEPO 0.3 1~CP 3 ULU LY8F8 A6P TRIOL 1S6 3.0 W81 HTO HEP2 0.3 11~1;CP 3 OLU A6P VAL2 -- 170 S.2 IN82 HT1 HEPO 0.3 111~CP 4 ~3LU A6P TYR PHE PA 176 '.2 IN62 HTli HEP4 1.0 167-CP 4 13LU A8P TYR PHE 6UL 176 2.7 INg2 HT6 HEP6 0.3 108-CP 3 ~3LU LY8FB A8P --- 1S6 3.2 IN6S 1.0 200-CP 3 GLU LY6H A6P PA 186 3.0 IN64 HTO HEPO 0.3 201-CP 3 OLU LY6F8 A6P 6ULFA1116 3.0 IN64 HTO HEPS 0.3 203-CP 4 ~3LU A6P VAL LY6 ~- 170 3.0 IN66 HT6 HEP 0.3 20~CP 3 GLU LY6 A6P ---- 186 3.0 IN64 HTO HEPO 0.3 206-CP 4 aLU ASP-TYR PHE 6ULFA 176 3.7 IN6~ HTO 0.6 208 3 OLUH LY6H A6PH N-HCOSHEOH80 8.0 WSO HTO HEPO 0.0 203-CP 4 13LU A6P-VAL LY6 SULFA 170 3.2 IN8~ HT4 HEP3 W 0.3 210-CP 4 ~lLU A8P-VAL LY8F11 6ULFA 170 3.0 IN64 HT4 HEP3 2.0213-CP 3 ULU-LY8 HIS 6ULFA 180 3.0 IN60 HTO HEPO 0.3 216-CP 3 aLU A8P aLY2 -- 180 6.6 IN8S HTO HEPO 0.3 2111-CP 4 13LU A6P-TYR PHE 6ULFA 176 3.0 IN64 HT4 HEP4 2.0 217 3 13LUA8PLY61DIET8TER) HEOH/ET3N 76 29.0 0.0 218-CP 4 aLU A8P-TYR PHE 6ULFA 176 .0 INSO HTO HEPO A 0.3 2111-CP 3 aLU-LY6-LEU6UL/POC13 180+ 8.6 INS2 HTO HEP2 0.3 220-CP 4 aLU ASP-TYR PHE 8ULFA 180 20.6 INS4 HT4 HEP6 0.3 221-CP 3 -A8P2 TYR PHE 8ULFA 180 22.0 IN82 HTO HEPO 0.3 222-CP 3 -LY6FB2 AR132 LEU 6ULFA 180 4.0 IN60 HTO HEP2 0.3224 CP 4 13LU A6P-TYR PHE- SU/PA 180 1).0 IN63 HTO HEPO 0.3 226-CP 3 MO~ER TYR 6ULF 180 3.6 IN62 HTO HEPO 0.3 221S <3K 4 OLU A6P TYR PHE 6ULF 180 4.0 IN83 HT4 HEP3 0.3 229 <3K 4 -13LU A6P TYR PHE 8ULF 180 6.6 IN8S HTO HEPO 0.3 230-CP 2 aLUTYR -- 180 4.0 W64 HTO HEPO 0.3 231-CP 3 ~3LU LY6FII PHE 6ULF 180 S.6 INS2 HTO HEPO 0.3 232-CP 3 aLU LEU ARa -- 180 4.0 IN60 HTO HEPO 0.3 2SS-CP 3 aLU LEU LY8H -- 180 4.0 IN60 HTO HEPO 0.3 23~CP 4 laLU A8P TYR PHE~ 6ULF 160 27.0 INS3 HTO HEPO 0.3 236-CP ' -laLU A6P) TYRlOPHE10 liULF 180 2ZO IN80 HTO HEPO 0.023~CP 3 OLU TYR LY8HCL --- 180 ZO IN60 HTO HEPO 0.3 237 <3K 4 13LU2LEU2LY8H2A8P -- 180 3.0 WBOHTOHEPO 0.1 238 <3K 4 aLU A6P TYR6 PHE6 8ULF 180 3.0 W60 HTO HEPO 0.1 2311-CP 3 ~LU A6P LEU 8UL-H 180 1.0 W83 HTO HEPO 0.0240-CP 3 laLU A8P) LEU 8UL-H 170 4.0 IN64 HTO HEPO 0.0 241 <3K 3 -laLU ASP) LEU 6UL-H 160 6.0 INS3 HTO HEPO 0.0242-CP 3 IOLU ASP LEU) 8UL-H 170 2.6 INSO HTO HEPO 0.1243-CP 6 pOI IIOA~ Y2LEU ~- 180 3.0 INSO HTO HEPO 0.1 244 CP 3 IGLU A8P) LEU --- 180 Z6 INSO HTO HEPO 0.0 246-CP 3 113LU A8P) LEU -- 170 1.0 INS3 HT1 HEPO 0.0 24~ <3K 6 OLU2 LY8HW PHE2 ASP ~- 180 o.O INS4 HT4 HEP4 0.0 247-CP 3 aLU ASP LEU -~ 170 6.0 IN80 HTO HEPO 0.0 ~o---rv: = morphou- o oll = v rjing I ~ = cook Om~ ch-nge SU~STITUTE SHEET

WO 93/2558~ ~ 1 3 8 1 4 6 PCr/~'S9?i/0572~

P-v- 7 o- 8 Prot-lnold ~tch--T-rnp n.... 6Vh-rlf l~tch 81~-8t No ~AA C ' AddWv- Q Ihr~ ~ 1~ OP rD_146 3 BLU A8P LY8FB PPA 186 0.0 HTO 0.0 14~ 3 OLU ASP VAL2 PPA 170 3.6 IN82 HTO 0.0 147 4 13LU A8P PHE ALA - - 170 S.O IN84 HT3 HEP4 0.0 148 4 13LU A8P TYR PHE PA 170 3.0 HTO HEP4 0.0 148 3 OLU A8P PHE2 170 a.o o.o 160 4 ~ILU A8P,LEU PHE PA 17024.0 HTO HEPO 0.0 161 4 OLU A8P TYR PHE PA 170 0.0 IN84 HT4 HEP4 0.0 162 4 OLU A8P TYR PHE PA 170 6.0 0.0 163 3 OLU LY8FB PHE PA 17024.0 0.0 164 4 ~ILU A8P TYR PHE PA 170 4.0 0.0 166 3 aLU2 TYR PHE PA 170 4.0 IN8, HT6 HEP3 0.0 168 3 ~lLU4 LY62 PHE -- 170 0.0 INSO HTO HEPc 0.0 167 3 OLU2 TYR LEU PA 170 6.0 INS2 HT1 HEOP 0.0 168 3 OLU2 PHE LEU PA 176 6.0 IN84 HTO HEP4 0.0 163 3 GLU3 PHE TYR PA 176 6.0 INS4 HT4 HEP4 0.0 160 4 OLU8 LY82 PHE TYR PA170 0.0 IN& HTe HEPe 0.0 181 4 OLU4 PHE2 TYR2 CY8 PA170 4.0 IN84 HT HEP 0.0 lo2 3 CLU2 TYR PHE PA 170 6.6 INI;3 HTO HEP2 0.0 183 3 aLU2 PHE TYR PA 170 6.0 INS3 HT2 HEP3 0.0 164 3 GLU2 PHE TYR PA 170 6.0 IN84 HT4 HEP4 0.0 186 4 CLU2 A8P PHE2 TYR2 PA170 :~.0 IN8~ HTO HEPO 0.0 10~1 3 OLU LY8FB POLU PA 170 7.0 0.0 167 4 OLU ASP TYR PHE PA 170 0.6 0.0 108 3 aLU A8P LYSFB PPA 186 1 72.0 HTO 0.0 1119 3 OLU A8P LY8FII WA 18672.0 HTO 0.0 170 3 GLU A8P LY8F8 -- 186 7.0 HT8 0.0 171 3 OLU LSYHa AU H.OIL 180 7.0 HTO 0.0 172 4 t~LU A8P TYR PHE PA 170 0.0 HT1 0.0 173 3 OLU LY8 A8P UIINERAL 0.186 3.0 A80RT 0.3 174 >OK 3 aLU LY8 A8PaLYCERlN 186 3.0 IN82 HT1 HEP3 0.3 176 >OK 4 OLU A8P TYR PHE PA 172 3.6 1.0 178 >OK 3 OLU LYS;2 LY8 -- 180 3.0 IN80 HTO HEPO 0.3 177 >ffK 3 CLU ARO A8P -- 180 3.2 IN80 HT2 HEPO 0.3 178 >OK 3 f3LU LY8 A8P --- 180 3.2 INSO HTO HEP1 0.11711 >OK 4 OLU ASP TYR PHE PA 176 4.0 1.0 180 >OK 4 OLU ASP TYR PHE PA 176 7.0 lif Not--. 1.0 181 >OK 3 OLU LY8 A8P --- 186 3.0 IN80 HTO 0.3 182 >ôK 4 OLU A8P TYR PHE PA 176 3.7 HT1 HEP1 0.3 183 4 Pt~LU A8P TYR PHE PA176 4.0 ABORTRETRY 0.3 184-CP 4 OLU ASP TYR PHE PA 176 3.6 HT2 HEP4 0.3 186-CP 4 ~3LU A8P TYR PHE PA 170 4.2 IN8 HT4 HEP4 1.0 187 3 A8P TYR PHE PA 170 .0 A,BORT 0.3 188-CP 3 A8P TYR PHE PA 16021.2 INSO HTO HEPO 0.3 188-CP 4 CLU ASP TYR PHE PA 17~f4.0 HT4 HEP6 1.0 180-CP 4 OLU ASP TYR PHE PA 176 4.0 HT8 HEP4 1.0 181-CP 3 ASP2 TYR PHE PA 16024.0 HTO HEPO 0.3 ~o~ rno-phou- o = oil = v-r~ing . , t = cook tirn- ch nv~
~a ", ~JTE SHEE~

wo 93/2558~ PCr/~lS9~/0572.~
?,,,3a~46 App ndh~ A
P-9- 8 ot Pmt~lnold 8 teh--T-mpTlm 6ph-~ 8 toh a~.
Bt No ~AA C Addh~v C Ihrl R-tln~ 1~ Op r D~018 2 1~LU2 A6P2 EQU ~ 1702.6 0.0 100 3 OLU A6P VAL2 ----- 1703.0 101 3 13LU A6P VAL2 ---- 1703.0 -102 3 OLU A8P VAL2 ~ 1703.0 -103 4 OLU A6P OLY VAL -- 1703.6 IN64 0.0 104 4 <lLU A6P VAL LEU ---- 1703.6 IN64 HT2 HEP6 0.0 106 3 tlLU A6P 13LY2 --- 1804.0 IN64 HT2 0.0 103 4 OLU A6P VAL LEU ---- 1706.0 0.0 107 4 13LU2 A6P2 OLY VAL2 --- 1703.0 IN66 0.0 108 4 13LU2 A6P2 OLY VAL2 -- 1704.0 IN64 NO AIIIIORPHO 0.0 103 4 OLU2 A6P2 13LY VAL2 --- 1704.0 IN84 HT1 0.0 110 4 ~LU2A6P2~1LY2VAL --- 1703.6 0.0 111 6 OLUA6POLYVALCYS -- 1703.0 -112 4 ~3LU A6P OLY HE --- 1704.0 IN64 HT3 HEP4 0.0 113 4 OLU A6P VAL2 13LY --- 1703.0 INS2 HTO 0.0 114 3 OLU A6P VAL -- 1703.0 INS4 HTO 0.0 116 3 OLU VAL TYR -- 1704.0 0.0 113 4 13LU A6P VAL LY6 -- 170'.0 0.0 117 3 OLU VAL TYR ---- 1703.0 IN86 0.0 118 2 OLUTYR ---- 1703.6 IN66 HTO HEPO 0.0 1111 2 13LU2 ASP2 EQU ----- 170 3.6 IN66 HT1 0.0 120 3 13LU A6P TYR ----- 1704.6 INI;O HTO EPO 0.0 121 4 OLU A6P TYR PHE -- - 1704.0 IN66 HT3 HEP4 0.0 122 4 ~ILU A6P VAL TYR --- 1703.0 IN63 HTO HEPO 0.0 123 1 OLU -- 1704.6 CAN T DRY 0.0 124 3 OLU TYR VAL --- 1703.6 IN64 HT3 HEP3 0.0 126 3 PI~LU VAL TYR ---- 1703.6 IN6J HT2 0.0 12B 4 OLU A6P VAL2 13LY ---~ 1704.0 IN61 0.0 127 4 ~3LU2 A6PA VAL2 PHE ----- 1704.0 INS3 HT2 HEP4 0.0 128 2 ~LU2 Al~P2 EQU -- 1703.6 HTO 0.0 128 2 aLU2 ASP2 EQU -- VARY 4.0 IN63 HTO 0.0 130 2 OLU2 A6P2 EQU ----- 2203.0 IN66 HTO 0.0 131 2 llLU2 LYI;FB ----- 1863.0 IN61 HTO 0.0 132 3 13LU A6P LY6F8 -- 1863.0 IN63 HT2 HEP2 0.0 133 3 ~3LU A6P LY6F8 PA 180IL2 INS6 HT1 HEP2 0.0 134 4 OLU A6P LY6 VAL PA 1863.0 0.0 136 3 13LU A6P LY6F8 13LYC 186 0.6 INS1 HT1 0 0 138 2 OLU2 ASP2 DQU ----- 1663.0 HTO 0.0 137 6 nl 117'- ~' EU THY VAL ---- 186 4.0 INSO HTO 0.0 138 4 13LU A6P VAL THY -- 1864.6 INS1 HT3 0.0 138 4 GLU AW VAL TYR PPA 180+ 7ZO INS2 HT3 0.0 140 3 OLU ASP LY6F8 PPA 120 + 7ZO IN66 HT2 HEP4 0.0 141 3 OLU LY6Fv'r~ --- 1868.0 0.0 142 3 13LU A6P LY6F8 PPA 120+ 7ZO IN61 HT1 o.o 143 3 13LU VAL TYR --- 1703.0 IN62 HT2 o.o 144 4 GLU2 A6P2 OLY VAL2 ----- 1703.0 INS1 HT1 o.o Glo---rt: = rnorphou- o = oi~ = v r~inv t - I = cook tirn- ch-nve SUBSTITUTE SHEET

` wo 93/25s8~ 2 1 3 8 1 9 6 PCr/~lS9~/0572~

App ndh A
~6 P~ o~

Pn~t lnoid i~h--T-mp Tln~- Sph-~ i~btch Sk-i3t No AA C ' Addi v- C Ihr) i~tlnu Op rO e 3~0 6 -ICLU A8P TYR PHE)Z ORN SULFOLAHE O O

Sph-r- R tlng O = Wo~t 6 = i~-t IHT = In ulln UT = on pt~
HEP = h-~dn SuiM = dh~bn-, n~-die-l gmd-Sdt- = Sdt = Sd = Slffobn-PA ~ i_phodc dd Equ - quibm~
13LYC = ol~d TRIOL -- Ubl~
PPA = F IW ~ Hdd hll Oil ~ ~d o ~n-r l oil Olo--~ = morphou~ o = oil ~ -r~nu I - ~ = coo~ tim- eh n~
SUBSTITUTE SHEET

WO 93/2558~PCr/~!S9~/0572~
~3~6 Appendix B
page 1 of 2 IEF TABLES
Protoinoid sortino, pKa and c~..",o tion Chomicol bosis for .~, ., ' u ODS

Matorial ID No. Co",,-l n Sphoro frac Sphoro pH Sphoro IEF Sphoro Max UV Max UV
~ no amp) numbor rongo ratina Matnx rating Frac. No. pH
-202B Glu2.4 Asp2Val2GLv no sphor -- INS4 HTO HEP3 -- ---210>1K Glu Asp Val Lvs 1420 2.3-4.4 2-3 INS4 HT4 HEP3 1419 4.43.0 213 > 1 k GIu Lyr FP HbFB 16-19 1.7-2.1 2 INSO HTO HEP0 11 -16 7.6-2.1 218<3k Glu A-p Tyr Pho 18-20 3.2-2.7 2 INSO HTO HEP0 1-7 12.2-9.3 129 Glu Asp Equ 10-18 2.5-4.4 3 INS3 HTO lE 18 2.9-2.5 214>1k GluL~FBAro noophor -- --- INSOHTOHEPO 1-4 11.8-9.2176 Glu2 Lyr~FB2 LVoFp no phor --- -- INSO HTO HEPO ~6 9-8.6 222-cp A-o2 Ly FB2 Ly FB 71-47 9.9-11.7 27 INSO HTO HEP2 2-6 8.5-11.5 202B Glu2.4 Ap 2 Val2 GlV 8-12 3.3-5 2,1-2 INS4 HTO HEP3 8-12 5-3.3 223-cp Aro2 LV FB2 Lou pGlu 1 10.3 2-3 INSO HTO HEP2 1 10.3 223-cp Ar~ 2 LV FB2 Lou pGlu 1-5 9.0-12 2 INSO HTO HEP2 3 10.7 170a Glu Asp LvsFB 16-20 2.3-5.0 2-3 HTS 1~20 5-2.3 2163K Glu Asp Tyr Pho~sul~ 1420 2.43.9 2 INS4 HT4 HEP4 1417 3.9-2.8 125 pGlu V-l Tyr 13-20 2.7-3.6 1-2 INS3 HT2 1420 3.2-2.7 228-H20 sul Val2 GIV2 pGlu no phor -- -- INSO HTO HEP0 9-13 5-3-4 228-AB ul Val2 GlV2 pGlu no sphor -- -- INSO HTO HEP0 16-18 3.8-3.3 177 Glu Asp Aro 1410 - 5.2-3.9 2-3 INSO HT2 HEP0 1419 5.2-3.9 118 Glu Tyr 12-14 5.2-6.0 1-2 INS5 HTO HEP0 12-19 6-4.2 153 Glu L~FB PHo no sphor -- -- 8-9 9.1-10.2 131 Glu2 Lvr~ no ophor -- -- INS1 HTO 1417 4.3-3.6 162 Glu2 Tyr Pho 16-17 4.1-3.7 2-3 INS3 HTO HEP2 16-17 4.1-3.7 156 Glu4 L~2 Pho no phor -- -- INSO 20 4.5 124 Glu Val Tvr no phor --- -- INS4 HT3 13-14 3.7-3.5 210>1K Glu Asp LVs Val 12-20 3.6-3.1 1-2 INS4 HT4 HEP3 12-19 3.6-3.2 156 Glu4 LVs2 Pho 1417 3.6-3.1 2 INSO 14 3.6 231 Glu LyrJ Pho sul no phor -- --- INS2 HTO HEP3 18 9.1 232 Glu Lou Lys no phor -- -- INSO HTO HEP2 6 10.9 233 Glu Lou Ar~ no phor --- --- INSO HTO HEP2 9 10blank 29~ - ", `~ '~t ~ -- --- --- -- -- ---216~3k Glu A-p wl Tyr Pho 1420 4.1-2.6 2 INS4 HT4 HEP4 18-20 3-2.6 230 > 1 k Glu Tyr 13-20 3.9-3 3 INS4 HT4 HEP0 15-20 3.3-3 170O Glu Asp Ly FB 15-20 3.9-2.2 2 HT5 18-19 3.3-2.6 236-cp Glu Tvr Lvo-HC1 19-20 3.0 0-1 INS2 HTO HEPO 19-20 3 216~3k Glu Asp oul TVrpho 1420 3.3-2.2 2 INS4 HT4 HEP4 20 2.2 216~3k Glu Asp sul Tvr Pho 16-20 3.9-2.3 1-2 INS4 HT4 HEP4 20 2-3 237-cp Glu2 Lou2 Lyr~2 Asp no phor --- -- INSO HTO HEP0 9-12 4.6-4 243-cs pGIuAro2Lvs7l ~ 4~ no ophor - _ INSO HTO HEP0 15-17 8.5-7 246-cs Glu2 Ly-H2 Pho2 Asp 17-20 4.1-2.2 2-3 INS4 HT4 HEP4 17-20 4.1-2.2 250-cs pGluAro2Lyr~H71. JA~ 2 no sphor --- --- INSO HTO HEP0 11 -14 8-6.8 249~3K Glu2 Lou2 Ly H2 pGlu no sphor -- -- INSO HTO HEP0 254cp r~ TvrPhoOrn.5 18 5.42.5 2 INS3 HT4 HEP4 1420 3.7-2.1 253-cp Glu Asp wl Tvr Pho 18-20 3-4 2 INSO HTO HEP0 1,4,19-20 11.5,3-3.5 235<3k G' ~ J'Tyr10Pho10 18-20P 2.6-3.2 1-2 INSO HTO HEP0 1-3 11 256-cp Glu2 LyrlH2 Pho2 pGlu 13-20 3.7-4.0 1-2 INSO HTO HEP0 2-5,1420 2.7-4,7-10 238~3K r'~A~, ~ITvr5Pho5 no-phor --- -- INS1 HT3HEP4 2 11.4 255-CP GluAspTv. ~I~Crn 13-20 5.3-3.1 2 INS1 HT3HEP4 1-6,19-20 11-9.3.6-3 251 <3K Glu2 Asp2sulTvr5Pho5 16-20 5.5-3.3 2 INS4 HT4 HEP4 17-20 5.8-3.8 SUBSTITUTE SHEET

WO 93~2558~ 2 1 3 8 1 ~ 6 PCr/~lS9~/0~72~

Appendix B
page 2 of 2 IEF TABLES
Protoinoid corano, pKc ond .,v"., : n Chomicol ba~ic for ".;~ , h .. ODS

Run No. Matoriol ID No. Cc~ n Sphoro frcc Sphoro pH Sphoro IEF Sphoro Max UV Max UV
~ no omp) numbor ronoo r-ino Motrix rotino Froc. No. pH

47 257-CP Glu Acp AroH OmH no cphor -- -- INSO HTO HEP0 19-20 5-3 48 257-CP Glu Aop AroH OmH- no ophor -- -- INSO HTO HEP0 15-17 8-9-8.5 49 258>3K GluAopAroH no-phor -- -- INSOHTOHEP0 1,17-20 9.8,42.5 262-CP GluOrnAcpL~FB 11-18 6.8-3.5 2 INSOHT1 HEP4 15 4.8 51 262-FILT Glu Orn Acp LycFB 411 7.7-5.4 1-2 in-O ntl hop4 1-2,12-20 9.4.6-1.8 52 267-cp Glu Ly-FB A-p L~BP no ophor -- -- INSa Htc HEPc 1420 6.3-3.8 53 268-cp c Glu Acp cul Tyr Pho 15-20 4.5-2.34 2-3 INS4OHT4OHEP4H 1-10,18-20 12-2.5 54 269-cp Glu OrnH Acp LycFB 17-20 2.91-1.4 1 INSc HTc HEPc 4,7,9 9-7.5 273-cp Glu Lou LycH Pho 17-20 3-1.2 1-2 INS2c MT2 HEP20 19 2 56 272/273 Glu Lou L~H no ophor -- -- -- 3-9,13-15 9.8.5,8-8 57 276 Glu Lou Aro Pho 12-18 3.57-1.4 1-2.2 INS2HT2HEP3 1-7,17-20 9-6,1.5-1 58 274 Glu Lou Aro Tyr no ophor -- -- INSc HTc HEPc 16-20 4.141.4 59 272 Glu Lou L~H Tyr no cphor -- -- INSc HTc HEPc 1-2 9.49.3 274A Glu Lou Aro no ophor -- -- -- oll trcc.
61 278 Glu LVc Pho oul 1~20 4.8-3.5 1-2 INSc HTc HEP4 ~ll troc. ---62 284E GluAcpT.,rP~ C ... 1420 3.8-2.1 1-2 INS4cHT4OHEP3 1~20 3.3-2.1 63 287-cp Glu Pho 10-20 3.55-2.3 2 INS3HT2HEP3 18-20,1-7 2.42.3,8 64 284E r~ ~A~ 2Tyr2Pho20rn 1420 3.95-1.6 2 w/oil INS40 HT4OHEP20 3-8 2.3-7.76 288-cp Glu Orn Pho no cphor -- -- INSc HTc HEPc17-20 2.7-1.02 66 293-cp Glu A-p oul Tyr Pho 1-8 1.9-3.9 1-2INS1 HT2 HEP1-67 290-cp Glu Aro Pho 1-7 1.05-3.8 1-2 INS1 HT1 HEP1 18-20 12.1-12.6 68 292-cp Glu Acp Aro no ophor -- --- INS HT HEP 16-20 3.19-1.5 69 300-cp Glu Om Acp Lyc Pho 15-19 4.05-1.5 1-1INS3 HT3 HEP3~ oll froc. --297-cp GLU ASP SUL TYR PHE 1-7 2.38-4.15 2-3INS2 HT4 HEP 1 -2 2.38-2 71 301 <3J GLU ASP SUL TYR PHE -- --- -- INS4 HT2 HEP3 1-20 72 303 GLU ASPSULTYR PHE1-8 2.83-3.76 2-3 INS4HT2HEP3D 1-3,18-20 2.88/1 73 299 GLU2 LYS2 PHE2 ASP 1-7 1.13-3.82 1INS4c HT4 HEP2c 3-7 1.58-3 74 305 PGLU ASP.5 TYR PHE 1-12 2.12-4.20 2-3INS3 HT2 HEP3 11-20 5.541 307-CF' GLN ASP TYR PHE 1-9 2.43-4.48 2-3 INS4c HT4 HEP4A 1 -13 2.43-7.0 76 305 PGLU ASP.5TYR PHE 1-6 2.05-5.56 2-3 INS3 HT2 HEP347 3 3-7.077 1241156 GLU TYR VAL/GLU2 LYS2 PHE -- -- -- 14 H3 H3/lOHOHO 1 10.58 79 319-CP SUL-U TYR PHE ASP PGLU 1-10 2.28-5.3 2-3c INS4OHT4hEP4u 2-8,19-20 2.3-4,12 314CP SUL-TYR PHE ASP PGLU 1-11 1.93-5.30 2 INS2OHT40HEP4c 18 8.95 81 320-CP SUL TYR PHE PGLU ASP 1-7 2,12-4.4 2c INS2cHT40HEP4A 16-20 9.3-1025 81 188~6K ASP2TYRPHE 1-6 1.85-5 OoINSOHTOHEPO 18-20 12-12.5 82 286-CP GLU ASP 1418 2.38-2.02 PARTICLES INSCHTCHEPC 1418 2.38 83 288/188 ASP2 TYR PHE/GLU ORN PHE 1417 4.9-3.1 1.2 INSCHTCHEPC 17-19 3.1-1.65 84 66 GLU2 GLY -- --- -- HTO 16-19 2.85-2.55 0112-2A GLU ASPTYR PHE 1-10 3.17-13.48 1,0-1 INS5OHT30HEP3O 14,2 920,3.32 SUBSTITUTE SHEE~

WO 93/2558~ PCr/~'S9?/0572~ -~3a~6 68 ~ o ~ C s + ~ . 1 5 . i ~ . j 5 . j t .; 0 ~ j 5 . j ~ . j t o j ~ - o _ c o ~ j t ~ j t o j " . ! o j 5 . j t . j ~ " o S C~ N o j ~ G j t . j o j ~ o j " i j ~ j j o . j ~ j j t j j o + r~; j t ~ j " . j "~ i i ~ i; N O; t . j L
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.s ~ 55 0C ~ 0 01` 0 ~ 0 " N N N _ . , ob O O o O O O
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N ~n U~ ~ ~ Z
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~UBSTITUTE SHEET

WO 93/2~i58 ~ 21 3 81 4 6 PCr/l,!S9?s/O~i~2:~

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Claims (35)

WHAT IS CLAIMED IS:

1. A proteinoid comprising a peptide polymer selected from the group consisting of:
i) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and ii) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid, and from at least one third monomer selected from the group consisting of lysine, arginine, and ornithine, said proteinoid being a microsphere or microcap-sule-forming proteinoid and being soluble within a selected pH
range.

2. The proteinoid of claim 1, said proteinoid having a molecular weight ranging between about 250 and about 2400.

3. The proteinoid of claim 2, said proteinoid having a molecular weight ranging between about 250 and about 400.

4. The proteinoid of claim 1, said proteinoid having between 2 to 20 amino acids.

5. The proteinoid of claim 4, said proteinoid having between 2 to 8 amino acids.

6. The proteinoid of claim 1, wherein said proteinoid is an acid-soluble proteinoid and said second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid, and said third monomer is selected from the group consisting of lysine, arginine and ornithine.

7. The proteinoid of claim 1, wherein said proteinoid is a base-soluble proteinoid and said second monomer is selected from the group of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid.

8. A proteinoid carrier comprising a proteinoid comprising i) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and ii) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and at least one third monomer selected from the group consisting of lysine, arginine, and ornithine, said proteinoid being a microsphere- or microcap-sule forming proteinoid and being soluble within a selected pH
range.

9. The proteinoid carrier of claim 8, wherein said proteinoid carrier comprises a proteinoid microsphere.

10. The proteinoid carrier of claim 8, wherein said proteinoid carrier comprises a proteinoid microcapsule.

11. The proteinoid carrier of claim 8, wherein said proteinoid is an acid-soluble proteinoid and said second monomer is selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid and said second monomer is selected from the group of lysine, arginine and ornithine.

12. The proteinoid carrier of claim 8, wherein said proteinoid is a base-soluble proteinoid and said second monomer is selected from the group of glutamic acid, pyroglutamic acid, glutamine and aspartic acid.

13. The proteinoid carrier of claim 8, wherein said proteinoid carrier having a diameter equal to or less than 10 microns.

14. The proteinoid carrier of claim 8, further encapsulating a cargo.

15. The proteinoid carrier of claim 14, wherein said cargo comprises a fragrance, cosmetic agent, dye, and water soluble vitamin.

16. The proteinoid carrier of claim 14, wherein said cargo is a biologically active agent.

17. The proteinoid carrier of claim 16, wherein said biologically active agent comprises an antigen, monoclonal antibody, calcitonin, erythropoietin, alpha interferon, heparin, insulin, growth hormone, atrial naturetic factor, factor IX, or interleukin-II.

18. A composition comprising a biologically active agent encapsulated within a proteinoid microsphere or microcap-sule, said microsphere or microcapsule comprising a proteinoid comprising i) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and ii) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and at least one third monomer selected from the group consisting of lysine, arginine, and ornithine, said proteinoid being a microsphere- or microcap-sule-forming proteinoid and being soluble within a selected pH
range.

19. The composition of claim 18, wherein said pro-teinoid is an acid-soluble proteinoid and said second monomer is selected from the group of lysine, arginine and ornithine.

20. The composition of claim 18, wherein said pro-teinoid is a base-soluble proteinoid and said second monomer is selected from the group of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid.

21. The composition of claim 18, wherein said biologi-cally active agent comprises an antigen, monoclonal antibody, calcitonin, erythropoietin, alpha interferon, heparin, insulin, growth hormone, atrial naturetic factor, factor IX, or interleu-kin-II.

22. A pharmaceutical preparation comprising an oral dosage form of calcitonin.

23. The pharmaceutical preparation according to claim 22, further comprising a microsphere- or microcapsule-forming proteinoid.

24. A pharmaceutical preparation comprising an oral dosage form of a monoclonal antibody.

25. The pharmaceutical preparation according to claim 24, further comprising a microsphere- or microcapsule-forming proteinoid.

26. A pharmaceutical preparation comprising an oral dosage form of erythropoietin.

27. The pharmaceutical preparation according to claim 26, further comprising a microsphere- or microcapsule-forming proteinoid.

28. A pharmaceutical preparation comprising an oral dosage form of alpha-interferon.

29. The pharmaceutical preparation according to claim 28, further comprising a microsphere- or microcapsule-forming proteinoid.

30. A pharmaceutical preparation comprising an oral dosage form of Factor IX.

31. The pharmaceutical preparation according to claim 30, further comprising a microsphere- or microcapsule-forming proteinoid.

32. A method for delivering calcitonin to a mammal which comprises orally administering the pharmaceutical prepara-tion according to claim 23.

33. A method for delivering erythropoietin to a mammal which comprises orally administering the pharmaceutical prepara-tion according to claim 27.

34. A method for delivering alpha-interferon to a mammal which comprises orally administering the pharmaceutical preparation according to claim 29.

35. A method for delivering Factor IX to a mammal which comprises orally administering the pharmaceutical prepara-tion according to claim 31.