CA1079721A - Polypeptide agents for blocking the human allergic response - Google Patents

Abstract

A B S T R A C T
A low molecular weight pentapeptide ASP-THR-GLU-ALA-ARG
blocks the allergic response. The "blocking" pentapeptide, the synthesis and use of the pentapeptide are described.

Description

~0~ 7Zl Backyround of the Invention -The symptons of human allergic disease or more properly the allergic syndrome, are brought about through the release into the organism of vasoactive amines, notably histamine.
The histamine is normally stored in special cells known as mast cells and basophil leucocytes distributed throughout the organism. The mast cells are dispersed throughout human tissue structures, while the basophils circulate with the blood in the body, i.e., within the vascular system.
The above-noted cells manufacture and store histamine within their internal structures, and the histamine remains therein unless a specialized se~uence of events occur to trigger the release of histamine from within the cell structures into the surrounding tissues and vascular system.
More specifically, histamine will be released in response to the presence of specific antigens (allergens) that gain entrance into the organism or may be released by the organism in response to some traumatic occurrence. However, the usual release of histamine from the mast cells or basophils is triggered by a necessary sequence of chemical and immunological events taking place on and in the mast cell and basoph.il structures.
Specifically, the allergen-mast cell (basophil) inter-action is mediated by a group of proteins known as immunoglo-buLin E (IgE) that are manufactured within the body. The IgE manufactured hy the human organism is a complex arrangement of polypeptide chains, each molecule of which may have certain variations in the sequence of amino acids in the polypeptide chain, but all of which in essence may be characterized as having a "Y" like structure, wherein the "tail" (actua:Lly t .

107972~L
base of the "Y") (Fc) polypeptide portion or fragment contains a fixed sequence of "constant region" of peptides along the chain. The "heads" (which are equivalent to the upper arms of the "Y" structure) may have regions wherein the polypeptide chain varies (the variable region of the E'ab) from molecule to molecule. Thus, the IgE molecules genera]ly have identical "tail" peptide sequences but may have a great number of different "head" peptide sequences.
The allergic or immunologic release of histamine within the organism from the specialized mast cells and basophils can occur only under the following circumstances:
All mast cells or basophils possess a number of receptor sites that are available for "lockiny" onko the constant region or Fc portion o IgE mol~cules. These "binding site~"
are specialized areas on the cell membranes wherein a special geometric or spatial molecular arrangement of molecules occurs, ~
thus enabling this "binding or receptor site" to "lock" into ;
the Fc fragment or a site in the constant region of the IgE
molecule.
Should a wandering IgE molecule find a ree "binding receptor site" on a mast cell or basophil, it locks or attaches at its Fc end onto the cell binding ~receptor) site to secure ~ i;, the Ige molecule to the mast cell or basophil.
When the Fc portion of the IgE molecule is secured to 2S the receptor "binding site", the upper arms of the "Y" shaped -molecule ~the F(ab) portion) are free to extend above the cell ~;
surface. These extended upper peptide chains in turn act as receptors to allergens which may be present in the organismls environment. If the polypeptide structure of the Fab portions are compatbile with a particular allergen, the allergen may . ,, . , ~

107~7Z~
attach to the outwardly extending Fab of the IgE polypeptide chain. Should such an attachment occur, the mast cell or basophil is automatically stimulated or "triggered" to release histamine from within its cell structure into the local environment of the mast cell or basophil. Once the histamine is released, the ~amiliar "allergic sympt:ons" are manifested.
The present state of therapy of allergic disease includes hyposensitization (repeated injections o offending allergens to produce "blocking antibodies"), systemic therapy with anti-histamines (which compete with histamines released during the allergic reaction) and disodium cromoglycate (which may lower the amount of histamine released by allergic reactions).
Cortocosteroids, isoprenaline and theoph,ylline as well as other medications are also utilized in the therapy of alleryy.
lS However, none of theqe a~orementioned drugs or techniques inter-fere with the basic IgE-mast cell (basophil) reaction itself, and all have significant limitations in usefulness.
Another course of therapy suggested by the analysis above of the allergen-IgE-mast cell (basophil) reaction would be the introduction into the organism of a drug that would "block"
the mast cell (basophil) receptor or binding sites against the attachment o~ the IgE molecule. Of equal importance would be a drug that would not only "block" the binding sites but in addit~on would displace IgE from binding sites to which the IgE was already attached. Any filling up or diminution in the binding sites available for IgE attachment would quite obviously reduce the number of allergen-IgE-mast cell (basophil~
reactions, and as a consequence, thereby reduce the release of histamine into the organism and thereby reduce or prevent the allergic reaction.

, ~7g7~1 Some prior attempts have been made to use this thera-peutic appraoch. For instance, in 1968 Stanworth, et al published in Lancet (July 6, 1968) a study wherein the whole Fc portion of the IgE as well as small proteolytic digestion fragments thereof were tested for their ability to suppress the allergic reaction. This study suggestea that only the complete IgE Molecule in inhibiting allergic reaction while the digestion fragments were ineffective. That is, any fraction of the Fc peptide chain less than the entire Fc polypeptide 10 was unable to prevent an induced allergic reaction. The Fc fragment itself cannot be used as a therapeutic agent or drug.
Description of the Invention The present invention is directed to novel low molecular weight polypeptides which are useful as therapeutic agents in 15 the treatment of allergic disease or the allergic syndrome.
More specifically, the present invention is directed to the pentapeptide ASP-THR-GLU-ALA-ARG and its salts, esters, amides, N-Acyl and O-acyl derivatives.
For convenience in describing this invention, -the 20 conventional abbreviations for the various amino acias are used. They are Eamiliar to those skilled in the art; but or clarity, those with which this invention is concerned are listed below. A11 chiral amino acid residues referred to herein are of the natural or L-configuration unless otherwise `~
25 specified. All peptide sequences mentioned herein are written according to the usual convention whereby the N-terminal amino acid is on the left and the C-terminal amino acid is on the right:
Asp = Aspartic Acid 30 Ala = Alanine Arg = Argine Asn = Asparagine Asx = Aspartic Acid or Asparagine (indicated uncertainty in degradation analysis) Cys = Cysteine Gly = Glycine Gln = Glutamine Glu = Glutamic acid Glx = Glutamic Acid or Glutamine (indicates uncertainty in clegradation analysis) His = Histidine Ile - Isoleucine Leu = Leucine Lys = Lysine Met = Methionine Phe = Phenylalanine Pro = Proline Ser = Serine Thr = Threonine Tyr = Tyrosine Val = Valine As used herein the term "salts'l refers to both salts of a carboxyl group of the polypeptide chain as well as acid addition salts of an amino group of the polypeptide chain. ;
Salts of a carboxyl group may be formed with either inorganic or organic bases. Inorganic salts include for example the alkali metal salts such as the sodium, potassium and lithium salts; the alkaline earth salts such as for example the calcium, barium, and magnesium salts; and the ammonium, ferrous, ferric zinc, manganous, aluminum, manganic salts, and the like.

~079721 Salts with organic amines include those ~ormed, for example, with trimethylamine, triethylamine, tri(n-propyl)amine, dicyclohexylamine, ~-(dimethylamino~ ethanol, tris(hydroxy-~
methyl)aminomethane, triethanolamine, B-(diethylamino) ethanol, arginine, lysine, histidine, N-ethylpepiridine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, : ~ .
theobromine, purines, piperazines, pipericlines, caffeine, procaine, and the like. ~ :
Acid addition salts include, for example salts, with mineral acids such as for example hydrochloric acid, hydro-bromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; and salts wikh organic acids such as for example, acetic acid, o~lic acid, tart~ric acid, succ:Lnic acid, maleic acid, um~ric acid, yluconic acid, citric acid, mal:lc acid, ascorbic acid, benzoic acid, and the like.
As used herein, the term "esters" refers to esters of a carboxyl group of the polypeptide formed with straight or : :
branched chain saturated aliphatic alcohols of from one to twelve carbon atoms, such as the methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-amyl, n-hexyl, octyl, decyl and dodecyl esters.
As used herein the term "amides" referes to amides of a carboxy group of the polypeptide Pormed with ammonia, or with primary or secondary amines having up to twleve carbon :~
atoms such as for example dimethylamine, diethylamine, di(n bu-tyl)amine, n-hexylamine, piperidine, pyrrolidine, morpholine, di(n-hexyl)amine, N-methylpiperazine and the like.
"N-acyl derivatives" refer to those derivatives of an amino group of the polypeptide formed with acyl moie-ties (e.g.
alkanoyl or carboxyclic aroyl groups) containing up to twelve ~97%~

carbon atoms, such as formamides, acetamides, benzamides, and the like.
"o acyl derivatives" refer to those derivatives of a hydroxyl group of the polypeptide chain formed with acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) containing up to twelve carbon atoms, such as formates, acetates, pro-pionates, benzoates, and the like.
While the compounds of the present invention are believed to act by "blocking" IgE binding sites as described hereln, it is not intended that the present invention be limited to any particular mechanism of action.
In the practice o~ the method of administering the present ~nvention, an e~fective amount of a polypeptide or derivative thereof, or a pharznaceutical composition containing lS same, as defined above, is administered via any of the usual and acceptable methods known in the art, either singly or in combination with another compound or compounds of the present invention or other pharmaceutical agents such as antihistamines, corticosteroids, and the like. These compounds or compositions can thus be administered orally, sublingually, topically (e.g.
on the skin or in the eyes), parenterally (e.g. intramuscularly, intravenously, subcutaneously or intradermally), or by inha-lation, and in the form of either solid, liquid or gaseous dosage including tablets, suspensions, aerosols, as discussed in more detail hereinafter. The administration can be conducted in single unit dosage form with continuous therapy or in single dose therapy ad libitum.
In one preferred embodiment, the method is practised when the relief of symptoms is specifically required or perhaps imminent; in other preferred embodiment, the method hereof is 1~7~72 IL
effectively practiced as continuous or prophylactic treatment.
In view of the foregoing as well as in consideration of the degree or severity of the condition being treated, age of subject, and so forth, all of which factors being determi-nable by routine experimentation by one skilled in the art, the effective dosage in accordance herewith can vary over a wide range. Since individual subjects vary in their IgE
content, an effective systemic dosage in accordance herewith can best be described as between 2xlO and 2xlO times the ~:
IgE content, on a molar scale. For an average subject this .
would be between about ~.5 and 500 mg/kg/day, depending upon the potency of the compound. Of course, for localized treat-ment, e.~., of the respiratory system, proprotionately less material will be required.
Useful pharmaceutical carriers for the preparation of the compositions hereof, can be solids, liquids, or gases;
thus, the compositions can take the form of tablets, pills, capsules, powders, enterically coated or other protected formu- :
lations (such as by binding on ion exchange resins or other carriers, or packaging in lipid-protein vesicles or adding additional terminal amino acids or replace a terminal amino acid in the L-form with one in the D-form), sustained release .' formulations, solutions (e.g. opthalmic drops), suspensions, elixirs, aerosols, and the like. The carrier can be selected from the various oils including those of petroleum, animal, ve:getable or synthetic origin, for example, peanut oil, soy- :
bean oil, mineral oil, sesame oil, and the like. Water, saline, aqueous dextrose, and glycols are preferred liquid carriers, particularly (when isotonic) for injectable solutions.
Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, 1~)79721 chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, gly-cerol, propylene glycol, water, ethanol, and the like. The compositions may be subjected to conventional pharmaceutical expedients such as sterilization and may contain conventional pharmaceutical additives such as preservat:ives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers, and the like. Suitable pharmaceu-tical carriers and their formulation are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will, in any event, contain an effective amount of the active compound together with A su.ikable amount o~
the active compound toyether with a suitable amount o~ carrier so as to prepare the proper dosage form Por proper adminis-tration to the host.
To be effective for the prevention or treatment of the allergic reaction, it is important that the therapeutic agents be relatively non-toxic, non-antigenic and non-irrita-ting at the levels in actual use. This has been demonstrated -.
ko be the case with all of the present compounds whose prepa-ration is described hereinbelow.
The polypeptides of the present invention may be syn-thesized by any techniques that are known to those skilled in the peptide art. An excellent summary of the many techniques so available may be found in J. Meienhofer, "~Iormonal Proteins and Peptides", Vol. 2, p. 46., Academic Press (New York), 1973 for solid phase peptide synthesis and E. Schroder and K. L~bke, "The Peptides", Vol. l, Academic Press (New York), 1965 for classical solution systhesis.
In general, these methods comprise the sequential ~ ;
_ g_ CJ 797~

addition to a growing chain of one or more amino acids or suitably protected amino acids. Normally, either the amino or carboxyl group of the first amino acid is protected, by a suitable protecting group. The protected or derivatized ~;
amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or aarboxyl) group suitably protected, under conditions suitable for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suit-ably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining pro~ecting groups (and any solid support) are removed sequentiall~ or concurrentl~, ~o afEor~ the final polypeptide. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected depeptide to form, after deprotection a pentapeptide.
Protecting groups should have the properties of being stable to the conditions of peptide linkage formation, while being readily removable without destruction of the growing peptide chain or racemization of any of the chiral centers contained therein.
Among the classes of amino protecting groups useful for stepwise synthesis of polypeptides are: (1) acyl type protecting groups illustrated by the following: formyl, trifluoroacetyl, phthalyl, toluenesulfonyl, (tosyl), benzensulfonyl, o-nitro- -phenylsulfenyl, tritylsulfenyl, o-nitrophenoxyacetyl, 1~7~7Zl chloroacetyl, acetyl, y-chlorobutyryl, etc.; (2) aromatic urethan type protecting groups illustrated by benzyloxycarbonyl and substituted benzyloxcarbonyl such as p-chlorobenzyloxy-carbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-(p-biphenyly:L)isopropyloxy-carbonyl, 2-benzoyl-1-methylvinyl; (3) aliphatic urethan pro-tecting groups illustrated by tert-butyloxycarbonyl, tert- .
amyloxycarbonyl diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; (~) cycloalkyl urethan type protecting groups illustrated by cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl; (5) thio urethan type protecting groups as illustrated by phenylthioGarbonyl; (6) alkyl type protecting groups as illustrated by triphenylmethyl (tri-tyl) and benzyl; and (7) trialkylsilyl groups such as trimethylsilyl.
Preferred protecting groups are tert-butyloxycarbonyl ~t-BOC), and tert-amyloxycarbonyl (AOC).
Among the classes of carboxyl protecting groups use:Eul for stepwise synthesis of polypeptides are: (1) substituted or unsubsituted aliphatic ester protecting groups such as methyl, ethyl, t-butyl, 2,2,2-trichloroethyl and t-buty} esters;

(2) aralkyl ester protecting groups such as benzyl, p-nitro-benzyl, p-methoxybenzyl, diphenylmethyl, or triphenylmethyl (trityl) esters; (3) N-substituted hydrazides such as t-butyloxycarbonylhydrazides and carbobenzyloxycarbonylhydrazides;
(4) amide protecting groups formed by condensation of a carboxyl moiety with e.g. ammonia, methylamine, ethylamine, diphenylmethylamine; and the like.
Hydroxyl groups of amino acids such as serine, threonine, and hydroxyproline may be protected as aralkyl ethers such as ~07~7'~1 benzyl ethers.
Suitable solid supports useful for the above synthesis are those materials which are inert to the reagents and reaction conditions of the stepwise condensation~deprotection reactions, as well as being insoluble in the media used.
Materials that may be used include, for example, crosslinked polystyrene divinylbenzene resins, crosslinked polyamide resins, polyethyleneglycol resins, appropriately f~mctionalized glass beads, and the like.
The first amino acid residue is linked to the solid support by forming a covalent bond with an active group on the resin. Suitable active groups for this purpose include, for example, chloromethyl, benzhydrilamino, hydroxymeth~:L/ phen-acyl halide, dehydroalanine and the like. The preerred active group is chloromethyl. The first amino acid may be coupled to the preferred chloromethyl resin by one of several base cata-lyzed processes wherein the triethylamine, tetramethylammonium or cesium (or similar) salt of the carboxylic acid is heated with the resin in a solvent such as ethanol, dioxane, dimethyl-formamide, and the like.
Suitable reagents that efect amide ~ormakion between carboxyl and amino groups are known in the art and include, -for example, (1) carbodiimides such as for example dicylcohexyl-carbodiimide (DCC), (2) a carbodiimide plus an additive such ;
as l-hydroxybenzotriazole or ethyl 2-hydroximino-2-cyanoace-tate; (3) alkyl chloroformates such as isobutylchloroformate or ethylchloroformate; (4) N-protected amino acids activated by formation of a suitable ester, for example, substituted phenyl esters, aryl or alkyl thio-esters, substituted 8~hydroxy isoquinoline esters, 2-thiopyridyl esters and similar esters ., ~ . . .
- : ' ;, ', . ~ ~, :, ~7~3i7z~
well known to those skilled in the art.
A preferred method for synthesizing the peptides of the present invention is the so-called "Merrifield" synthesis technique which is well known to those skilled in the art and is set forth in detail in the article entitled "Synthesis of a Tetrapeptide" by R.B. Merrifield, Journal of the American Chemical Society, Vol. 85, pp. 2149-2154 (1963) as well as Meienhofer, cited above.
In this preferred method a peptide of any desired length and of any desired sequence is produced through the stepwise addition oE amino acids to a growing peptide chain which is bound by a covalent bond to a solid resin particle.
In the preferred application oE this method, the C-terminal end of the growing peptide chain is covalently bound to a resin particle and amino acids having protected amino groups are added in the stepwise manner indicated above. A
preferred amino protecting group is the t-BOC group, which is stable to the condensation conditions and yet is readily removable without destruction of the peptide bonds or race-mization of chiral centers in the peptide chain. At the end of the procedure, the final peptide is cleaved from the resin, and any remaining protecting groups are removed, by treatment under acidic conditions such as, for example, wi-th a mixture of hydrobromic acid and trifluoroacetic acid or with hydro-fluoric acid, or the cleavage from the resin may be effected under basic conditions, for example, with triethylamine, the protecting groups being then removed under acid conditions.
The cleaved peptides are isolated and purified by means well known in the art such as, for example, lyophilization followed by either exclusion or partition chromatography 1079~1 on polysaccharide gel media such as Sephadex G~25 (trademark) or countercurrent distribution. The composition of the final peptide may be confirmed by amino acid analysis after degra-dation of the peptide hy standard means.
Salts of carboxyl groups of the pepticLe may be prepared in the usual manner by contacting the peptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as for example sodium carbonate or sodium bicarbonate, or an amine base such as for example triethylamine, triethanolamine, and the like.
Acid addition salts of the polypeptides may be prepared by contacting the polypeptide with one or more e~ulvalents of the desired inorganic or organic acid, such ag, ~or example, hydrochloric acid.
Esters of carboxyl groups of the polypeptides may be prepared by any of the usual means known in the art for convert-ing a carboxylic acid or precursor to an ester. One preferred method for preparing esters of the present polypeptides, when using the Merrifield synthesis kechnique described above, is to cleave the completed polypeptide from the resin in the presence of the desired alcohol either under basic or acidic conditions, depending upon the resin. Thus the C-terminal end of the peptide when freed from the resin is directly esteri-fied without isolation of the free acid. -Amides of the polypeptides of the present invention may also be prepared by techniques well known in the art for converting a carbolyxic acid group or precursor, to an amide. ;
A preferred method for amide formation at the C-termina:L
carboxyl group is to cleave the polypeptide from a solid -14~

... . . . : .

~L0~ 37Zl support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by amino-lysls with the desired amine.
N-acyl derivatives of an amino group o~ the present polypeptides may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected peptide. O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O- ac~lation may be carried out together, iE de~irecl.
The coupling, deprotection/aleavacJe reactions and prepa-ration o~ derivatives of the subject polypep-tides are suitably carried out at temperatures between about -10 and ~50C., most preerably about 20-25C. The exact temperature for any ~articular reaction will of course be dependent upon the subs-trates, reagents, solvents and so forth, all being well within the skill o~ the practitioner. Illustrative reaction conditions for these processes may be gleaned ~rom the examples.
The following examples are given to enable those skilled in the art to more fully understand and practice the present invention. They should not be construed as a limitation upon :
, the scope of the invention, but merely as being illustrative and representative thereof.

,, ~C~7~721 EXAMPI,E 1 Preparation o the Tripeptide Asp-Pro-Arg . 1.6 G. (5 mmoles) of t-30c-nitroarcJinine are reacted - with lO g. of chloromethyl resin (beaded copolystyrene-2 divinyl benzene containing 0.5~1 meq. of chloromethyl groups per gram of resin) in a mixture of 1.4 ml. (lO
mmoles) of triethylamine and lO0 ml. of ethanol for 24 hours at 22C. with constant stirring. The argininated resin is then washed thoroughly, successively, with acetic acid, absolute ethanol, water with increasing amounts o ethanol, then methanol and finally methylene chloride. The resin i5 then thoroucJhly dried in v~cuo, Analysis rcve~l~d 0.05 n~ol~ ~ry/y. resin. 2,5 G. of the r~sin SQ pr~pared is placed in a Merr1ield solid phase reaction vessel equipped for agitation and is put through the following DEPROTECT.ION CYCLE: .
(a) with agi-tation, and at 22'C., the t-Boc group is cleaved with lO ml. of 4 N. HCl in dioxane for 30 minutes, (b) two washes with lO ml. of dioxane, ~c) two w~shes with lO ml. of methylene chloride, (d) two washes with lO ml. of chloroform, , (e) the hydrochloride is neutralised with lO ml. of ~, triethylamine/chloroform (5:95), ' (f) two washes with lO ml. of methylene chloride, (g) two washes with lO ml. of chloroform, The resin is then subjected to the SYNT~IESIS CYCLE AS `
follows: a ten-fold excess, of t-Boc-proline (1.2S mmoles) in methylene chloride solution is,added followed by 258 mg~
(1.25 mmoles) of dicyclohexylcarbodiimide (DCC) and the mixture is shaken for 2 hours at 22C. The resin is then .

1079q~

washed three times each with 10 ml. portions of dioxane, chloroform, and methylene chloride, respectively.
The dipeptide resin is then subjected to the deprotection cycle and is reacted with a four-fold excess of t-BOC ~-benzyl aspartate (0.5 mmoles) as described above in the synthesis cycle. An O.S g. portion of the resin is then removed from the reaction vessel and subjected to the Cl,EAVAGE PROCESS as follows:
The tripeptide resin (0.5 g.) is suspended in dry trifluoroacetic acid (5 ml.) and a slow stream of anhydrous HBr is bubbled through the solution for 90 minutes.The resin is filtered off and washed twice with 5 ml. of trifl~oroacetic acid. 'rhe combined filtrates are concentra-ted in v~cuo and excess HBr is removed Erom the peptide by repeated evaporations of methanol-water (1:1) solutions. The peptide is finally dissolved in water and lyophilised yielding aspartyl-prolyl--nitroarginine. The nitro group is then removed by hydroge-nation in a Parr low pressure shaker hydrogenation apparatus as follows: The nitro protected tripeptide is dissolved in mixture of methanol-acetic acid water (10:1:1), about 10-20 mg./ml., and an equal weight oE a 5~ palladium on BaSO4 catalyst is added and the mixture is shaken overnight at a hydrogen pressure of about 50 psi. The catalyst is removed by ~iltration and the filtrates are concentrated in vacuo. The peptide residue is chromatographed in a column o~ Sephadex G-25 (trademark). The yield of the purified tripeptide as established by conventional amino acid anaLysis is approxi-mately 24~ based on the arginine incorporated in the resin.
A portion of the product was hydrolysed with 5.7 N.~IC:L in water and assayed on an amino acid analyser, which indicated 1C)79'7~1 a ratio of ~sp 1.05, Pro 0.95, Arg 1.00.
Purity was determined by paper electrophoresis in the standard manner at a numher of l-l's.

~07g~Zl EXAMPI,~ 2 Prep~ration of the Tetrapepticle Ser-~sp-Pro-~rg The tripeptide resin from Example 1, not used in the synthesis of the t~ipeptide, was put through the deprotection cycle (see Example 1) and then was allowed to react with 0.111 g. of t-Boc~O-benzyl serine and 0.13 g. of dicyclo-hexylcarbodiimide in 20 ml. of methylene chloride as described in the synthesis cycle (Example 1).
A portion oE the resin was then subjec-ted to the cleavage and hydrogenation processes as described in Example 1 and recovered in the same manner as in Example 1 yielding Ser-Asp-Pro-Arg in a 20~ yield based Oll arcJ;inine esterified to the resin. ~fter hydrolysis with ~ICl, a sample oE the recovered tetrapeptide was assayecl on the amino acid analyser, whlch indicated a ratio of Ser 0.79, Asp 1.18~ Pro 1.02, and Ary 1.01. ~Serine is partly destroyed during the acid hydrolysis.) Purity was determined by paper electrophoresis in the standard manner at a number of pH's.

~4)7972~ .

Pre aratlon of the Pentapeptid~ ~sp-Ser-Asp-Pro-Arg P
A. The uncleaved tetrapeptide resin from Example 2 was subjected to the deprotection cycle (Example 1) and the synthesls cycle using 0.152 g. of t-Boc-~-benzylaspartate.
The resln portion had the pentapeptide cleaved therefrom with HBr in trifluoroacetic acid ln the same manner as noted previously. The recovered polypeptlde was dried in vacuo, thoroughly washed with water and then lyophilised. An analysis revealed a 16% yield based upon the arginine.
The pentapeptide product was hydrolyzed with HCl and assayed on an amino acid anaLyser, which indicated ~ ratio of Asp 2.12, ~Ser O . i~, Pro 1.12, ancl ~rcJ 1.01.
B. The pentapeptide is also prepared by a modifica~
tion of the procedures of Examples 1-3A~
To a solution of-3.02 g. (6.82 mmoles) o ~-t-amyloxycarbonyl-N -tosyl-L-arginine (t-Aoc-tosyl-Arg) in 15 ml. of ethanol and 6 ml. of water is added dropwise a solution of caesium bicarbonate (1.4 g. in 3 ml. ~l2O) until th~ p~l of the solution is 7Ø The solution is concentrated in vacuo to a foam which is thoroucJhly dried in high vacuum over P2O5. To this residue is added 25 ml. of dry dimethyl-formamide (DMF) and 4.5 g. of chloromethylated resin (beaded Z5 copolystyrene-l~ divinyl benzene containing 1.10 meq. of chloro-methyl group/g. of resin) and the mixture is shaken at 50C. for 3 days. The resin is filtered and washed with DMF (5 ~ 20 ml.), 90~ DMF/H2O (3 x 20 ml.), DMF (2 x 20 ml.) and EtO~I (2 x 20 ml.) and is then dried in vacuo over P2O5 giving 5.54 g. of argininated resin (ca. 50~ incorporation) ~ 0797Zl This resin i5 then subjected to four cycles oE
deprotection and synthesis using 4 equivalents of the appropriate t-Boc-amino acid at each chain elongation step giving the protected pentapeptide resin material.
'' 5 This resin material is then placed in,an HF
resistant reaction vessel, 8 ml~ of anisole is a~ded and the vessel is attached, to an HF line. Approximàtely 70 ~7.
of HF is distilled ~nto the reaction vessel at 0C. and the mixture is stirred for a further 30 minutes at 0C. The HF is pumped o~ and the xesin is washed with ether (5 x 30 ml.) and then extracted with water ~5 x 30 ml.).
The aqueous layer is lyophilised to a yellow glassy powder , which is purlfied according to Example, 1 thereby giving the ' pentapeptide Asp-Ser-Asp-Pro-Arg.
` 15 The pentapeptide prepared above,exhibits an '`', ' " ~a~20 = -78.6n ~c=l, H20). ,Purity,was,determined by - ,'`,`,~;,,';,,, ,`:' ~paper electrophoresis in the stan ard m nner at a number ' ~ ''o'f'p~'s. '' ~ '', ;~,'; ~,~' i - - ' ~' ': : ': ' '~:
" ~
.. .

' ~ . ,. , .. ~ .::

E~IPLE 4 Preparatidn of the h~peptide Al~-~sp-Ser-~sp-Pro-Ar~
Another batch of arginated-resin ~0.20 mmoles) was taken throu~h 5he procedures of Examples 1-3A except that ~ :
after the attachment of the second aspartic acid residue and deprotection an equivalent amount of t-BOC-alanine was coupled on with dicycloheY.ylcarbodiimicIe in the usual' manner.
The resin was then subjected to t~e cleavage and hydrogenation processes as described in Example 1 and r.ecovered in the same manner as in Example 1 yielding .. ~ `
~la-~sp-Ser-~sp-Pro-Ar~ in a 0.026 mmol~, or 13~,yield.
The recovered polypeptide was assayed on an amino ~ ::
acid analyser, whichindicated an amino acid ratio of ALa 0.95, Asp 2.05, Ser 0.80, Pro 0.98, and Arg 1.00.
Purity was determined by.paper electrophoresis in~
; the standard manner at a number of pH's.

~, ' .. ,,;. .

...

. ", ~,.

, ' '" ' ' ':

;-' ' '; :~''`'',':

lQ79721 ,` ~ ;EXAMPLE 5 Utilizing similar synthesis procedures to those described in Examples 1-4 above, the following polypeptide . .
may be prepared:

Asp-Val-Asp~Leu-Ser Thr-Ala-Ser-Thr-Glu Asp-Val-Asp-Leu-Ser-Thr-Ala-Ser-Thr-Glu Leu-Ser-Glu-Lys-His '.

Ala-Pro-Ser-Lys-Gly-Thr .

) Ala-Ser-Gly-Lys-Pro Ala-Phe-Ala-Thr-Pro-Glu-Trp-Pro-Gly-Ser Ala-Phe-Ala-Thr-Pro Glu-Trp-Pro-Gly-Ser Pro-Asp-Ala~Arg-His-Ser Ala-Ser-Pro-Ser-Glu including the pentapeptide Asp-Thr-Glu-Ala-Arg .
of this invention.

i; ,,; - ,, .- .. .

- "
~0797; :1 Preparation of Metallic and Amine Salts A. The pentapeptide Asp-Ser-Asp-Pro-Arg is converted to its sodium salt as follows~
A solution of the pentapeptide (0.05 mmoles) in water is carefully treated with exactly 1 equivalent of 0.1 N. NaOH
and the monosodium salt of the peptide is isolated by lyo- ;
philisation. By the use of exactly 2 or 3 equivalents of ~`
0.1 N. NaOH the corresponding di- and tridosium salts are obtained respectively.
Similarly, this peptiae may be converted to other metallic salts, e.g., potassium, lithium, calcium, barium, ;
magnesium, ammonium, ferrous, ferric, zinc, manganous, man-ganic, and aluminum 9alts, by substitution o the appro-priate base.
B. The pentapeptide Asp-Ser-Asp-Pro-Arg is converted to its triethylamine salt as follows:
The careful addition of 1, 2 or 3 equivalents of triethylamine to the solution of the peptide in methanol, followed by careful evaporation of the solvent, yields the mono-, bis- and tris-triethylammonium salts respectively.
Similarly, this pentapeptide may be converted to other amine salts, e.g., triemethylamine, tri~n-propyl)amine~
dicyclohexylamine, ~-(dimethylamino)ethanol, ~-(diethyl- -amino)ethanol, triethanolamine, tris(hydroxymethyl)amino-methane, arginine, lysine, histidine, N-ethylpiperidine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglycamine, theobromine, purine, piperazine, piperidine, caffeine and procaine salts, by substitution of the appro-priate amine.

:: : . : , ~ : . .......................... . . . .
, C. In a similar manner, the other peptides of Examples 1, 2, 4 and 5 may be converted to their corresponding metallic and amine salts.
. _ . . .

, ' .'.
:

~0797Z~L

EX~IPLE: 7 The penta~eptide Asp-~er-Asp-Pro-Arg is converted to its hydrochloride acid addition salt as follows:
Careful neutralisation of a solution of the peptide in either water or tnethanol wi~h exactly 1 or 2 equiva.lents of hydrochloric acid gives the mono- and dihydrochloride salts respectively. The salts are isolated either by lyophilisation of an aqueous solution or by precipitation with ether from a methanolic solution.
Similarly, this peptide may be converted to other acid addition salts, e.g., the hydrobromide, sulfate, phosphate, nitrate, acetate, oxalate, tartrate, succinate, maleate, fumarate, gluconate, citrate, malate, ascorbate, and benzoate salts, by substituting the approp.ri~te acid ~or hydrogen chloride.
In a sirnilar mannerl the other peptides of Examples 1, 2, 4 and 5 may be converted to their corresponding acid addition salts.

~379~Z3L
EXAMPL~ 8 Preparation of Esters A. The appropriate peptide resin from Example 5 (1.0 g.) is suspended in anhydrous methanol (40 ml./g. of resin), triethylamine (50 mmoles) is added and the mixture is stirred at 22C for 20 hours. The resin is removed by filtration and the combined filtrates are concentrated in vacuo. The residue is dissolved in ethyl acetate, saturated with hydrogen chloride (5 ml.) and the solution is stirred at 22C for 30 minutes. The product is pre-cipitated by the addition of ether giving a hydrochloride salt of the peptide. The O-benzyl ether protecting groups oE Ser or Thr are removed by hydrogenolysis using Pcl/BaSo4 as described in E~ample l or the removal oE the nitro group in nitroarginine derivatives, thereby giving Ala-Pro-Ser-Lys-Gly-Thr-OMe, Ala-Ser-Gly-Lys-Pro-OMe, Ala-Phe-Ala-Thr-Pro-OMe respectively.
By substituting other alcohols for methanol and ra~sing the reaction temperature to 45-80C. and the reaction time to 45-90 hours there are obtained the corresponding ethyl, propyl, butyl, hexyl, octyl, decyl and dodecyl esters.
B. In this procedure a different type o~ anchoring bond is used for the attachment of the arginine residue, namely the resin-~-CH2-CH2-C(CH3)2-OCONHNH2 bond described by S. Wang and R.B. Merrifield in J. Amer. Chem. Soc. 91, 6488 (1969). Also, in this procedure, N -2-(p-biphenylyl)-isopropyloxycarbonyl (Bpoc) protecting groups are used ~'' ` ' ' , .

~` 107~72~.
instead of t-Box for ~-amino protection since the Bpoc group can be removed at each cycle of the synthesis with very mild acid under conditions where the anchoring bond is stable. The Bpoc-N~-nitro-Arg is attached to the resin by the DCC method and the synthesis is carried out essentially as described in Examples 1-3 except that 1% trifluro-acetic acid (TFA) / CH2C12is used in the deprotection cycle in order to cleave the Bpoc group. The ultimate amino acid incorporated is protected as a N -benzyloxy-carbonyl derivative (Z) so that the N-terminus remains protected during the cleavage of the protected peptide from the resin. The cleavage is done as follows: 500 mg.
of the peptide resin is suspended in 12 ml. oE 50~ TF~ in CEI2C12 and the mixture is shaken at room temperature ~or 30 minutes. The resin i5 removed by filtration, washed with CH2C12 (2 x 10 ml.) and the combined filtrates are concentrated in vacuo giving Z-~-benzyl-Asp-O-benzyl-Ser-~-benzyl-Asp-Pro-N~-nitro-Arg-NHNH2 as a white powder.
A solution of the protected peptide hydrazide (0.2 mmoles) in DMF ~1 ml.) is cooled at -20C and 3.35 N.
~Cl is dioxane (0.5 mmoles) is added. The bath is warmed to -15C and t-butylnitrite (0.03 ml.) is added and the mixture is left at -10C for 10 minutes giving the peptide-azide derivative. An excess of methanol is then added at -15C. followed by ethyl diisopropylamine (0.5 mmoles) and the mixture is kept at 0C. for 24 hours. During the first 5 hours, 5 ~1. of the base are added every hour. The protected peptide is then precipitated by pouring the mixture into ice cold 1% acetic acid (15 ml.) and the precipitate is collected and washed by filtration. The . ~ ; ' ', , ~

~0797;Z~

benzyl based protecting groups are then removed by hydro-genolysis, as described in Example 1, and the product is -~
purified by partition chromatography on Sephadex G-25 (trade-mark) or by countercurrent distribution giving Asp-Ser-Asp-Pro-Arg-OMe.
By replacing methanol in this procedure by other alcohols there are obtained the corresponding ethyl, propyl, ;~
butyl, hexyl, octyl, decyl, and dodecyl esters.
C. Utilizing similar procedures to those described in A and B, the corresponding esters of the polypeptides of Examples 1, 2, 4 and 5 may be prepared.

, . . .

~079~7%~L
Example 9 Preparation of Amides A. The products of Example 8A and 8B are treated with a saturated solution of ammonia in methanol at room temperature for 2 days. The solvent is removed in vacuo to afford Ala-Pro-Ser-Lys-Gly-Thr-NH2, Ala-Ser-Gly-Lys Pro-NH2, Ala-Phe-Ala-Thr-Pro-NH2, and Asp-Ser-Asp-Pro-Arg-NH2, respectively.
B. The peptide-azide of ~xample 8B is reacted with ammonia in DMF solution under the cond:itions described Ln ~xample 8B Eor reaction with methanol. The protected peptide-amide is isolated and deprotected as described earlier giving Asp-Ser-Asp-Pro-Arg-NH2.
C. The protected peptide resin product of Example 3A
is suspended in a saturated solution of ammonia in methanol and the mixture is agitated at room temperature for 2 days.
The resin is removed by filtration, washed with methanol and the combined ~iltrates are concentratecl in vacuo giving t-Box-As.-O-benzyl-Ser-Asn-Pro-N~-nitro-Arg-NH2.
The t-Boc group and the N6-nitro group are then removed by acidic hydrolysis and hydrogenolysis respectively, as described above, giving Asn-Ser-Asn-Pro-Arg-NH2.
By replacing ammonia with other amines, using DMF as solvent where appropriate and increasing the reaction temperature and time as necessary, there are obtained, for example, the corresponding dimethyl, diethyl, di(n-butyl), n-hexyl, piperidyl, pyrrolidinyl, morpholinyl, ~L0797~Z~

di(n-hexyl) and N-methylpiperazinyl amides, D. Utilizing similar procedures to those described in ~, B and C, the corresponding amides of the other poly-peptides of Examples 1, 2,4 and 5 may be prepared.

~.~797231 EX~r~l~'LE 10 Preparation of N-ac~l derivatives N -Acyl derivatives of Asp-Ser~~sp-Pro-Arg are prepared by replacing the terminal t-Boc-amino acid (t-Boc-~-benzyl-aspartate) ~ith the appropriate Na-acyl amino acid (e.g.
Na-acetyl-~-benzylaspartate). All other steys in the deprotection, synthesis and cleavage cycles remain the same.
Thus, there may be prepared Na-Acetyl-Asp-Ser-Asp-Pro-Arg Na-Butryl-Asp-Ser-Asp-Pro-Arg Na-Hexanoyl-Asp-Ser-Asp-Pro-Arg NC~-Octanoyl-Asp-Ser-Asp-Pro-Ary Na-Decanoyl ~sp-S~r-Asp-Pro-~rcJ
Na-Dodecanoyl-Asp-Ser-Asp-Pro-~rcJ
Similarly, the corresponding NCl-acyl derivatives o~ -other peptides mentioned in Examples 1, 2, 4 and 5 may be prepared.

~97;~i~
EXAMPLE ll Preparation of O-Acyl Derivatives . . .
In order to prepare the protected p~ntapeptide resin material in which the hydroxyl group of serine is unpro-tected, the following modification of the solid phase synthesis method is used.
The tripeptide resin material from Example l is sub-~ected to the deprotection cycle and is then allowed to react with t-Boc-serine-N-hydroxysuccinimide ester giving t-Boc-Ser-~-benzyl-Asp-Pro-N -nitro-Arg-resin which is then deprotected and coupled with p-nitrophenyl t-Box-~-benzyl-aspartate under standard conditions, thereby yiving t-Boc-~-benzyl-Aso-Ser-~ benzyl-~sp-Pro-N~-nitro-Arg-resin.
0.5 Mmoles of this protected peptide resin mat~rial is washed thoroughly with CHC13 and CH2Cl2 and 1.5 mmoles of hexanoic acid dissolved in l:l DMF/CHCl3 is added followed by 1.5 mmoles of carbonyl diimidazole dissol~ed in the same solvents. The mixture is rocked in the Merrifield reaction vessel at room temperature for 2 hours and the peptide is then cleaved from the resin as described earlier. The N~-nitro group is removed hydrogenolytically and the peptide is purified as described in earlier examples giving Asp-O-hexanoyl-Ser-Asp-Pro-Arg.
By replacing hexanoic acid with acetic acid, butyric acid, octanoic acid, decanoic acid and dodecanoic acid, the corresponding O-acetyl, butyryl, octanoyl, decanoyl and dodecanoyl compounds may be prepared.
Similarly, the corresponding O-acyl derivatives of the other peptides having side chain hydroxyl groups, mentioned in Examples 2, 4 and 5 may be prepared.

:, . , ," , . . . . ,- .

10797'~.
,,~.
E:~A~li'l~. 12 The ~ollowing illustrates typical pharmaceutical compositions oE the compounds hereoE, exemplified by Asp-Ser-Asp-Pro-Arg:

S Aerosol Formulation (per dose) Asp-Ser-Asp-Pro-Arg 10 mg.
Sodium chloride 8 mg.
Water to make 1.0 ml.

Injectable Formulation (per dose) Asp-Ser-Asp-Pro-Arg 10 mg.
Sodium chloride 8 mg.
Methylparaben 0.25 mg.
Propylparaben 0.14 mg.
Water to mAke 1.0 ml.

Dry Powder Formulation for Inhalation with device such as Spinhaler~ (per dose) Asp-Ser-Asp-Pro-Arg 10 mg.
Lactose 30 mg.

~ - - , . . .

10797;Z1 The "blocking" activity of the polypeptides of the invention can be assayed by utilization of the classic Prausnitz-Kustner (P-K) reaction. In this classic method, a known allergic serum i.e., one that contains IgE specific for a known antigen or allergen is in~ected intradermally into a human volunteer. After waiting a period of time, e.g. 20 or more hours, the injected sites are then challenged :
with a prick or injection of a solution of an antigen that is specific for the IgE in the injected serum. Within the ne~t 10 to 30 minutes a positive reaction is evidenced :by the development of a wheal (and flare) at the injected site. The more extensive the diameter of the wheal the more intensive is the allergic reaction, That is, a more extensive wheal indicates a greater release of histamine into the tissues at the injected site. Conversely, the development of wheals of lesser diameter or the absence of any wheal at all indicates diminished allergic reaction and/or no allergic reaction at all. The P-K reaction as noted above is a classic test and is universally known and utilitzed by allergists.
As noted above, the classic P-K reaction is utilized to assay the "blocking" abilities of the polypeptides utilized in the present invention.
The following describes assays of a number of poly-peptides and in particular the pentapeptide of the present invention, the synthesis of which was described hereinabove.
All of these assays were performed using a single proven safe P-K donor serum that contains IgE specific for guinea pig allergens.

Peptide solutions were either injected intradermally 1 to 24 hours prior to the P-K serun, or mixed with dilutions of the P-K serum for simultaneous injection.
Initial tests were performed using the P-K serum at from 1:4 to 1:200 dilutions. Further studies were run at a fixed P-K dilution of 1:32 while the peptide solutions were varied to contain from about 1 mM to 2 M of the peptide being tested.
Injected sites on the volunteers were challenged by prick-puncture of guinea pig BCA 1:40w/v (purchased from Berkeley Biologicals, Inc.).
~Iuman volunteexs were chosen who had serum IgE levels below 100 U/ml (242ng/ml) which levels have been previously shown to assure successul P-K reactivity. ~n addition, ~or the purpose o these -tests, the individuals were chosen who had a negative direct skin test to guinea pig antigen. P-K and skin tests were performed on the back and/or forearm. Multiple test sites of approximately 25 mm diameter were circled with a marking pen and all injections were made within the circled skin areas.
A typical sequence of events was intradermal injection o 0.1 ml o the peptide solution or control bufered saline diluent solution; followed in 1 to 2~ hours by intradermal injection of 0.05 ml of P-K serum into each of the previously injected sites. After 20 to 24 hours has elapsed, each site was prick-punctured with the antigen solution, blotted dry in 5 minutes and measurements of the wheal and flare in both their narrowest and widest diameter were made three times, usually 15, 20 and 25 minutes after prick-punctures.
Blocking activity assays were undertaken with the ~97~1 following polypeptides: Asp-Pro-Arg; Ser-Asp-Pro-Arg;
Asp-Ser-Asp-Pro-Arg; and Ala-Asp-Ser-Asp-Pro-Arg. Also Asp-Thr-Glu-Ala-Arg and tosyl-L-arginine sarcosine methylester (TASME), were synthesized and tested.
The above-noted polypeptides were assayed as noted above on six different individuals. Results were as follows:
For Asp-Pro-Arg, the average % inhibition was 15%, with an individual range from as low as 0% to as high as 38%.
For Ser-Asp-Pro-Arg, the average inhibition was 18%, with an individual low of 0~ and a high of 50%.
For Asp-Ser-Asp-Pro-Arg, the average inhibition was 72~, with an individual low of 60%, and a high of 89~.
For ~ Asp-Ser-Asp-Pro-Ar~, the average inhibition wa~ ~6~, with an individual low of :L0~, and a high of 61~.
For Asp-Thr-Glu-Ala-Arg, the average inhibition was 58%, with an individual low of 30%, and a high of 80%.
For TASME, the average inhibition was 24%, with a low of 0% and a high of 40~.
The results, as noted above, present the average of measuremenks at three time intervals, in duplicate, for each reaction in each individual, substracted from the average control wheal measurements, and divided by the average measurement of each individual's control wheal.
Control wheals in different individuals varied from 8 to 2~ 40 mm wi~h a mean of 17 mm . Each peptide was utilized -at approximately 6 ~g/ml dilution and 0.1 ml. was injected at each site, followed by 0.05 ml. of diluted P-K serum _g containing 0.2 ng. of IgE. Thus 10 M of the peptide was competing with 10 M of the IgE for the binding sites ~0797Z~ :
:
on mast cells, or a ratio of one IgE molecule to lO pep-tide molecules. ~rom the above assays, it appears that the pentapeptide, i.e., Asp-Ser-Asp-Pro-Arg, exhibits the strongest "blocking" activity, with the hexapeptide, i.e., Ala-Asp-Ser-Asp-Pro-Arg, exhibiting somewhat less activity.
The tetrapeptide, Ser-Asp-Pro-Arg and the tripeptide Asp-Pro-Arg, exhibited the least activity.
The pentapeptide Asp-Thr-Glu-Ala-Arg was prepared and assayed along with the other peptides as described above.
This particular polypeptide exhibits a high activity in the assay test.

-3~-1~7972~
_XAMPLE 14 It has also been determined that the active poly-peptides appear to have the ability to "displace" IgE from mast cell sites as well as to prevent the binding of IgE to these sites. In a single test, an individual known to have extreme sensitivity to guinea pig antigens, that is a person with a high natural concentration of guinea-pig-antigen-sensi-tive IgE, was injected with polypeptides similar to this invention, and his reaction to guinea pig antigen was noted.
Specifically, approximately 2nM of each of Asp-Ser-Asp-Pro-Arg and Ala-Asp-Ser-Asp-Pro-Arg were each intrader-mally injected into 3 marked sites. For comparison, TASME, as well as a con-trol of the bufer diluent along, was also each injected into 3 marked sites. At one, five and twenty-our hours subsequent to the polypeptide and control injection, one of each peptide and one diluent site were prick-puncture challenged with guinea pig antigen. No inhibition of the wheal and flare reaction was observed at any site at the one and five hour intervals. ~owever, at the twenty-four hour challenge, the wheal at the Asp-Ser-Asp-Pro-Arg site was approximately ~5% smaller, while at the Ala-Asp-Ser-Asp-Pro-Arg site, the wheal was approxi-mately 23% smaller. No reduction in the size of the wheal was observed at the TASME site compraed to the buffered saline diluent site.
It thus appears that, at least the most active of the peptides will "displace" IgE already bound to mast cell sites, thus inhibiting a natural allergic reaction. As has previously been demonstrated in Example 13 this same pentapeptide is ex-tremely effective in inhibiting a passively transferred (P-K) allergic reaction.

~L0797;~

, Acute toxicity was determined as follows:
DBA white mice (average weight 15 g.) were each injected with 1.4 ml. of a solution of the peptide in phosphate buffered saline, pH 7.4, as follows:
0.1 ml. x 3 intradermally ~
0.1 ml. x 3 subcutaneously 0.2 ml intravenously 0.6 ml. intraperitoneally 24 to 72 hours post-injection the mice (all still living) were killed and autopsied.
The peptides and concentrations used wexe as ollows:
Ala-Asp-Ser-Asp-Pxo-Arg ~Example 4) S ~g/ml (375 mg/]cg) - 6 mice Asp-Ser-Asp-Pro-Arg (Example 3) 10 ~g/ml (1 mg/kg~ - 8 mice Asp-Ser-Asp-Pro-Arg (Example 3) 13 ~ug/ml (1.3 mg/kg) - 8 mice.
Post-mortem gross and microscopic examination of tissues and organs indicated no local or systemic toxico-logical abnormalities.

..

Claims (2)

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

1. A method for the preparation of the pentapeptide ASP-THR-GLU-ALA-ARG, or a pharmaceutically acceptable, non-toxic salt, ester, amide, N-acyl or O-acyl derivative thereof which method comprises:
(a) condensing a first amino acid or peptide which may optionally be covalently bound to a solid support and/
or have a protected carboxyl or amino group with a second amino acid or peptide, optionally having a protected carboxyl or amino group, there being five amino acid residues in said first and second amino acids and/or peptides and of the kind to provide by said condensation reaction the following sequence of amino acid residues ASP-THR-GLU-ALA-ARG.
(b) sequentially or concurrently cleaving any protecting groups and/or solid support from the product of step (a), (c) optionally converting the product of step (a) or (b) to a salt, (d) optionally converting the product of steps (a) or (b) to an ester, or (e) optionally converting the product of step (a) or (b) to an amide, (f) optionally converting the product of step (a) or (b) to an N-acyl derivative, or (g) optionally converting the product of steps (a) or (b) to an O-acyl derivative.

2. A biologically active pentapeptide ASP-THR-GLU-ALA-ARG
and the pharmaceutically acceptable non-toxic salts, esters, aminides, N-acyl and O-acyl derivatives thereof when prepared by the process of claim 1.