Note: Descriptions are shown in the official language in which they were submitted.
~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
--2--
. ,, . , ~
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.
--3--
,
~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
--4--
7972
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.
--5--
~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
--6--
~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
--7--
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,
--10--
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
--11--
~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
-12-
., ~ . . .
- : ' ;, ', . ~ ~, :,
~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
-13-
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
-16-
.
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
-17-
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~ .
EXNMPLE 3
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)
-20-
~ 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
- - ' ~' ': : ': ' '~:
" ~
.. .
' ~ . ,. , .. ~ .::
1079721
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.
~, ' .. ,,;. .
...
. ", ~,.
, ' '" ' ' ':
;-'
-22-
'
'; :~''`'',':
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
EXAMPLF 6
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.
-24-
:: : . : , ~ : . .......................... . . . .
,
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
-27-
~'' ` ' ' , .
~` 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
-28-
. ~ ; ' ', , ~
~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,
-30-
~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.
-34-
~ - - , . . .
10797;Z1
EXAMPLE 13
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.
-35-
1079721
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
-36-
~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
-37-
~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.
-39-
~L0797;~
EXAMPLE 15
,
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.
-40-
..
