Note: Descriptions are shown in the official language in which they were submitted.
10~717~
Background of the Invention
The symptoms 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
into 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 sequence of events occur to
trigger the release of histamine from within the cell structures
into the surrounding tissues and vascular system.
' ' '
. .
10871q~
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 t~aumatic 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 basophil structures.,
Specifically, the allergen-mast cell (basophil) inter-
action is mediated by a group of proteins known as immuno-
globulin E (IgE) that are manufactured within the body.
The IgE manufactured by 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" ~actually the base of the l'Yn) (Fc) polypeptide
portion or fragment contains a fixed sequence or "constant
region" o~ peptides along the chain. The "heads" (which
are equivalent to the upper arms of the "Y" structure) may
ha~e regions wherein the polypeptide chain varies (the
variable region of the Fab) from molecule to molecule.
Thus, the IgE molecules generally have identical "tail"
peptide sequences but may have a great number of different
nhead" 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 th,at are available for "locking" onto the
2--
- lOB7171
constant region or Fc portion of IgE molecules. These
"binding sites" are specialized areas on the cell membranes
wherein a special geometric or spatial molecular arrange-
~ ment 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 free "binding
receptor site" on a mast cell or basophil, it locks or
attaches at its Fc end onto the cell bindlng ~receptor)
site to secure the IgE molecule to the mast cell or basophil.
When the Fc portion of the IgE molecule is secured
to 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 organism's environment. If the polypeptide structure
of the Fab portions are compatible with a particular
allergen the allergen may attach to the outwardly extending
Fab of the IgE polypeptide chain. Should such an attach-
ment 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 reIeased, the
familiar "allergic symptoms" are manifested.
The present state of therapy of allergic disease
includes hyposensitization (repeated injections of offending
allergens to produce "blocking a~tibodiesn), systemic
therapy with anti-histamines (which compete with histamines
released during the allergic reaction) and disodium cromo-
glycate ~which may lower the amount of histamine released
~` 1087171
by allergic reactions). Corticosteroids~ isoprenaline and
theophylline as well as other medications are also utilized
in the therapy of allergy. However, none of these afore-
mentioned drugs or techniques interfere 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 of the IgE molecule. Of
equal importance would be a drug that would not only "block"
the binding sites, but in addition 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 obvLously reduce the number
of aliergen-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.
Some prior attempts have been made to use this
therapeutic approach. 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 proteo-
lytic digestion fragments thereof were tested for their
ability to suppress the allergic reaction. This study
suggested that only the complete Fc fragment of IgE was as
effective as the intact IgE Molecule in inhibiting allergic
reaction while the digestion fragments were ineffective.
That is, any fraction of the Fc peptide chain less than
-
1~87171
the entire Fc polypeptide 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 the treatment of allergic disease or the allergic
syndrome.
More specifically, the present invention is directed
to polypeptides containing from 3 to 10 amino acid residues
which have the property of blocking the human allergic
response. These relatively short chain polypeptides
correspond to sequences occuring in the second (C-2),
third (C-3), and fourth (C-4) domains of the constant (Fc)
region of the epsilon (~) peptide chain of the IgE molecule.
The amino acid sequence of the entire E chain has been
recently determined by Bennich and his coworkers and
reported in ~Progress in Immunology II-Vol. 1: Immunochemical
Aspects", July, 1974, pp. 49-58, North-Holland Publishing
Company, Amsterdam, 1974. The sequence of the Fc region
in which the amino acid sequences of the present invention
occur is as follows, with the marginal numbers indicating
the numerical position in the sequence of the amino acid
to the right thereof:
265-(Met)-Asp-Val-Asp-Leu-Ser-Thr-Ala-Ser-Thr-Glu-Ser-Glu-
Gly-Glu-Leu-Ala`Ser-Thr-Glu-Ser-Glu-Leu-Thr-
289- Leu-Ser-Gln-Lys-His-Trp-Leu-Ser-Asp-Arg-Thr-Tyr-Thr-
Cys-Glu-Val-Thr-Tyr-Glx-Gly-His-Thr-Phe-Glx-
313- Asx-Ser-Thr-Lys-Lys-C~s-Ala-Asp-Ser-Asp-Pro-Arg-Gly-
Val-Ser-Ala-Tyr-Leu-Ser-Arg-Pro-Ser-Pro-Phe-
--
108717~
337- Asp-Leu-Phe-Ile-Arg-Lys-Ser-Pro-~hr-Ile-Thr-Cys-Leu-
Val-Val-Asx-Leu-Ala-Pro-Ser-Lys-Gly-Thr-Val-
361- Asn-Leu-Thr-Trp-Ser-Arg-Ala-Ser-Gly-Lys-Pro-Val-Asx-
His-Ser-Thr-Arg-Lys-Glu-Glu-Lys-Gln-Arg-Asn-
385- Gly-Thr-Leu-Thr-Val-Thr-Ser-Thr-Leu-Pro-Val-Gly-Thr-
Arg-Asx-Trp-Ile-Glu-Gly-Glu-Thr-Tyr-Glx-Cys-
409- Arg-Val-Thr-His-Pro-His-Leu-Pro-Arg-Ala-Leu-Met-Arg-
Ser-Thr-Thr-Lys-Thr-Ser-Gly-Pro-Arg-Ala-Ala-
433- Pro-Glu-Val-Tyr-Ala-Phe-Ala-Thr-Pro-Glu-Trp-Pro-Gly-
Ser-Arg-Asp-Lys-Arg-Thr-Leu-Ala-Cys-Leu-Ile-
457- Gln-Asn-Phe-Met-Pro-Glu-Asp-Ile-Ser-Val-Gln-Trp-Leu-
~is-Asn-Glu-Val-Gln-Leu-Pro-Asp-Ala-Arg-His-
481- Ser-Thr-Thr-~Gln-Pro-Arg-Lys-Thr-Lys-Gly-Ser-Gly-Phe-
Phe-Val-Phe-Ser-Arg-Leu-Glu-Val-Thr-Arg-Ala-
505- Glu-Trp-Gln-Glu-Lys-Asp~Glu-Phe-Ile-Cye-Arg-Ala-Val-
His-Glu-Ala-Ala-Ser-Pro-Ser-Gln-Thr-Val-Gln-
529- Arg-Ala-Val-Ser-Val-Asn-Pro-Gly-Lys
The novel compounds of the present invention are poly-
peptides comprising between 3 and 10 amino acids in sequence,
said sequence selected from a portion of the above amino
acid sequence; as well as the salts, esters, amides,
N-acyl and O-acyl derivatives thereof.
As set forth above and for convenience in describing
this invention, the conventional abbreviations for the
various amino acids are used. They are familiar to those
skilled in the art; but for clarity, those with which this
invention is concerned are listed below. All chiral amino
acid residues referred to herein are of the natural or L-
configuration,unless otherwise specified. All peptide
3~ sequences mentioned herein are written according to the
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. .
` 1087171
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
Ala = Alanine
Arg = Arginine
Asn = Asparagine
Asx = Aspartic Acid or Asparagine
(indicates uncertainty in degradation analysis)
Cys = Cysteine
Gly = Glycine
Gln = Glutamine
Glu = Glutamic acid
Glx = Glutamic Acid or Glutamine
(indicates uncertainty in degradation analysis)
His z Histidine
Ile = Isoleucine
Leu - Leucine
. Lys = Lysine
Met = Methionine
Phe = Phenylalanine - -
Pro = Proline
Ser = Serine
Thr = Threonine
Trp = Tryptophan
Tyr = Tyrosine
Val = Valine
- As used herein the term "salts" 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
--7--
ios7~7~1
.
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, man-
ganic salts, and the like. Salts with organic amines
include those formed, for example, with trimethylamine,
triethylamine, tri(n-propyl)amine, dicyclohexylamine, ~-(di-
methylamino) ethanol, tris(hydroxymethyl)aminomethane, tri-
ethanolamine, 3-(diethylamino) ethanol, arginine~ lysine,
histidine, N-ethylpiperidine, hydrabamine, choline, betaine,
ethylenediamine, glucosamine, methylglucamine, theobromine,
purines, piperazines, piperidines, 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 with organic acids such as for example
acetic acid, oxalic acid, tartaric acid, succinic acid,
maleic acid, fumaric acid, glucon-ic acid, citric acid, malic
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,
. 25 isopropyl, n-butyl, t-butyl, n-amyl, n-hexyl, octyl,. decyl,
and dodecyl esters.
As used herein the term "amides" refers to amides of
a carboxy group of the polypeptide formed with ammonia, or
with primary or secondary amines having up to 12 carbon atoms
such as for example dimethylamine, diethylamine, di(n-butyl)-
lOB7171
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 moieties
(e.g. alkanoyl or carbocyclic aroyl groups) containing up to
12 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 car~ocyclic aroyl groups) con-
taining up to 12 carbon atoms, such as formates, acetates,
propionates, benzoates, and the like.
Preferred polypeptides of this invention are those
which have amino acid sequences that are non-analogous with
comparable regions in other immunoglobulins. In this regard,
the following peptides may be especially mentioned:
266 - Asp-Val-Asp-Leu-Ser
271 - Thr-Ala-Ser-Thr-Glu
266 - Asp-Val-Asp-Leu-Ser-Thr-Ala-Ser-Thr-Glu
289 - Leu-Ser-Gln-Lys-His
319 - Ala-Asp-Ser-Asp-Pro-Arg
320 - Asp-Ser-Asp-Pro-Arg
321 - Ser-Asp-Pro-Arg
322 - Asp-Pro-Arg
354 - Ala-Pro-Ser-Lys-Gly-Thr
367 - Ala-Ser-Gly-Lys-Pro
437 - Ala-Phe-Ala-Thr-Pro-Glu-Trp-Pro-Gly-Ser
437 - Ala-Phe-Ala-Thr-Pro
442 - Glu-Tr~-Pro-Gly-Ser
476 - Pro-Asp-Ala-Arg-His-Ser
_9_
1087171
521 - Ala-Ser-Pro-Ser-Gln
as well as salts, esters, amides, ~-acyl and O-acyl deriva-
tives thereof.
A particularly preferred polypeptide is Asp-Ser-Asp-
Pro-Arg.
The above list is not intended to be exhaustive and
additional peptides having shorter sequences than the above,
or having sequences with additional amino acids therein, or
sequences taken from other regions of the C-2, C-3 or C-4
domains, are of importance.
In a second aspect, the present invention is direrted
to a method useful for preventing or relieving symptoms
associated with allergic manifestations such as are brought
about by antigen-antibody ~allergic) reactions. The method
hereof serves to block (i.e., inhibit or prevent) the effects of
the allergic reaction when the subject polypeptide is admin-
. istered in an effective amount. Thus this aspect of the
present invention relates to a method useful for preventing
or inhibiting the effects of allergic reaction which com-
prises administering to a mammalian subject ~preferably a
human) an effective amount of a polypeptide or derivative
thereof as hereinabove described.
While the compounds of the present invention are
believed to act by "blocking" IgE binding sites as described
herein, it is not intended that the present invention be
limited to any particular mechanism of action.
~he present invention, in a third aspect, is directed
to pharmaceutical compositions useful for blocking (i.e. prevent-
--10--
1087171
ing or inhibiting~ the effects of the allergic reaction comprising
an effective amount of a polypeptide or derivative thereof,
as described hereinabove, in admixture with a pharmaceuti-
cally acceptable non-toxic carrier.
In the practice of the method of the present invention,
an effective amount of a polypeptide or derivative thereof,
or a pharmaceutical composition containing 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 composi-
tions can thus be administered orally~ sublingually~ topi-
cally (e.g. on the skin or in the eyes), parenterally (e.g.
intramuscularly, intravenously, subcutaneously or intrader-
mally), or by inhalation, and in the form of either
solid, liquid or gaseous dosage including tablets, sus-
pensions, and aerosols, as discussed in more detail herein-
after. 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 of the present
invention is practiced when the relief of symptoms i9
specifically required or perhaps imminent; in another pre-
ferred embodiment, the method hereof is 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
determinable by routine experimentation by one skilled in
--11--
.
.
.
0~717~ -
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 2x103 and 2X106
times the IgE content, on a molar scale. For an average
subject this would be between about 0.5 and 500 mg/kg~day,
depending upon the potency of the compound. Of course, for
localized treatment, e.g., of the respiratory system, pro-
portionately 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
formulations (such as by binding on ion exchange resins or
other carriers, or packaging in lipid-protein vesicles or
adding additional terminal amino acids or replacing a te m inal
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, vegetable or synthetic origin, for
example, peanut oil, soybean 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, chalk, silica gel, magnesium
stearate, sodium-stearate, glycerol monostearate, sodium
chloride, dried skim milk, glyceroi, propylene glycol,
water, ethanol, and the like. The compositions may be sub-
-12-
.
` -- lOB7171
jected to conventional pharmaceutical expedients such as
sterilization and may contain conventional pharmaceutical
additives such as preservatives, stabilizing agents, wetting
or emulsifying agents, salts for ad~usting osmotic pressure,
buffers,and the like. Suitable pharmaceutical
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 suitable amount of carrier so as
to prepare the proper dosage orm for proper administration
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-
irritating at the levels in actual use. This has been -
demonstrated to be the case with all of the present compounds
whose preparation is described hereinbelow.
The polypeptides of the present invention may be
synthesized by-any techniques that are known to those
skilled inthe peptide art. An excellent summary of the many
techniques so available may be found in J. Meienhofer,
"Hormonal Proteins and Peptides", Vol. 2, p. 46., Academic
Press (New York), 1973 for solid phase peptide synthesis
and E. Schroder and K. Lubke, "The Peptides", Vol. l,
Academic Press (New York), 1965 for classical solution
synthesis.
In general, these methods comprise the sequential
addition to a growing chain of one or moxe amino acids or
suitably protected amino acids. Normally, either the amino
or carboxyl group of the first amino acid is protected,
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.. . . .
.
10~717~
by a suitable protecting group. The protected or deriva-
tized 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 carboxyl) 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 (suitably protected)
is then added, and so forth. After all the
desired amino acids have been linked in the proper sequence,
any remaining protecting groups (and any solid support) are
removed sequentially or concurrently, to afford the final
- polypeptide. By simple modification of this general pro-
cedure, it is possible to add more than one amino acid at
lS a time to a growing chain, for example, by coupling (under
conditions which do not racemize chiral centers) a pro-
tected tripeptide with a properly protected dipeptide 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, toluen~sulfonyl (tosyl), benzen-
sulfonyl, o-nitrophenylsulfenyl, tritylsulfenyl, o-nitrophen-
oxyacetyl, chloroacetyl, acetyl, y-chlorobutyryl, etc.;
3~ (2) aromatic urethan type protecting groups illustrated by
.
:
1087~7~
benzyloxycarbonyl and substituted benzyloxycarbonyl such as
p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-
bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-(p-
biphenylyl)isopropyloxycarbonyl, 2-benzoyl-1-methylvinyl;
~3) aliphatic urethan protecting groups illustrated by tert-
butyloxycarbonyl, tert-amyloxycarbonyl diisopropylmethoxy-
carbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxy-
carbonyl; (4) cycloalkyl urethan type protecting groups
illustrated by cyclopentyloxycarbonyl, adamantyloxycarbonyl,
cyclohexyloxycarbonyl; (5) thio urethan type protecting
groups such as phenylthiocarbonyl; (6) alkyl type protecting
groups as illustrated by triphenylmethyl (trityl) 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 useful
for stepwise synthesis of polypeptides are: (1) substituted
or unsubstituted aliphatic ester protecting groups such as
methyl, ethyl, t-butyl, 2,2,2-trichlorethyl and t-butyl
esters; (2) aralkyl ester protecting groups such as benzyl,
p-nitrobenzyl, p-methoxybenzyl, diphenylmethyl or triphenyl-
methyl (trityl) esters; (3) N-substituted hydrazides such
as t-butyloxycarbonylhydrazides-and carbobenzyloxycarbonyl-
hydrazides; (4) amide protecting groups formed by condensa-
tion of a carboxyl moiety with e.g. ammonia, methylamine,
ethylamine, diphenylmethylamine; and the like.
Hydroxyl groups of amino acids such as serine, threo-
nine and hydroxyproline may be protected as aralkyl ethers
such as benzyl ethers.
Suitable solid supports useful for the above synthesis
-15-
' , . : . .' ~,
.
- 1087171
are thos~ 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 function-
alized 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, hydroxymethyl,
phenacyl halide, dehydroalanine and the like. ~he preferred
active group is chloromethyl. The first amino acid may be
coupled to the preferred chloromethyl resin by one of
several base catalyzed 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, dimethylformamide, and the like.
Suitable reagents that effect amide formation between
carboxyl and amino groups are known in the art and include,
for example, ~1) carbodiimides such as for example dicyclo-
hexylcarbodiimide (DCC), t2) a carbodiimide plus an additive
- such as l-hydroxybenzotriazole or ethyl 2-hydroximino-2-
cyanoacetate; (3) alkyl chloroformates such as isobutyl-
chloroformate 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-thio-
pyridyl esters and similar esters well known to those skilled
in the art.
-16-
,
1087~7~
A preferred method for synthesizing the peptides of
the present invention is the so-called "Merrifield" syn-
thesis 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 Societyc 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 of amino acids to a growing peptide chain
which is bound by a covalent bond to a solid resin particle.
In the preferred application of 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 indica~e~
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 racemization 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, with a mixture of hydrobromic acid and trifluoro-
acetic acid or with hydrofluoric acid, or the cleavage from
the resin may be effected under basic conditions~ for example,
with triethylamine, the protecting groups then being
removed under acid conditions.
The cleaved peptides are isolated and purified by
means well known in the art such as, for example, lyo-
philization followed by either exclusion or partition
.
- 10~7~71
chromatography on polysaccharidegel media such as
Sephadex G-25*, or countercurrent distribution. The compo-
sition of the final peptide may be confirmed by amino acid
analysis after degradation of the peptide by standard means.
Salts of carboxyl groups of the peptide may be pre-
pared 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 pre-
pared by contacting the polypeptide with one or more
equivalents of the desired inorganic or organic acid, such
as, for example, hydrochloric acid.
Esters of carboxyl groups of the polypeptides may be
prepared by any of the usual means known in the art for
converting a carboxylic acid or precursor to an ester. One
preferred method for preparing esters of the present poly-
peptides, when using the Merrifield synthesis technique
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 esterified 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 carboxylic acid group or precursor, to
*trade mark -18-
.: . : . . ' .
.
1087~71
an amide. A preferred method for amide formation at the
C-terminal carboxyl group is to cleave the polypeptide
from a solid support with an appropriate amine, or to cleave
in the presence of an alcohol, yielding an ester, followed
S by aminolysis with the desired amine.
N-acyl derivatives of an amino group of the present
polypeptides may be prepared by utilizing an N-acyl pro-
tected 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- acylation may be carried out together, if
desired.
The coupling, deprotection/cleavage reactions and
preparation of derivatives of the subject polypeptides
are suitably carried out at temperatures between about
-10 and +50C., most- preferably about 20-25C. The exact
temperature for any particular reaction will of course
be dependent upon the substrates, reagents, solvents and
so forth, all being well within the skill of the practi-
- - tioner-. Illustrative reaction-conditions for these
processes may be gleaned from 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.
--19--
.: . . - .
. .
7171
EXAMPLE 1
Preparation of the Tripe~tide Asp-Pro-Arg
1.6 G. (5 mmoles) of t-Boc-nitroarginine are reacted
with 10 g. of chloromethyl resin tbeaded copolystyrene-2
divinyl benzene containing 0.5-1 meq. of chloromethyl
groups per gram of resin) in a mixture of 1.4 ml. (10
mmoles) of triethylamine and 100 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 of ethanol,
then methanol and finally methylene chloride. The resin
is then thoroughly dried in vacuo. Analysis revealed
0.05 mmole Arg/g. resin. 2,5 G. of the resin so prepared
is placed in a Merrifield solid phase reaction vessel
equipped for agitation and is put through the following
DEPROTECTION CYCLE:
(a) with agitation, and at 22C., the t-Boc group is
cleaved with 10 ml. of 4 N. HCl in dioxane for 30 minutes,
(b) two washes with 10 ml. of dioxane,
(c~ two washes with 10 ml. of methylene chloride,
(d) two washes with 10 ml. of chloroform,
~e) the hydrochloride is neutralised with 10 ml. of
triethylamine/chloroform (5:95),
(f) t~o washes with 10 ml. of methylene chloride,
(g) two washes with 10 ml. of chloroform,
The resin is then subjected to the SYNTHESIS CYCLE as
follows: a ten-fold excess of t-Boc-proline (1.25 mmoles)
in methylene chloride solution is,added followed by 258 m~.
(1.25 mmoles) of dicyclohexylcarbodiimide (DCC) and the
mixture is shaken for 2 hours at 22C. The resin is then
-20-
OB7171
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 to t-Boc
~-benzyl aspartate (0.5 mmoles) as described above in the
synthesis cycle. An 0.5 g. portion of the resin is then
removed from the reaction vessel and subjected to the
CLEAVAGE 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 tri-
fluoroacetic acid. The combined filtrates are concentrated
in vacuo and excess HBr is removed from 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 hydrogenation in a Parr low pressure
shaker hydrogenation apparatus as follows: The nitro
protected tripeptide is dissolved in a mixture of methanol-
acetic acid-water (10:1:1)" about 10-20 mg./ml., and an equal
weight of 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 filtration and
the filtrates are concentrated in vacuo. The peptide
residue is chromatographed on a column of Sephadex G-25*.
The yield of the purified tripeptide as established by con-
ventional amino acid analysis is approximately 24~ based on
the arginine incorporated in the resin. A portion of the
product was hydrolysed with 5.7 N. HCl in water and assayed -
* trade mark -21-
,~ -
. , ' ~ ~ : '
10i37171
on an amino acid analyser, which indicated a ratio of
Asp l.OS, Pro 0.95, Arg 1.00.
Purity was determined by paper electrophoresis in
the standard manner at a ~umber of pH's.
~ ~ '
.
~0~717~
EXAMPLE 2
PreParation of the Tetrapeptide Ser-Asp-Pro-Arg
The tripeptide resin from Example 1, not used in the
synthesis of the tripeptide, was put through the deprotection
cycle (see Example l) 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 of the resin was then subjected to the
cleavage and hydrogenation processes as described in
Example 1 and recovered in the same manner as in Example l
yielding Ser-Asp-Pro-Arg in a 20% yield based o~ arginine
esterified to the resin. After hydrolysis with HCl, a
sample of the recovered tetrapeptide was assayed on the
amino acid analyser, which indicated a ratio of Ser 0.79,
Asp 1.18, Pro 1.02, and Arg l.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.
.
. . ' ' . . .; : . : ' ::
- , . . . .
. :: : . . .
, ' .
1087171
EXAMPLE 3
Preparation of the Pentapeptide Asp-Ser-Asp-Pro-Arg
A. The uncleaved tetrapeptide resin from Example 2
was subjected to the deprotection cycle (Example 1) and
the synthesis cycle using 0.152 g. of t-Boc-~-benzylaspartate.
The resin portion had the pentapeptide cleaved
therefrom with HBr in trifluoroacetic acid in the same
manner as noted previously. The recovered polypeptide 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
a ratio of Asp 2.12, 5er 0.74, Pro 1.12, and Arg 1.01.
B. The pentapeptide is also prepared by-a modifica-
tion of the procedures of Examples 1-3A:
- To a solution o~-3.02 g. (6.82 mmoles) of ~-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. H2O) until
the pH of the solution is 7Ø The solution is concentrated
in vacuo to a foam which is thoroughly 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
cop~lystyrene-1~ 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 x 20 ml.), 90% DMF/H2O (3 x 20 ml.), DMF (2 x 20 ml.)
and EtOH (2 x 20 ml.) and is then dried in vacuo over
P2O5 giving 5.54 g. of argininated resin (ca. 50% incorporation).
- -24-
10~7~7~
This resin is then subjected to four cycles of
deprotection and synthesis using 4 equivalents of the
appropriate t-Boc-amino acid at each chain elongation step
giving the protected pentapeptide resin material.
This resin material is then placed in an HF
resistant reaction vessel, 8 ml. of anisole is added and
the vessel is attached to an HF line. Approximately 70 ml.
of HF is distilled into the reaction vessel at 0C. and the
mixture is stirred for a further 30 minutes at 0C. The
HF is pumped off and the resin 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 purified according to Example 1 thereby giving the -
pentapeptide Asp-Ser-Asp-Pro-Arg.
The pentapeptide prepared above exhibits an
[a]20 = -78.6 (c=l, H2O). Purity was determined by
paper electrophoresis in the standard manner at a number
of pH's.
,
- . . . .
~, ~, -, . - . . . ..
,
. .
10~37~71
- EXAMPLE 4
: Preparation of the hexapeptide Ala-Asp-Ser-Asp-Pro-Arg
Another batch of arginated-resin (0.20 mmoles) was
taken through the 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 dicyclohexylcarbodiimide in the usual
manner. -- ~
The resin was ~hen subjected to the cleavage and
hydrogenation processes as described in Example 1 and
j recovered in the same manner as in Example 1 yielding
; Ala-Asp-Ser-Asp-Pro-Arg in a 0.026 mmole, 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 p~'s.
. ~ .
,
.
, ' ' ; -
1~87171
EXAMPLE 5
. Utilizing similar synthesis procedures to those
described in Examples 1-4 above, the following polypeptid~
may ~e prepared: . , .
Asp-Val-Asp-Leu-Ser
Thr-Ala-Ser-Thr-G,lu
Asp-val-Asp-Leu-3~-lrhr-Ai~- ~c-r-Thr-Glu
Leu-Ser-Glu,-Ly,s-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
Asp-Thr-Glu-Ala-Arg
, - , : . - :. -, - .
., , . - . .. ~ . . . : . . : . -
, , . ,. . ~ ,, ,. .. ,, ~,, .. . . . , ~.
:-- ':' . ~ : - . -
..
1~87171
EXAMPLE 6
Preparation of Metallic and Amine ~alts
_ _ _ _ _ _ _ _ _A ___ _ ___ _ _ _ .
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 e~uivalents of
0.1 N. NaOH the corresponding di- and trisodium salts are
obtained respectively.
Similarly, this peptide may be converted to other
metallic salts, e.g., potassium, lithium, calcium, barium,
magnesium, ammonium, ferrous, ferric, zinc, manganous, man-
ganic, and aluminum salts, by substitution of 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,
20 ~ -followed-by careful evaporatiQn-of the-solvent, yields
the mono-, bis- and tris-triethylammonium salts respectively.
Similarly this pentapept-i-de--may be-converted to other ----- --
--- amine salts, e.g., trimethylamine, tri(n-propyl)amine, -
. dicyclohexylamine, ~-tdimethylamino)ethanol, ~-(diethyl- _- -
amino)ethanol, triethanolamine, tris(hydroxymethyl)amino-
met~ane,arginine, lysine, histidine, N-ethylpiperidine,
hydrabamine, choline, betaine, ethylenediamine, glucosamine,
methylglucamine, theobromine, purine, piperazine, piperidine,
caffeine and procaine salts, by substitution of the appro-
priate amine.
C. ID a similar manner, the other peptides of Examples 1,
--28--
` 1087171
. ' .
-: 2, 4 and 5 may be converted to their corresponding metallic
and amine salts.
. . ,
, . . . . . . . . .
., ., . . . .: . . . : . . :
,, ~
1t)~37171
EXAMPLE 7
The pentapeptide Asp-Ser-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 methanol with exactly 1 or 2 equivalents
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 appropriate acid for hydrogen
chloride.
In a similar manner~ the other peptides of Examples 1,
2, 4 and 5 may be converted to their corresponding acid
addition salts.
.
'. ' ' ,
- ' '
,
1087171
EXAMPLE 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
of Ser or Thr are removed by hydrogenolysis using Pd/BaSO4
as described in Example 1 for the removal of the nitro
-15 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
- - -- - respecti~ely.
By substituting other alcohols for methanol and
raising the reaction t~per~ture to 45-~0~C.-*nd.the-~
reaction time to 45-90-hours-~here-are-~btained--the--~
corresponding ethyl, propyl, butylr.hexyl, ~ctyl-~ decyl
and dodecyl esters.
B. In this procedure a different type of anchoring
~ond is used for the attachment of the arginine residue,
namely the resin-0-CH2-CH2-C(CH3)2-OCONHNH2 bond described by
S. Wang and R.B. Merrifield in J. Amer Chem. Soc. 91,
6488 (1969J. Also, in this procedure N-2-(p-biphenylyl)-
isopropyloxycarbonyl (~poc) protecting groups are used
-31-
,
.
108717~
instead of t-Boc for -amino protectlon 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~ trifluoro-
- acetic acid (TFA) /CH2C12 is used in the deprotection
- cycle in order to cIeave the Bpoc group. The ultimate
amino acid incorporated is protected as a Na-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. of 50% TFA in
CH2C12 and the mixture is shaken at room temperature for
30 minutes. The resin is 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 soluti-on-~-f--the-protected--peptide--hydrazide--~
(0.2 mmoles) in DMF (1 ml.) is cooled to -20C. and 3.35 N.
HCl in 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 lO 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
6 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 filtràtion. The
-32-
7171
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 or
by countercurrent distribution gi~ing 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.
., .:
';
,
.,, ~' . , ' ' `
., ' ' ' ' ~
' ' ' '~
..
.
7171
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
; 5 temperature for 2 days. The solvent is removed in vacuo
to afford
Ala-Pro-Ser-Lys-Gly-Thr-NH2,
Ala-Ser-Gly-I-ys-Pro-NH2,
Ala-Phe-Ala-Thr-Pro-NH2, and
Asp-Ser-Asp-Pro-Arg-NH2,
respectively.
B. ~he peptide-azide of Example 8B is reacted with
ammonia in DMF solution under the conditions described in
Example 8B for reaction with methanol. The protected
. : -peptide-amide-ls isolated and deprotected as described ...
earlier giving Asp~Ser-Asp-Pro-Arg-NH2.
. C. The protected peptide.resin_product_~f~Example 3A__
is suspended in.a saturated solution of ammonia in methanol
;.......... .... ...and .the mixture is agi-.ta-ted~at.~oo~;temperatu~e_or.~2_days.
The resin is removed by filtration, washed with methanol
_._ _ _=. and the combined filtrates-are concentrated i~-vacuo ..
- giving t-Boc-Asn-O-benzy~-Ser-Asn-Pro-N~-nitro-Arg-NH2. - -~
. The t-Boc-group:and::the.;.'~.~-nitro group are then.remQved by--~
' acidic hydrolysis and hydrogenolysis respectively, as
des'cribed 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,
3Q di(n-butyl), n-hexyl, piperidyl, pyrrolidinyl, morpholinyl,
-34-
1~7171
ditn-hexyl) and N-methylpiperazinyl amides.
D. Utilizing similar procedures to those described
in A, B and C, the corresponding amides of the other poly-
peptides of Examples 1, 2,4 and 5 may be prepared.
.. . . . ~ : -
, . ~: ' ' ~ - '
.: - , . .
,~` "' ~.
1~87171
EXAMPLE 10
Preparation of N-acyl derivatives
.
N -Acyl derivatives of Asp-Ser-Asp-Pro-Arg are prepared
by replacing the terminal t-Boc-amino acid (t-Boc-~-benzyl-
S aspartate) with the appropriate Na-acyl amino acid (e.g.
Na-acetyl-~-benzylaspartate). All other steps 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
Na-Octanoyl-Asp-Ser-Asp-Pro-Arg
Na-Decanoyl-Asp-Ser-Asp-Pro-Arg
N -Dodecanoyl-Asp-Ser-Asp-Pro-Arg
Similarly, the corresponding Na-acyl derivatives of
other peptides mentioned in Examples 1, 2, 4 and 5 may be
prepared.
7ln
EXAMPLE 1 1
. .
Preparation of O-Acyl Derivatives
In order to prepare the protected pentapeptide 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 1 is sub-
jected 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 a p-nitrophenyl t-Boc-~-benzyl-
aspartate under standard conditions, thereby giving t-Boc-
~-benzyl-Asp-Ser-~-benzyl-Asp-Pro-N~-nitro-Arg-resin.
0.5 Mmoles of this protected peptide resin material
is washed thoroughly with CHC13 and CH2C12 and 1.5 mmoles
of hexanoic acid dissolved in 1:1 DMF/CHC13 is added
followed by 1.5 mmoles of carbonyl diimidazole dissolved -
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.
-37-
lOB71~1
EXAMPLE 12
.
The following illustrates typical pharmaceutical
compositions of the compounds hereof, exemplified by
Asp-Ser-Asp-Pro-Arg:
.
Aerosol Formulation (per dose)
Asp-Ser-Asp-Pro-Arg 10 mg.
Sodium chloride 8 mg.
Water to make 1.0 ml.
Injec_able 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 Spinhale ~ (per dose)
Asp-Ser-Asp-Pro-Arg 10 mg.
Lactose 30 mg.
-38-
, .
1087~71
EXAMPEE 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 injected 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
next 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 r-elease 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 al-lerg-ic--reaction-at all-.-~-The-~-P-K-r,eaction as --- _ -
noted above is a classic test and is universally known and
utilized by allergists.
As noted above, the classic P-~-reaction is utilized - --
to assay--the "blocking" abil-i~ties-of the--polypept~des - ---- ----~ =----
utiliz,ed in the present invention.
~he following describes assays of a number of poly-
peptides useful in 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.
-39-
: ~ ,
10~7171
Peptide solutions were either injected intradermally
1 to 24 hours prior to the P-K serum, 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 invention peptide solutions
were varied to contain from about 1 mM to 2 M of the peptide
being tested. Injected sites on the volunteers were chal-
lepged by prick-puncture of guinea pig BCA 1:40w/v (pur-
chased from Berkeley Biologicals, Inc.).
Human volunteers were chosen who had serum IgE levels
below 100 U/ml (242ng/ml) which levels have been previously
shown to assure successful P-K reàctivity. In addition,
for the purpose of these tests, the individuals were
chosen who had a negative direct skin test to guinea ~ig
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
- of 0.1 ml of the peptide solution or control buffered
saline diluent solution; followed in 1 to 24 hours by - -
intradermal injection of-~0.05 ml.-of P-X-serum into each -
of the previously injected sites. After 20 to 24 hours
had 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
-40-
.
10~7171
following polypeptides: Asp-Pro-Arg; Ser-Asp-Pro-Arg;
Asp-Ser-Asp-Pro-Arg; and Ala-Asp-Ser-Asp-Pro-Arg. For
comparison testing, Asp-Thr-Glu-Ala-Arg and tosyl-L-arginine
- sarcosine methylester (TASME), were also 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 Ala-Asp-Ser-Asp-Pro-Arg, the average inhibition
was 46%, with an individual low of 10%, 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
- measurements at three tLme intervals, in duplicate, for
each reaction in each individual, subtracted from the -
- - _ average control wheal measurements, and divided by the - ~~ ~
- - a~erage measurement of each individual's control wheal.
- -25 Control wheaIs in different individuals varied from 8 to
40 mm2 with a mean of 17 mm2. Each peptide was utilized
at approximately 6 ~g/ml dilution and 0.1 ml. was injected
at each site, followed ~y 0.05 ml. of diluted P-K serum
containing 0.2 ng. of IgE. Thus 10 M of the peptide
was competing with 10 M of the IgE for the binding sites
-41-
. .
.
10~7171
on mast cells, or a ratio of one IgE molecule to 10 pep-
tide molecules. From 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 tripeotide
A`sp-Pro-Arg, exhibited the least activity.
It should be noted that the pentapeptide Asp-T~r-Glu-
Ala-Arg was prepared and assayed along with the other
peptides as described above. This particular polypeptide
does not have an analogous sequence of amino acids appearing
in the C~2, C~3 or C~4 domains of the IgE molecule, yet it
exhibits a high activity in the assay test.
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EXAMPLE 14
. It has also been determined that the active poly-
peptides of the invention appear to have the ability to
Ndisplace" 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 concentra-
. ..tion of guinea-pig-antigen-sensitive IgE, was injected
with polypeptides in accordance with the invention, and
his reaction to guinea pig antigen was not.ed.
Specifically, approximately 2nM 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 control of the buffer diluent alone, was also
- - -15 . each injected -into 3-marked--sit;es. -At one, five and '
- twenty-four hours subsequent to the-polypeptide and control --
- - injection, one of each peptide and.. Qne_diluent site were .
prick-puncture challenged with guinea pig antigen. No
- - :: ... ~inh-ibition of the wheal and--f.lare reaction was--observed~
at any site at the one and five hour intervals. However,
at the twenty-four hour challenge, the wheal at the Asp-
Ser-Asp-Pro-Arg site was approximately 45% smaller; while
at the Ala-Asp-Ser-Asp-Pro-Arg site,~the-whealwas--approxi- - ~-
. ..... mately 23% smaller. No reduction in the size of the wheal
was.observed at the TASME site compared to the buffered
saline diluent site.
It thus appears that, at least the most active of the '
peptides of the invention, will "displace" IgE already
bound to mast cell sites, thus.inhibiting a natual allergic
reaction. As has previously been demonstrated in Example 13
-- . , .
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10137171
this same pentapeptide is extremely effective in inhibiting
apassively transferred (P-K) allergic reaction.
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EXAMPLE 15
Acute toxicity was determined as follows:
DEA 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:
O.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 were as
follows:
Ala-Asp-Ser-Asp-Pro-Arg ~Example 4) 5 ~g/ml (375 mg/kg)- 6 mice
Asp-Ser-Asp-Pro-Arg ~Example 3) lO ~g~ml (1 mg/kg) - 8 mice
Asp-Ser-Asp-Pro-Arg (Example 3) 13 ~g/ml (1.3 mg/kg)- 8 mice
Post-mortem gross and microscopic examination of
, tissues and organs indicated no local or systemic toxico-
I logical abnormalities.
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