Note: Descriptions are shown in the official language in which they were submitted.
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UTSD:083
ANTIPYRETIC AND ANTI-INFLAMMATORY PEPTIDES
This invention relates to a new pharmaceutical com-
position useful for the treatment of pyrexia and inflamma-
tion. More particularly, this invention relates to a
tripeptide sequence contained in alpha-Melanocyte
Stimulating Hormone and ACTH which has been identified as
an antipyretic and anti-inflammatory agent.
There are two classes of agents presently in common
usaye as antipyretic agents, the salicylates and the
para-aminophenol derivatives. The salicylates,
characterized by acetylsalicylic acid (i-e- Aspirin), are
the most extensively employed antipyretic agents. Aspirin*
is the prototype for both the salicylates and other drugs
with similar efects and is the standard of reference for
comparison and evaluation of these agents. The anti-
pyretic effect of Aspirin is usually rapid and effective
in febrile patients. The salicylates act to reset the
"thermostat" for normal temperature.
Although Aspirin is generally well-tolerated by most
individuals, a number of toxic side effects are associated
with its use. Of particular concern is salicylate-induced
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gastric ulceration and sometimes hemorrhage. Exacerbation
of peptic ulcer symptoms (heartburn, dyspepsia), gastro-
intestinal hemorrhage, and erosive gastritis have all been
reported in patients taking Aspirin*. Other less common
side effects include tinnitus and hearing loss, changes in
acid-base balance and electrolyte pattern, and respiratory
alkalosis. Although generally such side effects are not
particularly dangerous, they tend to reduce patient
compliance. Other salicylate derivatives, which are
generally employed for their analgesic and/or anti-
inflammatory activity, demonstrate increased toxicities
relative to Aspirin.
The para-aminophenol. derivativesr acetaminophen and
phenacetin, are alternatives to Aspirin for its analgesic
and antipyretic uses. Acetaminophen has somewhat less
overall toxicity and is generally preferred over phenace-
tin. Because acetaminophen is well-tolerated and lacks `
many of the undesired effects of Aspirin, it has been
gaining favor as the "common household analgesic." How-
ever, its suitability for this purpose is questionable: inacute overdosage, acetaminophen can cause fatal hepatîc
necrosis. In addition, phenacetin may cause methemo-
globinemia and hemolytic anemia as a form of acute
toxicity, but more commonly as a consequence of chronic
overdosage. These a~ents are about equipotent with As-
pirin in the treatment of pyrexia.
Recent advances in the study of alpha-Melanocyte
Stimulating Hormone (hereinafter referred to as "MSH")
have demonstrated that this protein is active in the
treatment of pyrexia. Alpha MSH is a 13-amino acid pep-
tide derived from Adrenocorticotropic Hormone ("ACTH").Both MSH and ACTH share a 13-amino acid sequence that is
effective in modulating body temperature. There is
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evidence that these neuropeptides can influence centrally
mediated processes, including central control of body
temperature. Both peptides lower core temperature of
afebrile rabbits when given peripherally or centrally in
sufficient dosages. Much smaller dosages reduce fever
without altering normal temperature.
Alpha MSH is found in brain regions that govern tem-
perature regulation, including the anterior hypothalumus
and the septum. The concentration of alpha MSH in the
septum rises during fever, and the concentration in the
arcuate nucleus tends to decline at the same time.
Studies comparing the antipyretic activity of centrally-
administered alpha MSH to the widely-used antipyretic,
acetaminophen indicate that alpha MSH is much more potent
in reducing fever than acetaminophen, and that alpha MSH
was ~ore than 2500 times more potent by weight than
acetaminophen in reducing fever. No endogenous substance
other than ACTH is known to have such potency in reducing
fever.
The antipyretic potency of alpha MSH and the fact
that this peptide reduces fever even when given peripher-
ally may have clinical significance. ACTH was used to
reduce clinical and experimental fever soon after it was
first described, but this peptide also stimulates cortico-
steroid release, and can, with repeated administration,
result in Cushing's syndrome. On the other hand, the
shorter alpha MSH molecule, which is derived from ACTH,
does not stimulate steriod release and there appears to be
no irreversible deleterious effects when given to rabbits
or to man.
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With respect to ACTH (amino acids 1-39 of proopio-
cortin), it has previously been known that due to its
corticosteroid stimulatory effect, this protein was active
in the treatment of inflammation. However, shorter ACT~
related peptides such as alpha-MSH (amino acids 1-13 of
ACTH) which do not exhibit corticotropic activity have not
been shown to have anti-inflammatory action, and there has
previously been no basis to suggest such a role for
peptides which correspond to amino-terminal portions of
ACTH yet which exhibit no corticotropic activity.
The present invention provides a pharmaceutical com-
position useful in the treatment of pyrexia and inflamma-
tion. The active component of this pharmaceutical compo-
sition is a peptide which includes an amino acid sequencecorresponding to amino acids 11 through 13 of alpha MSH,
lysine-proline-valine ("lys-pro-val"). In its most
general scope, the invention is directed to peptides of
from 3 to 13 amino acids, which peptides have sequences
corresponding to that of alpha-MSH and include at least
the lys-pro-val sequence thereof. In more particular
embodiments, the invention is directed to the tripeptide
itself.
Preferably, the tripeptide itself is administered to
achieve maximal benefits in accordance herewith, prefer-
ably in a biologically "protected" form. When the
tripeptide is "protected" through acylation of the amino
terminus and/or amidation of the carboxyl terminus, the
resulting tripeptide demonstrates an increase in pharma-
cologic activity. Similarly, when the tripeptide, whether
"protected" or "unprotected", is co-administered with
copper ion, a further increase in antipyretic activity is
observedO
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The present invention provides a method for treating
pyrexia and/or inflammation in an individual in need of
such treatment in which an effective dose of a peptide
which includes the tripeptide sequence is administered to
the pyretic individual. Moreoverl such peptides may be
used in the treatment of both generalized or localized
inflammation and, therefore, is a useful alternative to
- steroidal and salicylate anti-inflammatory agents. Anti-
inflammatory activity is observed following administration
of the tripeptide to animals at doses approximately equal
or greater than those used to demonstrate antipyresis, and
testing the reactivity of treated and control animals to
inflammatory challenge. Therefore, the peptides of the
present invention may be used both as an antipyretic and
as an anti-inflammatory agent when administered at a
selected dose to a patient in need.
In particular embodiments herein, doses of the
tripeptide effective for the expression of anti-
inflammatory activity, for example, for the reduction ofinflammation-associated swelling and/or capillary perme-
ability, are shown to be roughly similar on a weight basis
to those of hydrocortisone, an accepted standard for
anti-inflammatory activity. The sensitive "skin-blueing"
assay was employed to test the ability of the tripeptide
to inhibit the capillary permeabilizing effects of
inflammatory agents such as histamine. In this assay, the
protected Lys-Pro-Val tripeptide exhibited antihistamine
effects, and in particular, a reduction in histamine-
mediated increases in capillary permeability, at intra-
venous dosages as low as 1.25 ug protected tripeptide/kg
body weight. Moreover, in the traditional carrageenan/rat
paw edema test, intraperitoneally-administered tripeptide
demonstrated an ability to inhibit carrageenan-induced
swelling of rat paws on a per weight basis commensurate
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with hydrocortisoneO Accordingly, from such observations
it can be concluded that the tripeptide lys-pro-val is an
effective anti-inflammatory when administered to a patient
at a dose ranging from as low as 1 to 10 ug/kilogram, to
preferred ranges of on the order of about 0.2 to about 3
mg/kg/day.
Figure 1. Amino acid sequence of alpha-MSH
Figure 2. Comparison of inhibition of paw swelling
caused by Ac-Lys-Pro-Val-NH2 (100 mg/kg) and hydro--
cortisone (100 mg/kg) in rats. Each score i5 the mean %
change in paw volume relative to the change in matched
control paw volume.
Peptides used in the practice of the present
invention include the sequence lysine~proline-valine.
This tripeptide sequence is characterized as follows.
In its naturally occurring form, the tripeptide
sequence (lysine-proline-valine) comprises amino acid num-
bers 11-13 of alpha-Melanocyte Stimulating Hormone
(hereinafter "MSH") and ~CTH. This finding may explain
the antipyretic activity of the amino-terminal portion of
ACTH (amino acid numbers 1-24 of proopiocortin) and MSH
(amino acid numbers 1-13 of ACTH and proopiocortin; see
Figure 1), both of which exhlbit the tripeptide sequence
within their structure. Therefore, both MSH and ACTH
represent potential naturally occurring sources from which
the antipyretic tripeptide can be obtained, or, in the
case of alpha~MSH, which can be used directly in accord-
ance with less preferred emhodiments.
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Due to the high corticotropic effect of ACTH, which
can lead to incidences of Cushing's Syndrome, the
invention is generally directed to the alpha-MSH sequence
and peptides thereof, so long as such peptides include at
least the lys-pro-val sequence. This is based on the
finding that alpha-MSH (amino acids 1-13 of ACTH) does not
exhibit the corticotropic effect of ACTH and, instead,
appear to exert their anti-inflammatory action directly
rather than through a corticosteroid intermediate.
1~
In preferred embodiments, the tripeptide can be
isolated from MSH. This can be accomplished by first
fragmenting the MSH protein into four smaller peptides
through total digestion with the proteolytic enzyme,
chymotrypsin. Paper electrophoresis of the digestlon
produots reveals four major products, one of which is the
tetrapeptide, glycine-lysine-proline-valine, which may be
used directly. However, the glycine residue can be
removed by partial acid hydrolysis to yield the
tripeptide.
Peptides in accordance with the invention can also be
obtained by chemical synthesis. This is accomplished by
way of peptide bond formation between the appropriate
amino acids. Amino acids are amphoteric molecules which
contain both an acidic (-COOH) moiety and a weekly basic
; (-NH2) moiety. Peptide bond formation (-CONH-), there-
fore, is accomplished through a nucleophilic attack of the
amine group on the carboxylic function.
In forming a peptide bond between two hypothetical
amino acids, X and Y, four possible dipeptides may be
produced: X-Y, Y-X, Y-Y, and X-X. Therefore, in order to
reduce the possible structures that may be formed in such
an interaction, the amino or carboxy terminus o~ the
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appropriate amino acid must be first "protected" so as to
preclude a reaction involving the "protected" moiety. For
example, if "c" represents a protectecl carboxy terminus
and "n" a protected amino terminus, then an interaction
involving cX and Yn could generate only one structure,
cX-Yn.
However, in order to be chemically useful for synthe-
tic purposes, the protecting groups must be removable. In
general, carboxy groups can be protected by esterification
or amidation of the -COOH to -COO-alkyl or -CONH2. The
preferred alkyl groups for the carboxy terminus include
methyl and benzyl residues, yet other alkyl groups, such
as ethyl, propyl, butyl, p-nitrobenzyl or p-methoxybenzyl
groups, can be utilized.
Likewise, the amino terminus is protected by acyla-
tion, introducing a carboxyl group such as an acetyl
group, t-butyloxycarbonyl group, t-amyloxycarbonyl group,
o-nitrophenylsulfenyl group, benzyloxycarbonyl group, p-
nitrobenzyloxycarbonyl group, tosyl or formyl group.
Protecting groups may also serve a function in
nature. Bioactive peptides which contain an acetyl group
bound to be amino-terminus of the peptide and an amido
function bound to the carboxy-terminus are less
susceptible to acid hydrolysis. Furthermore, it has been
speculated that such groups play a role in reducing the
susceptibility of the "protected'l peptide to enzymatic
attack and degradation. Accordingly, a "protected"
tripeptide has been synthesized which contains these
protecting groups. This protected tripeptide is more
active pharmacologically than the unprotected tripeptide.
The various distinct pharmacological actions of the
tripeptide, i.e., anti-inflammation and antipyresis, are
demonstrated herein through the use of accepted pharma-
cological assays. For example, antipyretic action is
demonstrated using an ln vivo rabbit pyresis assay in
which increasing amounts of a protected tripeptide
(acetyl-lys-pro-val-NH ) was administered to pyrogen-
induced rabbits. In this assay, an effective dose range
of on the order of 10 ug to lO0 mg/kg for the unprotected
tripeptide was observed, resulting in fever reductions
over control of on the order of about 25% to about 70%, in
a generally dose dependent fashion. Moreover, the
protected tripeptide (diacetyl-lys-pro-val-N~2) exhibited
an activity approximately twice the activity of the
unprotected species on a weight basis.
The anti-inflammatory action of the tripeptide is
demonstrated employing accepted in vivo assays designed to
test the ability of a test agent in inhibiting various
symptomology of inflammation, lncluding tissue swelling
(e.g., localized edema) and capillary permeability.
In one assay, the skin-blueing test, the tripeptide
was tested for its ability to inhibit the capillary
permeabilizing effects of histamine by its action in
blocking the effects of exogenous histamine. Using this
assay, which has been found by the present inventors to be
sensitive to low amounts of the agent, it was found that
dosages as low as about 1 microgram of the protected
3a tripeptide/kilogram elicited a demonstrabIe efféct as
measured by reduction in histamine-mediated increase of
vascular permeability to vital dyes.
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In a second test, referred to in the art as the
carrageenan/rat paw edema assay, the tripeptide is shown
to achieve an anti-inflammatory action roughly equivalent
to that of a well-known anti-inflammatory agent, hydro-
cortisone. In this assay, rats were first admlnisterede~ual intraperitoneal doses of either control (saline), of
tripeptide or of hydrocortisone. The paw of the rat was
then challenged with an antigenic substance, generally
carrageenan, and the resultant swelling measured and data
compared. From such assays, it was found that approxi-
mately equal weights of the protected tripeptide
(diacetyl-Lys-Pro-Val-NH2) and hydrocortisone resulted in
a roughly equivalent overall response in reducing the
degree of carrageenan-induced swelling.
Based on the foregoing and additional observations,
it is found that dosages on the order of 0.2 to about 3 mg
of protected tripeptide per kilogram body weight per day
will result in advantages in accordance with the present
invention in terms of effective anti-inflammatory action.
Generally, it will be preferred to administer doses of on
the order of 0.35 and about 1.5 mg of protected
tripeptide/kg body weight/day to achieve the greatest
degree of anti-inflammatory benefit. These dose ranges
are derived from the aforementioned observation of
approximately equipotentcy of the tripeptide and hydro-
cortisone, and the general knowledge in the art regarding
effective dose ranges of hydrocortisone (see, e.g.,
Goodman et al. (1985), The Pharmacoloqical Basis of
Therapeutics, 7th Edition).
In accordance with the inv2ntion, it will generally
be preferred to administer the tripeptide parenterally,
for example, intramuscularly or intravenously. However,
due to its small size, membrane permeability and
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relatively acid-stable structure, it will be recognized
that the tripeptide may be administered orally, albeit at
somewhat higher doses. In this regard, it is believed
that doses of on the order of about 0.2 to about 3.5
S mg/kg/day will achieve benefits in accordance herewith.
Pharmaceutical preparations of the tripeptide,
preferably a protected tripeptide such as diacetyl-Lys-
Pro-Val-NH2, comprise generally the active agent in
combination with pharmaceutically acceptable buffers,
diluents, stabilizers and the like. For a fairly complete
listing of various techniques, including a variety of
agents and additives useful in the preparation of accept-
able pharmaceutical compositions, one may wish to refer to
Reminqton's Pharmaceutical Sciences, 16th ed., 1980, Mack
Publishing Co.
In a preferred pharmaceutical composition, approxi-
mately 100 to 500 mg of diacety-Lys-Pro-Val-NH2 is
dispersed in about 1 to 7 cc. of sterile isotonic saline,
including a pharmacologically accepted buffer to maintain
a pH of about neutral. For intravenous administration,
for example, to a patient suffering from arthritis, or
severe allergic reaction or various other diseases
involving inflammatory processes, it will generally be
desirable to administer about 0.2 to about 3.5 mg/kg/day
of the agent by slow infusion over a period of time (up to
several hours~. Where infusion is impractical, the agent
is administered in the form of an intramuscular injection,
preferably in combination with a lipophilic carrier and at
somewhat higher doses. For the treatment of mild to
severe arthritic episodes, it is generally recommended
that a parenteral dose of on the order of about 0.3 to 1.5
mg/kg/day, preferably about 0.5 to about 0.6 mg/kg/day.
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However, for severe allexgic reactions, higher doses on
the order of about 2.5 to up to about 4 mg/kg/day may be
indicated.
It is believed that many changes may be made in the
S amino acid sequence o~ the peptides of the present
invention and still obtain a protein which exhibits a
biologically functional equivalent pharmacologic activity.
For example, it has been found by Kyte et al. (1982), J.
Mol. Biol., 157:105, that certain amino acids may be
substituted for other amino ~ids having a slmilar hydro-
pathic index, and still retain the biolo~ic activity of
the protein. As displayed in the table below, amino acids
are assigned a hydropathic index on the basis of their
hydrophobicity and charge characteristics. It is believed
that the relative hydropathic character of the amino acid
determines the secondary structure of the resultant
protein, which in turn defines the interaction of the
protein with its receptor.
In the case of the present peptides, it is believed
that biological functional equivalents may be obtained by
substitution of amino acids having similar hydropathic
values. As used herein, a biological functional
equivalent is defined as a protein that is functionally
equivalent in terms of biolo~ical functional equivalent is
defined as a protein that is functionally equivalent in
terms of biological activity. Thus, for example,
isoleucine of leucine have a hydropathic index of +4.5 and
+3.8, respectively, can be substituted for valine (+4.2),
and still obtain a proteln having like biological-
activity. Alternatively, at the other end of the scale,lysine (-3.9) can be substituted with arginine (-4.5), and
so on. In general, it is believed that amino acids can be
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successfully substituted where such amino acid has a
hydropathic score of within about +/- 1 hydropathic index
unit of the replaced amino acid.
Amino acid~ydropathic Index
Isoleucine 4.5
Valine 4.2
Leucine 3.8
Phenylalanine 2.3
Cysteine/cystine 2.5
Methionine 1.9
Alanine 1.8
Glycine ~0~4
Threonine -0.7
Tryptophan -0.9
Serine -0.8
Tyrosine -1.3
Proline -1.6
Histidine -3.2
; Glutamic Acid -3.5
Glutamine -3.5
Aspartic Acid -3.5
Asparagine -3.5
Lysine -3.9
Arginin~ -4.5
The following examples illustrate experiments
conducted by the present inventor to illustrate the
production of the preferred tripeptide, as well as various
"protected" species, and use of the tripeptide in various
accepted ln vivo assays which demonstrate its activity.
It will be appreciated that these examples are
illustrative only and variations may be made in light
thereof and in light of the level of skill in the art.
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Thus, for example, where peptides having different
sequences, or longer or shorter peptidyl length, are
desired, it will be apparent to those of skill in the art
that the procedures generally as set forth below may be
employed. Accordingly, where the sequence arg-pro-val is
desired (a biologically functional equivalent of lys-pro-
val), it will be apparent that dibenzyloxycarbonyl-
confugated arginine ("Z-arg") should be employed in the
place of "Z-lys"). Moreover, wherel for example, gly-
lys-pro-val is desired, it will be apparent that "Z-gly"
should be employed as the starting reagent and synthetic
steps employed as set forth to sequatically add the lys,
pro and val residues, respectively. These and all other
modifications to achieve the various peptides are well
known and will be apparent to those of skill.
EXAMPLE I: CHEMICAL SYNTHESIS OF L-LYSINE-
L-PROLINE-L-VALINE
The tripeptide was custom synthesized by Bachem,
Inc., Torrance, Ca., as follows:
l. Z-Lys-Pro-OMe PreParation
50 mmoles ~20.7 ~rams) of dibenzyloxycarbonyl-
conjugated lysine ("Z-lys") in 200 ml of methylene
chloride was combined with 50 mmoles (8.3 grams) of
proline methyl ester (pro-OMe) in 100 ml dimethyl forma-
mide. The mixture was added to a conical flask and cooledto -5C with stirring. 50 mmoles (5.5 ml) of N-methyl
morpholine was added, followed by the addition of 10.3
grams of dicyclohexyl-carbodiimide in 20 ml of methylene
chloride and the reaction mixture was stirred overnight.
The mixture was then filtered from urea and the filtrate
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concentrated in vacuo. The re~idue was taken up in ethyl
acetate and washed successively with sodium bicarbonate
solution, water, lN hydrochloric acid, and water. Ethyl
acetate was removed in vacuo and the oily product was
saponified without purification.
2. Removal of the -OMe Carboxy Terminus
"Protectinq" Grou~.
The oily product from the previous experiment was
dissolved in methanol (200 ml) and treated with 2N sodium
hydroxide (25ml) for an hour. Methanol was removed under
reduced pressure and the residue was taken up in water and
acidified with 6N hydrochloric acid. The product was
extracted with ethyl acetate, and the organic layer was
washed with water and dried over sodium sulphate. Ethyl
acetate was removed in vacuo and the residue was tri-
turated with hexane. The product was checked by thin
layer chromatography using chloroform: methanol: acetic
acid (95:4:1).
3. Preparation of Z-L~s-Pro-Val-OBe
The next step in the synthesis of the tripeptide
involved the addition of a carboxy-protected valine resi-
due (Val-OBe). The protecting group in this instance was
a benzyl ester.
37 mmoles (19 grams) of Z-Lys-Pro obtained from step
2 above was dissolved in 200 ml distilled tetrahydrofuran.
This solution and 4.1 ml N-methyl morpholine were mixed
together and cooled to -l5~C with stirring. Isobutyl-
chloroformate (5)(ml) was added and the mixture stirred
for 5 minutes at -10C. Concurrent with the above, 35
mmoles (13.2 grams) of valine benzyl ester tosylate was
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dissolved in 10Q ml dimethyl formamide. The mixture was
cooled to -10C and neutralized with N-methyl morpholine
(4ml). This was added to the above mixed anhydride and
stirred overni~ht. The mixture was then filtered from
urea and the filtrate con~entrated ln vacuo. The residue
was taken up in ethyl acetate and washed successively with
sodium bicarbonate solution, water, lN hydrochloric acid,
and water. The crude product was purified on a silica gel
column using chloroform-methanol (95:5~. Fractions
containing the pure product were determined by thin layer
chromatography utilizing the same solvent as for step 2.
The appropriate fractions were pooled.
4. Removal of the Protectina Grou~s Z- and -OBe
9 grams of the protected tripeptide produced in step
3 above was hydrogenated in an acetic acid-water-methanol
mixture in the presence of Pd/BaSO4 overnightO It was
filtered from the catalyst and the filtrate was evaporated
in vacuo to give an oily residue. This was triturated
with absolute ethanol and absolute ether to yield 3 grams
of the crystalline product. The product was checked by
thin-layer chromatography using a solvent system composed
of butanol: acetic acid: water: pyridine ~20:6~ 24).
Chemical SYnthesis of diacetyl-L-Lysyl-L-Prolyl~ Valyl-
NE2
The protected tripeptide, diacetyl-L-Lysyl-L-Prolyl-
L-Valyl-NH2 can also be prepared by the chemical tech-
niques described above in steps 1-3. For example, in step
l, the starting material would be diacetyl conjugated
lysine. In step 3, the valine-benzylester is substituted
with valyl-amide.
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EX~MPLE II: ANTIPYRETIC ACTIVITY OF
L-LYS-L-PRO-L-VAL
Production of Leukocvtic Pyroqen
Leukocytic pyrogen is a molecule capable of producing
transient fever in mammals which is produced by incubating
rabbit leukocytes with Salmonella ty~hosa endotoxin. More
specifically, to produce leukocytic pyrogen, donor rabbits
were first sacrificed by decapitation. Blood was
collected in a heparinized pryrogen-free beaker.
Heparinized 50-ml glass centrifuge tubes were filled 3/4
full with whole blood, saline was added to fill each tube,
and thé solution was gently mixed. The tubes were then
centrifuged at low speed for 20 min. The buffy coat was
drawn off and placed in pyrogen-free flasks. Lactate
Ringer's solution equal in volume to one-half that of the
red cell layer, was added along with Salmonella tvPhosa
endotoxin (Difco, No. 0901) also in Ringer's solution ~1
ug/ml~, to the buffy coat. The mixture was incubated at
38C in a shaking water bath for 4 hours. The solution
was centrifuged, filtered ~Nalgene* 0.20 microns), and the
leukocytic pyrogen-containing filtrate was stored at 4C.
Samples of leukocytic pyrogen were heated to 73C for 2
hours and injected intravenously to test for endotoxin
contamination. Only characteristic leukocytic pyrogen
fevers occurred and no prolonged fevers were observed,
; indicating that the leukocytic pyrogen was free of
endotoxin and other heat-stable pyrogens.
Injections were 50 ul in volume and were followed by
a 20 ul saline flush. Intravenous injections were made
via the marginal ear vein. Intravenous leukocytic pyrogen
injections were 0.07 ml of a stock solution made up of a
mixture of leukocytic pyrogen derived from 4 donors. When
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injected, leukocytic pyrogen stock solution was diluted
with nonpyrgenic isotonic saline. Injections were made
with commercial nonpyrogenic syringes. Glassware was
washed with chromic acid, rinsed with deionized water and
heated to 200C for a minimum of 2 hours to insure that it
was pyrogen free.
Animal Procedures
Adult New Zealand white rabbits were used for the
development of an antipyretic assay. The rabbits were
housed individually in a 21-23C environment with a 12
hour light/dark cycle; food and water were available ad
libitum. Central nervous system injections of the anti-
pyretic agents were performed as follows: The animals
were pretreated with ketamine hydrochloride and promazine
(Ketaset Plus* Bristol Labs, 0.4 ml/kg. intramuscularly)
and anesthesia was induced and maintained by inhalation of
methoxyfluorane (Metafane, Pitman-Moore, Inc.) and an
N2O-O2 mixture. Rabbits were placed in a Kopf rabbit
stereotaxic instrument and a stainless steel cannula (No.
201, David Kopf Instruments) was inserted into a lateral
ventricle at a point l.0 mm anterior to the bregma and 2.7
mm lateral to the midline. The cannula was lowered until
cerebrospinal fluid appeared inside the well of the
cannula. Stainless steel screws and dental acrylic were
used to anchor the cannula to the caluarium. Benzathine
penicillin G (Bicillin, Wyeth ~aboratories) was given
post-operatively (150,000 units intramuscularly).
Experimental rabbits that were to be used in the
antipyresis study were restrained in conventional holders
and a thermistor probe (Yellow Springs International, No.
701) was inserted about lO0 mm into the rectum and taped
to the tail. In certain experiments, another thermistor
* Trade Marks and/or Trade Designations
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probe (Yellow Springs International, No. 709) was attached
to the dorsal surface of the ear. Temperature recordings
were made every 10 minutes via a MINC 11 online computer
connected to a digital temperature recorder (Datalogger*
United Systems Corp.) At least 1 hour was allowed after
the probes were inserted before injections were made, and
all experiments were separated by at least 48 hours.
Experiments were run in an environmental chamber at 23C.
The average thermal response, the mean change in
temperature (C) over the duration of the response mea-
sured in hours, was calculated for each response and the
paired t-test was used for statistical analysis of the
data. The time period over which the experimental anti-
pyretic temperature response was determined was generally
set by the duration of the control response for the
individual animal. The control response begins with the
first deviation from baseline temperature and continues
until the temperature returns to baseline or to the point
nearest the baseline within S hours. The mean change in
temperature for each 10 minute intervals during this time
period are summed and divided by the total number of 10
minute periods.
Assay Protocol and Results
Immediately after the tripeptide, L-Lys-L-Pro-L-Val,
was synthesized as described above, it was dissolved in
sterile non-pyrogenic isotonic saline and stored frozen in
aliquots until just prior to use. Before any injections
of the tripeptide were given, leukocytic pyrogen was
tested in each animal in order to establish its sen-
sitivity to the pyrogen and to test for endotoxinactivity.
* Trade Mark and/or Trade Designation
~L3~ 2
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To induce fever in the test animals, 0.15 ml of a
stock solution of leukocytic pyrogen was injected into a
marginal ear vein. Pyrogen from several batches was used,
but each animal received pyrogen from the same batch
throughout each series of experiments.
Injections of the tripeptide were given 30 minutes
after the pyrogen, into the intraventricular cannula.
Centrally administered tripeptide resulted in an observed
antipyresis. Decreases in fever, calculated as percent
reduction of the area under the control fever curve over
the apparent duration of action of the peptide (1.5
hours), were 24%, 31~, and 48~ for the 0.5, 1.0, and 2.n
milligram doses, respectively. Similarly, intravenous
administration o~ 2, 20 and 200 milligrams of the tri-
peptide reduced fever 34, 27 and 67% , respectively,
during the 1.5 hour period after injection of the leuko-
cytic pyrogen. Control saline injections, centrally
administered, caused no significant reduction in body
temperature. Similarly, when injections of 200 milligrams
were given to afebrile rabblts, no reduction in body
temperature was observed.
EXAMPLE III: ANTIPYRETIC ACTIVITY
OF THE PROTECTED TRIPEPTIDE
DIACETYL-L-LYS-I.-PRO-L-VAL-NH2
Central administration of the acetylated and amidated
tripeptide diacetyl-L-Lysine-L-Proline-L-Valine-NH2
resulted in an increase in the observed antipyresis as
well as an increase in the duration of action. Fever
induced by intravenous administration of leukocytic
pyrogen was reduced more than 50% by 0.5 mg of the pro
tected peptide. The duration of action was at least four
hours compared to 1.5 hours observed for the unprotected
tripeptide (see Example I~. Smaller doses of the pro-
tected tripeptide resulted in correspondingly lower
antipyresis.
EXAMPLE IVo ANTIPYRETIC ACTIVITY
OF DIACETYL-L-LYS-L-PRO-L-VAL-NH2
AND COPPER ION
When 0.5 mg of the protected peptide was given
centrally and copper ions ~l-lQ mg of the cupric chloride
salt) were given either centrally or peripherally, the
antipyretic effect was greatly augmented and hypothermia
developed that was similar to that that has been observed
with large doses of the parent alpha-MSH peptide. The
protected peptide thus appears to be at least four times
more potent than the unprotected tripeptide. The addition
of copper ions, in doses that have no effect on normal
temperature, markedly enhanced the antipyretic, and
hypothermic, effects of the protected peptideO
EXAMPLE V: ANTI-INFLAMMATORY ACTIVITY
The anti-inflammatory activity of the tripeptide was
demonstrated through the use of an animal model developed
by Sparrow and Wilhelm (1957), J. PhYsiol., 137:51-65.
This model relies on the principal that localized,
subcutaneous injections of histamine will result in a
localized increase in capillary permeability. When the
test animal has been pretreated with blue dye
intravenously, the localized histamine injections will
elicit blue~colored "weals" around the injection site.
Thus, by preadministration of an effective anti-
inflammatory agent, the blue color of the histamine-
~3(~
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induced weals will be much less pronounced, with the
amount of color reduction being dependent on the relative
amount and/or potency of the anti-inflammatory agent used.
Non-moulting New Zealand white rabbits were used for
the Sparrow/Wilhelm assay. The skin of the rabbits back
was closely clipped 1-2 days previous to the experiment,
but not dipilated, and the rabbits were kept warm until
tested. Various amounts of the protected tripeptide
(diacetyl-L-lys-l-Pro-L-Val-NH2) were injected intra-
venously into an ear vein approximately 15 minutes prior
to intravenous injection of blue dye. Control rabbits
received sham injections. Fifteen minutes following
injection of the agent or sham, the rabbits received
approximately 30 mg/kg of Pontamine blue dye as a 2.S%
solution in 0.45% saline, into an exposed vein.
Immediately following dye injections, histamine was
injected intradermally in a 0.10 ml volume (1.25 mg
histamine /0.1 ml volume) at several sites on each side of
the spine. In all, one vertical row of si~ injections
were made on each side of the spine. The relative inten-
sity of the resultant blue weals were scored by an inde-
pendent observer 30 minutes after histamine injection.
The results are displayed in Table I below.
~3~snz
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TABLE I: Anti-inflammatory Activity
of the TripePtide
S _ . _
No. Animals Tripepticle
_ Tested Dose + Result
_ . .
3 (2E, lC)* 5 E lighter than C
2 (lE, lC) 10 E lighter than C
15 2 (lE, lC) 5 E lighter than C
2 (lE, lC) 1.25 E lighter than C
2 ~lE, lC) 0.625 No difference
observed
* E = experimental; C = control
+ Dosages in ug of protected tripeptide per kg body
weight, administered intravenously.
?;~:
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As will be appreciated from the results displayed in
Table I, intravenous doses down to 1.25 ug tripeptide per
kg body weight resulted in an appreciable reduction in
histamine-induced blue weal formation and is thus indica-
tive of an effective anti-inflammatory action. At doses
of 5 and 10 ug/kg, the observecl response was even more
pronounced. Also as will be appreciated, the anti-
inflammatory effect of the tripeptide is observed at
relatively lower doses as compared to its anti-pyretic
effect.
EXAMPLE VI: CARRAGEENAN/RAT PAW EDEMA ASSAY
A second ln vivo bioassay for anti-inflammatory
activity was conducted in which the action of the
tripeptide was compared to that of hydrocortisone. In
this assay, the two agents were given at similar doses and
tested for their independent ability to inhibit
carrageenan-induced swelling of rats paws. This assay,
the rat paw edema test, was conducted generally as it is
typically performed in the art, for example, as described
by Winter et al. ~1962), Proc. Soc. ExP. Biol. Med.,
544 or in U.S. Patent 4,150,137.
Briefly, the assay was performed as follows. Each of
twenty-four male Sprague-Dawley rats was assigned to one
of four groups: tripeptide treatment and controls
(matched according to body weight and initial paw volume),
hydrocortisone treatment and matched controls. The volume
of the right rear paw of the test and control animals was
determined using standard procedures and a mercury
displacement volumetric technique. An intraperitoneal
injection of the tripeptide (Ac-Lys-Pro-Val-NH2, 100
mg/kg, N=6), of hydrocortisone (100 mg/kg, N=6), or saline
~3~S~;~
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tmatched volume/ N=12) was given to each rat. One hour
later 0.5 ml of 1% lambda carrageenan in saline solution
was injected into the right rear paw of the animals and
the paw volume was again recorded (baseline measure).
Thereafter paw volume-was measured each hour for our
hours. For comparison of the effects of the two treat-
ments, paw volume of experimental animals measured at
hourly intervals was expressed as a percentage of the
volume change of their respective matched controls.
The results of this experiment is shown in Figure 2.
As will be appreciated from this data, except for the
first hour when hydrocortisone markedly inhibited swelling
(p <0.05, Mann-Whitney test), there was no significant
difference in the inhibition of paw edema caused by the
tripeptide and hydrocortisone (p >0.20). These results
indicated that the tripeptide inhibits inflammation as
well as the classic anti-inflammatory agent, when given in
an equal dose by weight, albeit with a slight difference
in time course. Based on the present results and the
known effects of hydrocortisone and inflammation in man,
it is concluded that the tripeptide Lys-Pro-Val can be
used to reduce inflammation in man, in dosage not markedly
different from that of hydrocortisone.
* * *
The foregoing invention has been described by way of
illustration and example and in terms of standard labora-
tory techniques employed by the applicant. It will be
apparent to those skilled in the art that certain changes
and modifications of these procedures may be employed
without departing from the spirit and scope of the inven-
~3~
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tion. For example, although chemically synthesized
tripeptide was utilized to demonstrate its antipyretic
activity, it is contemplated by the inventor that tri-
peptide isolated ~rom natural sources will function
equally well. Moreover, it will be apparent that admini-
stration of any copper salt, whether it be the sulfate,
chloride, or some other similar copper salt, should be as
active as the chloride salt in augmenting the activity of
the tripeptide~ Similarly, although activity was demon-
strated using either intravenous or centrally administered
tripeptide, orally administered trlpeptide, at higher
doses, should be active in reducing fever. It will be
apparent to those skilled in the art that these and other
modifications and changes are within the scope of the
appended claims.
