Note: Descriptions are shown in the official language in which they were submitted.
7 7
(Serl7, Leul8)GRF DERIVATIVES
Field of Invention
This invention relates to derivatives of the
growth hormone-releasing factor (GRF). More speci-
fically, this invention concerns synthetic peptides
having growth hormone-releasing activity, to proces-
ses for producing the peptides, to pharmaceutical
compositions of the peptides and to methods of using
the peptides in agriculture and medicine.
Background of the Invention
GRF causes the release of growth hormone (GH)
into the blood and as such plays an important role
in animal growth. The source for its first isola-
tion and characterization was an islet tumor from a
human pancreas, see R. Guillemin et al., Science,
218, 585 (1982). GRF now has been isolated from the
hypothalamus of humans and various animal species.
The structures of the GRF's from the various species
are similar, differing slightly in their amino acid
sequence.
The human GRF, conventionally designated by the
acronym "hGRF", has the following structure:
3o
1340077
l 5 lO
H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-
Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-
25 30 35
Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-
Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH2
Porcine growth hormone-releasing factor (pGRF)
has the same structure as hGRF except that the amino
acid residues at positions 34, 38 and 42 are re-
placed with Arg, Gln and Val, respectively. Bovine
growth hormone (bGRF) has the same structure as pGRF
except that the amino acid residues at positions 28
and 41 are replaced with Asn and Lys, respectively.
Ovine growth hormone-releasing factor toGRF) has the
same structure as bGRF except that the amino acid
residue at position 13 is replaced with Ile.
The intrinsic biological activity of hGRF has
been found to reside in the N-terminal portion of
the peptide, see N. Ling et al., Biochem. Biophys.
Res. Commun., 123, 854 (1984); and both the carboxy
terminal acids and carboxy terminal amides of the
full length GRF's of the aforementioned species, as
well as the corresponding fragments thereof contain-
ing about the first 27 amino acids, display growth
hormone-releasing activity. According to conven-
tion, the position numbers of the amino acid
1'340077
residues present in the GRF free acid or amide
forms, or their fragments are indicated by numbers
in parentheses following "GRF". Thus, the amide
fragment of hGRF containing the first 29 amino acids
of the N-terminal portion thereof is designated as
"hGRF(1-29)NH2" and the corresponding free acid as
"hGRF(1-29)OH."
The importance and potential use of GRF and its
active fragments are well documented; for example,
see F.X. Coude et al., Trends in Biotechnology, 2,
83 (1984). As a result, numerous analogs of GRF
have been made and tested for the purpose of finding
more potent, and hopefully, less expensive replace-
ments for GRF. For instance, see D.H. Coy et al.,
J. Med. Chem., 28, 181 (1985), J.S. Tou et al.,
Biochem. Biophys. Res. Commun., 139, 763 (1~86),
U.S. patent 4,518,586, May 21, 1985 and European
patent application, publication number 177819,
published April 16, 1986.
The present application discloses new GRF pep-
tides derivatives which are characterized by having
the order of the amino acid sequence at positions 17
and 18 rearranged (i.e. Ser at position 17 and Leu
at position 18). The new derivatives possess en-
hanced growth hormone-releasing activity and can be
prepared economically. These attributes render the
derivatives useful agents for ~edicine and agricul-
ture.
~,
13 l~O77
--4--
Summary of the Invention
The GRF peptide derivatives of this invention
are represented by formula 1
Rl-R2-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-
R3-Leu-R4-Gln-Ser-Leu-Ala-Arg-R5-Leu-Leu-
R6-Asp-Ile-R7-Y-Z
wherein Rl is Tyr, (lower alkanoyl)-Tyr, des-
NH2-Tyr, His or (lower alkanoyl)-His,
R2 is Ala or D-Ala,
R3 is Val or Ile,
R4 is Gly, Ala, Leu, Ile, Sar, Val or Aib,
R5 is Lys or Glu,
R6 is Gln, Lys, Ser or Glu,
R7 is Met, Nle, Nva or lle,
Y is des-Y or an amino acid sequence select-
ed from the group of R8-Arg-Gln-Gln-Gly-Glu-R9-
Asn-Gln-Glu-R10-Gly-Ala-R11-R12-Arg-Leu
wherein R8 is Ser or Asn, R9 is Ser or Arg,
R1O is Arg or Gln, Rll is Arg or Lys and
R12 is Ala or Val, and fragments thereof wherein
from one to sixteen amino acid residues may be
deleted serially from the carboxy terminus, and
Z is hydroxy, amino, lower alkylamino or
Gly-OH;
or a therapeutically acceptable salt thereof.
1340077
- 4a -
A first preferred group of GRF peptide
derivatives, namely a human growth hormone-
releasing factor (hGRF) or a bovine growth
hormone-releasing factor ~bGRF), is
represented by formula 1
~n which R is Tyr, (lower alkanoyl)-Tyr,
des-NH2-Tyr, His or (lower
alkanoyl)-His,
R is Ala or D-Ala,
R is Val or Ile,
R is Gly, Ala, Leu, Ile, Sar, Val
or Aib,
R is Lys or Glu,
R is Gln, Lys, Ser or Glu,
R is Met, Nle, Nva or Ile,
Y is des Y or an amino acid
sequence R8-Arg-Gln-Gln-Gly-Glu-R9-Asn-Gln-
Glu-Rl0-Gly-Ala-Rll-Rl2-Arg-Leu wherein R8 is
Ser or Asn, R9 is Ser or Arg, Rl~ is Arg or
Gln, Rll is Arg or Lys and Rl2 is Ala or Val,
or
Y is a fragment selected from the
group of: Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-
Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu,
Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-
Gly-Ala, Ser-Arg and Asn-Arg, and
Z is hydroxy, amino, lower
alkylamino or Gly-OH; or a therapeutically
acceptable salt thereof.
13 iO077
A preferred group of peptide derivatives of
this invention is represented by formula l wherein
Rl to R7, inclusive, are as defined herein-
above, Y is des-Y, or an amino acid sequence select-
ed from the group of Ser-Arg-Gln-Gln-Gly-Glu-Ser-
Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu, Ser-Arg-
Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala, Ser-Arg
and Asn-Arg, and Z is hydroxy, amino or Gly-OH; or a
therapeutically acceptable salt thereof.
A more preferred group of peptide derivatives
is represented by formula l wherein R1, R3 and
R6 are as defined hereinabove, R2 is Ala, R4
is Gly, Ala, Leu, Ile or Val, R5 is Lys, R7
is Met or Nle, Y is Ser-Arg or Asn-Arg, and Z is
amino or Gly-OH; or a therapeutically acceptable
salt thereof.
A most preferred group of peptide derivatives
is represented by formula l wherein Rl is Tyr or
AcTyr, R2 is Ala, R3 is Val, R4 is Gly or Ala,
R5 is Lys, R6 is Gln, Lys, Ser or Glu, R7 is
Met or Nle, Y is Ser-Arg and Z is NH2; or a thera-
peutically acceptable salt thereof.
Included within the scope of this invention is
a pharmaceutical composition for stimulating the
release of GH in an animal comprising an effective
amount of a peptide derivative of formula 1, or a
3o
1~007~
--6--
therapeutically acceptable salt thereof, and a
pharmaceutically or veterinarily acceptable car-
rier.
The administration of the peptide derivatives
to animals, including humans, stimulates the release
of GH in the animals. Thus, included within the
scope of this invention is a method of promoting
growth in animals, and/or treating growth related
disorders caused by a deficiency of GH in the
animals, which comprises administering thereto an
effective amount of the peptide derivative of formu-
la 1, or a therapeutically acceptable salt thereof.
Processes for preparing the peptide derivatives
are described hereinafter.
Details of the Invention
For convenience, the peptide derivatives of
this application hereinafter are designated simply
as peptides.
The term 'residue' with reference to an amino
acid means a radical derived from the corresponding
~-amino acid by eliminating the hydroxyl of the
carboxyl group and one hydrogen of the ~-amino
group.
In general, the abbreviations used herein for
designating the amino acids and the protective
groups are based on recommendations of the IUPAC-IUB
1~0~77
Commission on Biochemical Nomenclature, see Biochem-
istry, 11, 1726-1732 (1972). For instance, Gln,
Ala, D-Ala, Gly, Ile, Arg, Asp, Phe, Ser, Leu, Asn,
Thr, Lys, Val, Met, His, Nle, Nva, Sar and Tyr
represent the 'residues' of L-glutamine, L-alanine,
D-alanine, glycine, L-isoleucine, L-arginine, L-
aspartic acid, L-phenylalanine, L-serine, L-leu-
cine, L-asparagine, L-threonine, L-lysine, L-valine,
L-methionine, L-histidine, L-norleucine, L-norval-
ine, sarcosine, and L-tyrosine, respectively.
The symbols "AcTyr", "des-NH2-Tyr" and "Aib"
represent the residues of N-acetyl-L-tyrosine, des-
amino-L-tyrosine and L-~-aminoisobutyric acid,
respectively.
The term "lower alkanoyl" as used herein means
straight chain alkanoyl radicals containing from two
to six carbon atoms and branched chain alkanoyl
radicals containing from four to six carbon atoms
and includes acetyl, l-oxopropyl, 2-methyl-1-oxopro-
pyl, l-oxohexyl and the like.
The term "pharmaceutically acceptable carrier"
as used herein means a non-toxic, generally inert
vehicle for the active ingredient which does not
adversely aff'ect the ingredient.
The term "veterinarily acceptable carrier" as
used herein means a physiologically acceptable
0 7 7
--8--
vehicle for administering drug substances to
domestic animals comprising one or more non-toxic
pharmaceutically acceptable excipients which do not
react with the drug substance or reduce its effec-
tiveness.
The peptides of this invention can be prepared
according to processes known for the preparation of
peptides. Suitable processes include exclusively
solid phase techniques, partial solid phase tech-
niques and/or fragment condensations, or classical
solution couplings. For example, the techniques of
exclusively solid phase synthesis are described by
J.M. Stewart and J.D. Young in the textbook 'Solid
Phase Peptide Synthesis', 2nd ed., Pierce Chem. Co.,
Rockford, Illinois, 1984. The fragment condensation
method is exemplified by the disclosure of Canadian
patent 1,178,950, issued December 4, 1984. Other
available syntheses are exemplified by U.S. patent
3,842,067, issued October 15, 1974, and U.S. patent
3,862,925, issued January 28, 1975.
The terminal amino acylated derivatives of the
peptides of formula 1, i.e. peptides of formula 1
wherein Rl is (lower alkanoyl)-Tyr or (lower
alkanoyl)-His, are obtained from the corresponding
free terminal amino peptides by treatment with a
suitable acylating agent; for instance, the appro-
priate acid chloride or acid anhydride in the pre-
sence of a strong organic base, e.g. l-oxobutyl
.. . . ~ ",
13 4 g) 0 7 ~
chloride with triethylamine. Alternatively, the
terminal amino acylated derivatives are obtained by
using the appropriate N~-acylated amino acid
residue while preparing the peptide by conventional
means.
A common feature of the aforementioned proces-
ses for the peptides is the protection of the labile
side chain groups of the various amino acid residues
with suitable protective groups which will prevent a
chemical reaction from occurring at that site until
the protective group is ultimately removed. Usually
also common is the protection of an a-aminO group on
an amino acid or a fragment while that entity reacts
at the carboxyl group, followed by the selective
removal of the ~-amino protecting group to allow
subsequent reaction to take place at that location.
An important intermediate in the processes for
preparing the peptides of this invention is the
intermediate of formula 2
xl -Rl(X2)-R2-Asp(X3)-Ala-Ile-Phe-Thr-
(X4)-Asn-Ser(X4)-Tyr(X2)-Arg(X5)-Lys(X6)-
R3-Leu-R4-Gln-Ser(X4)-Leu-Ala-Arg(X5)-R5-
(X6orX3)-Leu-Leu-R6(X7)-Asp(X3)-Ile-R7-
yl_zl
~3'~0~77
--10 -
wherein Rl to R7, inclusive, are as defined
hereinbefore; Xl is hydrogen, an a-amino protec-
tive group, preferably t-butyloxycarbonyl, or des-
Xl when R1 is (lower alkanoyl)-Tyr, des-NH2-
Tyr or (lower alkanoyl)-His; x2 is a protective
group for the hydroxyl of Tyr, (lower alkanoyl)-Tyr
or des NH2-Tyr,preferably benzyl or 2-chloro-
benzyloxycarbonyl, or a protective group for the
imidazolyl group of His or (lower alkanoyl)-His,
preferably 2,4-dinitrophenylsulfenyl or tosyl; X3
is a protective group for the ~-carbonyl of Asp or
Glu, preferably, benzyl, 2,6-dichlorobenzyl or
cyclohexyl; X4 is a protective group for the
hydroxyl of Thr or Ser, preferably benzyl; X5 is
a protective group for the guanidino group of Arg,
preferably tosyl or nitro; x6 is a protective
group for the amino group of Lys, preferably 2-
chlorobenzyloxycarbonyl or tosyl; X7 is des-X7
when R6 is Gln, or is the protective group X6,
X4 or X3 when R6 is Lys, Ser or Glu, respec-
tively; yl is des-Yl or an amino acid sequence
selected for the group of R8(X8)-Arg-Gln-Gln-
Gly-Glu-R9(X4 or X5)-Asn-Gln-Glu(X3)-R10~
(X9 )-Gly -Al a-Rll( X5 o r x6 )-R1 2 -Arg -
Leu wherein R8 to R12, inclusive, and X3,
X4, X5 and x6 are as defined hereinbefore,
x8 is a protective group for the hydroxyl of Ser
or is des-R8 when R8 is Asn, and X9 is a pro-
tective group for the guanidino group of Arg or is
des~R9 when R10 is Gln, and fragments of the
13 1~)07~
amino acid sequence thereof wherein from one to six-
teen amino acid residues may be deleted serially
from the carboxy terminus; and Z1 is hydroxy,
amino, lower alkylamino, Gly-OH, OCH2-(resin sup-
port), NH-(resin support) or Gly-OCH2-(resin sup-
port).
The radicals OCH2-(resin support) and NH-
(resin support) represent radicals derived from
solid polymeric supports of the type used in solid
phase peptide synthesis and may optionally include a
spacer or linking group, such as 4-methylphenyl-
acetamido present in ~-(phenylacetamido)benzyl
resins (PAB-resins), between the radical and the
peptide portion. Such radicals are broadly con-
sidered as protective groups.
Hence, the peptides of formula l can be pre-
pared by forming the intermediate of formula 2 by
the stepwise coupling of the appropriate protected
amino acid residues or peptide fragments in the
order of the sequence of the intermediate to obtain
the intermediate of formula 2, followed by depro-
tecting the intermediate, including cleaving the
solid resin support, if present, to give the corres-
ponding peptide of formula l; and if desired, trans-
forming the peptide of formula l into a thera-
peutically acceptable salt.
In an embodiment of the exclusively solid phase
method, the protected intermediate of formula 2 in
~&~77
-12-
which Zl is amino is prepared as follows: an
a-amino protected amino acid (with side chain pro-
tection, if desired), corresponding to the first
amino acid of the carboxy terminus, is coupled to a
cross-linked benzyhydrylamine (BHA) resin in the
presence of potassium fluoride or cesium chloride to
give the corresponding solid support resin having
the first amino acid (in protected form) linked
thereto. Alternatively, a benzhydrylamine solid
resin support with the incorporated protected amino
acid may be obtained commercially and used as the
starting material. In either event, the next step
is the removal of the a-amino protective group of
the incorporated amino acid to give the free a-amino
group. In the instance where the a-amino protective
group is a t-butyloxycarbonyl, trifluoroacetic acid
in methylene chloride or chloroform, or hydrochloric
acid in dioxane, is used to effect the deprotection.
The deprotection is carried out at a temperature
between about 0~ C and room temperature. Other
standard cleaving reagents and conditions for remov-
al of specific a-amino protective groups may be used
as described by E. Schroder and K. L~bke, in "The
Peptides", Vol. 1, Academic Press, New York, 1965,
PP. 72-75. After removal of the a-aminO protective
group from the last mentioned intermediate, the
remaining a-amino protected amino acids are coupled
stepwise in the desired order to obtain the corres-
ponding protected intermediate of formula 2 in which
3o zl is NH-(benzhydrylamine resin). Each protected
~1340 077
-13-
amino acid is introduced into the reaction system in
a one to four-fold excess and the coupling is car-
ried out in a medium of methylene chloride, di-
methylformamide, or mixtures of dimethylformamdie
and methylene chloride. In cases where incomplete
coupling has occurred, the coupling procedure is
repeated before removal of the ~-amino protective
group, prior to the coupling of the next protected
amino acid. The success of the coupling reaction at
each stage of the synthesis is monitored by the
ninhydrin reaction as described by E. Kaiser et al.,
Anal. Biochem., 34, 595 (1970).
The preceding intermediate of formula 2 there-
after is simultaneously cleaved from the resin and
deprotected by treatment with liquid hydrogen fluor-
ide to give the corresponding peptide of formula 1
in which Z is amino.
Alternatively, when it is desired to prepare
the peptides in the form of their free acids (I, Z
= OH), the peptide can be prepared by the solid
phase method using a chloromethylated resin or a PAB
resin, and incorporating into the process the
cleavage of the resulting resin-coupled peptide and
any required deprotection according to known
procedures such as described by Stewart and Young,
supra.
Again, alternatively, the latter resin-coupled
peptide can be separated from the resin by trans-
esterification with a lower alkanol, preferably
methanol or ethanol, in the presence of trietyla-
mine. Thereafter, the recovered ester is purified
by chromatography. The collected fraction may be
subjected to treatment with ammonia or a (lower
7 7
-14-
alkyl)amine to convert the lower alkyl ester to the
correspondinK carboxy terminal amide (the interme-
diate of formula 2 wherein Zl is amino or lower
alkylamino); note that the 2,4-dinitrophenyl-
sulfenyl or tosyl, if present on a histidyl
residue, will be cleaved under these conditions.
The remaining protective groups then are removed,
for example with sodium in liquid ammonia or with
hydrogen fluoride, to give the corresponding peptide
of formula ] in which Z is amino or lower alkyl-
amino. Still again, alternatively, the latter
resin-coupled peptide, i.e. intermediate of formula
2 in which zl is OCH2-(resin support), can be
cleaved with ammonia to give the corresponding
intermediate of formula 2 in which z1 is amino,
which in turn, is deprotected to give the desired
peptide of formula 1 in which Z is amino.
The peptide of formula 1 of this invention can
be obtained in the form of therapeutically accept-
able salts.
In the instance where a particular peptide has
a residue which functions as a base, examples of
such salts are those with organic acids, e.g.
acetic, lactic, succinic, benzoic, salicyclic,
methanesulfonic or p-toluenesulfonic acid, as well
as polymeric acids such as tannic acid or carboxy-
methyl cellulose, and salts with inorganic acids
such as hydrohalic acids, e.g. hydrochloric acid, or
13'1G~7-i
sulfuric acid, or phosphoric acid. If desired, a
particular acid addition salt is converted into
another aeid addition salt, sueh as a non-toxic,
therapeutieally aeeeptable salt, by treatment with
the appropriate ion exehange resin in the manner
deseribed by R.A. Boissonnas et al., Helv. Chim.
Aeta, 43, 1849 (1960).
In the instance where a particular peptide has
one or more free carboxyl groups, examples of sueh
salts are those with the sodium, potassium or cal-
cium cations, or with strong organic bases, for
example, triethylamine or N-ethylmopholine.
In general, the therapeutically acceptable
salts of the peptides of formula 1 are biologically
fully equivalent to the peptides themselves.
The outstanding effectiveness of the peptides
of formula 1 in stimulating or causing the release
of growth hormone can be demonstrated in four-day
old rat anterior pituitary cells according to the
method of P. Brazeau et al., Proe. Natl. Aead. Sei.
U.S.A., 79, 7909 (1982). For example, when tested
aecording to this method, (Serl7, Leul8)-
hGRF(1-29)NHC) was found to be four times more
potent than hGRF(1-29)NH2, described by N. Ling et
al., Biochem. Biophys. Res. Commun., 123, 854 (19-
84) at concentrations of 0.5 and 2.0 fentamoles.
3o
1340077
-16-
The effectiveness of the peptides of this
invent.on, or their therapeutically acceptable
salts, to stimulate the release of GH in animals,
incluaing humans, render them useful for treating
growth hormone deficiencies and for augmenting the
desirable effects of GH. More explicitly, the
peptides or their therapeutically acceptable salts
are useful for treatinB growth related disorders due
to insufficient production of endogeneous GH in
animals, for example prepubertal growth hormone
deficiency in humans; for nealing wounds; for
improving milk production in dairy herds, such as
cows and goats; 40r improving the quality of meat in
meat-producing animals ti.e. increasing the ratio of
meat to fat); for increasing wool growth; and for
improving feed efficiency in meat-producing animals
and dairy cows. The peptides also can be used
diagnostically to evaluate pituitary function.
When the peptides of this invention, or their
therapeutically acceptable sai~s, are used to effect
the release of GH, they usually are administered
systemically to warm-blooded animals, e.g. humans,
cattle or pigs, in combination with pharmaceutically
or veterinarily acceptable carriers, the proportion
of which is determined by the solubility and chemi-
cal nature of the peptide, chosen route of adminis-
tration and standard biological practice.
For systemic administration, the peptides of
formul~ l are administered ~y either intravenous,
-
~0~77
subcutaneous or intramuscular injection, in composi-
tions with pharmaceutically acceptable vehicles or
carriers. For administration by injection, it is
preferred to use the peptides in solution in a
sterile aqueous vehicle which may also contain other
solutes such as buffers or preservatives, as well as
sufficient quantities of pharmaceutically acceptable
salts or of glucose to make the solution isotonic.
Examples of suitable excipients or carriers for
human or veterinary use are found in standard phar-
maceutical texts, e.g. in "Remington's Pharmaceuti-
cal Sciences", 16th ed, Mack Publishing Company,
Easton, Penn., 1980.
The dosage of the peptides will vary with the
form of administration and the particular compound
chosen. Furthermore, it will vary with the particu-
lar host and the particular condition to be treated.
Generally, treatment is initiated with small dosages
substantially less than the optimum dose of the
compound. Thereafter, the dosage is increased by
small increments until the optimum effect under the
circumstances is reached. In general, the peptides
of this invention are given at a concentration which
affords effective results without causing deleteri-
ous side effects.
Appropriate dosages can be determined by
relating the amount of the peptide to be administer-
,., , . , . ~
13~0077
-18-
ed to give the desired concentration of circulating
GH. Hence, when treating GH deficiencies, a suffi-
cient amount of the peptide is given systemically
e.g. subcutaneously or intravenously, to the host to
maintain the amount of circulating GH at levels
usually associated with normal growth.
Usually a dose of 0.05 mcg to 10 mcg per kilo-
gram of body weight per day is employed although the
aforementioned variations will occur. However, a
dosage in the range of from about 0.1 mcg to 10 mcg
per kilogram of body weight per day is most desir-
ably employed in order to achieve effective re-
sults.
Accordingly, a method of treatment for growth-
related disorders due to growth hormone deficiency
in an animal is provided which comprises administer-
ing to the animal an effective amount of the peptide
of formula 1, or its therapeutically acceptable
salt, to stimulate the endogenous production of
growth hormone to levels associated with normal
growth.
When the peptides of formula 1, or their thera-
peutically acceptable salts, are employed to stimu-
late growth activity in livestock, e.g. to increase
milk production in dairy herds or to improve and
increase meat production, the dosage will generally
be greater than that used to obtain normal levels of
1 3~ OB 77
--19--
GH in growth hormone deficiency states. In live-
stock, the systemic dosage generally ranges from 0.1
mcg to 20 mcg per kilogram of body weight per day,
preferably 0.5 to 20 mcg per kilogram of body weight
per day. The dosage is preferably administered sub-
cutaneously.
Thus, there also is provided a method of accel-
erating the growth rate of non-human animals and/or
increasing milk production in female species thereof
which comprises administering to the animal an
effective amount of the peptide of formula 1, or a
therapeutically acceptable salt thereof, to stimu-
late the production of GH at a level greater than
that associated with normal growth.
It is sometimes desirable to administer the
peptides of this invention continuously over pro-
longed periods of time in long-acting, slow-relase,
or depot dosage forms. Such dosage forms may either
contain a pharmaceutically acceptable salt of the
respective peptide having a low degree of solubility
in body fluids, for example one of those salts
described above, or they may contain the peptide in
the form of a water-soluble salt together with a
protective carrier which prevents rapid release and
decomposition of the peptide. Examples of such
formulations are found in standard pharmaceutical
texts, e.g., in "Remington's Pharmaceutical Sci-
ences", cited above. Long-acting, slow-release
3o
0077
-20-
preparations of the peptides of this invention may
also be obtained by microencapsulation in a pharma-
ceutically acceptable coating material, for example
gelatine, polyvinyl alcohol or ethyl cellulose.
Further examples of coating materials and of the
processes used for microencapsulation are described
by J.A. Herbig in "Encyclopedia of Chemical Technol~
ogy". Vol. 13, 2nd ed., Wiley, New York, 1967, pp.
436-456. Such formulations, as well as suspensions
of salts of the peptide which are only sparingly
soluble in body fluids, are designed to release from
about 0.1 mcg to 20 mcg of the peptide per kilogram
body weight per day, and are preferably administered
by intramuscular injection.
The following examples illustrate further this
invention. Abbreviations used in the examples in-
clude BOC: t-butyloxycarbonyl; TFA: trifluoro-
acetic acid; DCC: N,N'-dicyclohexylcarbodiimide;
HOBT: l-hydroxybenzotriazole monohydrate; HF:
hydrofluoric acid; and HPLC: high performance
liquid chromatography. Solution percentages are
calculated on a volume/volume basis unless stated
otherwise. The following terms are trademarks:
Pharmacia, Vydac and Waters.
Finally, with reference to formula 1, the
symbols Tyr, des-NH2-Tyr and His for R1 include
the terminal hydrogen. In other words, for example,
Tyr- has the same meaning as H-Tyr- with reference
to Rl
13llO077
Example 1
Preparation of (Serl7,Leu18)hGRF(1-29)NH2
having the formula:
H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-
Val-Leu-Gly-Gln-Ser-Leu-Ala-Arg-Lys-Leu-Leu-Gln-
Asp-Ile-Met-Ser-Arg-NH2
The title compound was synthesized by the
solid-phase technique of B. Merrifield, J. Amer.
Chem. Soc., 85, 2149 (1963). The synthesis of the
fully protected linear peptide having the correct
sequence of amino acids was conducted on a benzhy-
drylamine resin. The following protocol was used:
a) BOC-deprotection: 45% TFA in chloroform (2
times, firstly for 5 minutes then for 25 minutes);
b) wash: chloroform (3 times for 2 minutes each);
c) neutralization: 5% diisopropylethylamine in
methylene chloride (2 times for 3 minutes each); d)
amino acid coupling: symmetrical anhydride or
active ester mediated methods using a three fold
molar excess of the preformed active ester or sym-
metrial anhydride and a reaction time of 3 to 5
hours, and e) wash: methylene chloride (3 times for
2 minutes each). With reference to the amino acid
coupling, each Asp and Ile residue was double
coupled by the symmetrical anhydride method. The
134~077
Arg residue was double coupled in methylene chlor-
ide/dimethylf'ormamide after activation of the cor-
responding amino-acid with DCC-HOBT. The Gln and
Asn residues were double coupled in dimethylforma-
mide via their corresponding 2-nitrophenyl esters.
The BOC group gave N~ protection for all
amino-acids. Side chain protection was as follows:
2-chlorobenzyloxycarbonyl for tyrosine, benzyl for
aspartic acid, benzyl for threonine, benzyl for
serine, tosyl for arginine and 2-chlorobenzyloxy-
carbonyl for lysine. After each coupling, a resin
sample was removed during synthesis for a ninydrin
test. On completion of the synthesis, the protected
peptide- resin was removed from the reaction vessel
and dried in vacuo over phosphorus pentoxide and
then sodium hydroxide.
The protected peptide resin was mixed with
dl-methionine (10:1, w/w) and the mixture was treat-
ed at 0~ C for one hour with an excess of anhy-
drous HF and anisole (9:1, v/v). After rapid re-
moval of the HF in vacuo, the resulting residue was
triturated thrice with diethyl ether. The suspended
material, a mixture of peptide and resin, was col-
lected by filtration and dried in vacuo. The dried
solid was extracted with TFA. The TFA extract was
concentrated in a rotary evaporator at 30~ C. The
residue was triturated with diethyl ether and the
resulting white solid, the crude peptide, was col-
lected by filtration.
~ 3~1Q~77
Purification of the white solid ~rude product was
effected by reversed-phase HPLC on a Pharmacia
octadecasilyl- silica (ODS) column (2.5 x 40 cm,
C-18, Vydac, 30 ~ particle size) using a gradient of
0.06% TFA in H20 and 0.06% TFA in MeOH. The
purity of collected fractions were monitored using
analytical reverse phase HPLC 'Waters). Pure
fractions were combined to give the title compound.
Reverse HPLC in two different buffer systems and
amino acid analysis confirmed t~,at the desired
peptide ;~as been obtained in a pure form.
Example 2
By following the procedure ~f example 1, but
using ~OC-Ala instead of BOC-Gly for introducing the
15th amino acid residue (determineà from the carboxy
terminus), (Alal5 Serl7, r eul 8 )hGRF(l-
29)NH2 having the formula:
H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-
Val-Leu-Ala-~ln-Ser-Leu-Ala-Arg-Lys-Leu-Leu-Gln-
Asp-Iie-Met-Ser-Arg-NH2, is obtained.
3o
-
'~
~ ~q~(J77
-24-
Example 3
By following the procedure of example 1, but
using N~-BOC-Nim-tosylhistidine instead of
N~-BCC-04-(2,6-dichlorobenzyl)tyrosine for
introducing the 29th amino acid residue, (Hisl,
Alal5,Serl7,Leul8)hGRF(1-29)NH2 having the formula:
H-His-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-
Val-Leu-Ala-Gln-Ser-Leu-Ala-Arg-Lys-Leu-Leu-Gln-
Asp-Ile-Met-Ser-Arg-NH2 is obtained.
Other examples of peptides within the scope
of this invention include (AcTyr ,Ser 7,Leu )bGRF-
1-40)0H, (AcTyrl,serl7,Leu18)hGRF(l_29)NH2,
(serl7~Leul8~Ile27)hGRF(l-29)NH2~ (AcTyr ,Ala ,-
Serl7,Leul8,Nle27)hGRF(1-29)NH2, ~Serl7,Leul8,-
Lys24)hGRF(1-29)NH2, ~Serl7,LeU18,ser24)hGRF(l_
NH2, (Ser 7,Leu 8,Glu24)hGRF(1-29)NH (AcTyrl
Ala 5,Ser 7,Leu 8)hGRF(1-29)NH , (Leu 5,Serl7,-
Leu 8)hGRF(1-29)NH2 and (AcTyr~,Leul5,Serl7,Leul8)-
hGRF(1-29)NH2.
