U.S. patent number RE30,548 [Application Number 06/071,559] was granted by the patent office on 1981-03-17 for peptides which effect release of hormones.
This patent grant is currently assigned to The Salk Institute for Biological Studies. Invention is credited to Marvin R. Brown, Jean E. F. Rivier, Wylie W. Vale, Jr..
United States Patent |
RE30,548 |
Vale, Jr. , et al. |
March 17, 1981 |
Peptides which effect release of hormones
Abstract
The present invention relates to peptides which possess
biological activity in respect to the inhibition of growth hormone,
insulin .[.secretion.]. and glucagon secretion .[.are provided.]..
The peptides have fewer amino acid components than somatostatin and
some of the peptides have dissociated activity. .Iadd.
Inventors: |
Vale, Jr.; Wylie W. (La Jolla,
CA), Rivier; Jean E. F. (La Jolla, CA), Brown; Marvin
R. (Del Mar, CA) |
Assignee: |
The Salk Institute for Biological
Studies (San Diego, CA)
|
Family
ID: |
22102109 |
Appl.
No.: |
06/071,559 |
Filed: |
August 31, 1979 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
781580 |
Mar 28, 1977 |
04105603 |
Aug 8, 1978 |
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Current U.S.
Class: |
525/54.11;
360/21; 514/806; 530/311; 530/334; 930/10; 930/160; 930/DIG.701;
930/DIG.706 |
Current CPC
Class: |
C07K
14/6555 (20130101); A61K 38/00 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/655 (20060101); A61K
38/00 (20060101); C08L 037/00 (); A61K 037/00 ();
C07C 103/52 () |
Field of
Search: |
;260/112.5S,8
;424/177 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Burgus et al., Proc. Nat. Acad. Sci., 70, No. 3,pp. 684-688 (1973).
.
Brazeau et al., Science 179, pp. 77-79 (1973)..
|
Primary Examiner: Phillips; Delbert R.
Attorney, Agent or Firm: Fitch, Even, Tabin, Flannery &
Welsh
Government Interests
The invention described herein was made in the course of work under
a grant or award from the Department of Health and Human Services
(formerly DHEW). .Iaddend.
Claims
What is claimed is:
1. A peptide selected from those of the formulae: ##STR3## wherein
R.sub.1 is selected from the group consisting of Asn and desR.sub.1
; R.sub.2 is selected from the group consisting of Trp and D-Trp;
R.sub.3 is selected from the group consisting of Phe and Thr;
R.sub.4 is selected from group consisting of Thr and desR.sub.4 ;
R.sub.5 is selected from the group consisting of Ser, Phe and
desR.sub.5, provided that at least one of R.sub.1, R.sub.4 and
R.sub.5 is deleted; X is selected from the group consisting of H,
and an alpha-amino protecting group; X.sup.1 and X.sup.6 are
selected from the group consisting of H and a protecting group for
Cys selected from S-p-methoxybenzyl, S-acetamidomethyl, S-trityl
and S-benzyl; X.sup.2 is selected from the group consisting of H
and a side chain amino protecting group; X.sup.3, X.sup.4 and
X.sup.5 are selected from the group consisting of H and a hydroxyl
protecting group selected from the group consisting of acetyl,
benzoyl, tert-butyl, trityl, benzyl and benzyloxycarbonyl; with the
proviso that at least one of X, X.sup.1, X.sup.2, X.sup.3, X.sup.4,
X.sup.5, and X.sup.6 is other than hydrogen; .[.and.]. R.sub.6 is
selected from the group consisting of hydroxy, methoxy, and an
anchoring bond used in solid phase synthesis linked to a solid
resin support selected from the group consisting of --O--CH.sub.2
-polystyrene resin support and O--CH.sub.2 -benzylpolystyrene resin
support.Iadd.; and Cys is either L-Cys or D-Cys.Iaddend..
2. A peptide in accordance with claim 1 wherein R.sub.1 is des
R.sub.1, R.sub.2 is D-Trp, R.sub.3 is .[.Phe.]. .Iadd.Thr.Iaddend.,
R.sub.4 is desR.sub.4 and R.sub.5 is desR.sub.5.
3. A peptide in accordance with claim 1 wherein R.sub.1 is
desR.sub.1, R.sub.2 is D-Trp, R.sub.3 is .[.Phe.].
.Iadd.Thr.Iaddend., R.sub.4 is DesR.sub.4, R.sub.5 is desR.sub.5
and .[.D-Cys is substituted for.]. Cys.sup.11 is D-Cys.
4. A peptide in accordance with claim 1 wherein R.sub.1 is
desR.sub.1, R.sub.2 is D-Trp, R.sub.3 is .[.Phe.].
.Iadd.Thr.Iaddend., R.sub.4 is .[.desR.sub.4 .]. .Iadd.Thr
.Iaddend.and R.sub.5 is desR.sub.5.
5. A peptide in accordance with claim 1 wherein R.sub.1 is Asn,
R.sub.2 is D-Trp, R.sub.3 is .[.Phe.]. .Iadd.Thr.Iaddend., R.sub.4
is desR.sub.4 and R.sub.5 is desR.sub.5.
6. A peptide in accordance with claim 1 wherein R.sub.1 is Asn,
R.sub.2 is Trp, R.sub.3 is .[.Phe.]. .Iadd.Thr.Iaddend., R.sub.4 is
Thr and R.sub.5 is desR.sub.5.
7. A peptide in accordance with claim 1 wherein R.sub.1 is Asn,
R.sub.2 is D-Trp, R.sub.3 is .[.Phe.]. .Iadd.Thr.Iaddend., R.sub.4
is Thr and R.sub.5 is des R.sub.5.
Description
The present invention relates generally to peptides having
biological activity in respect to the inhibition of growth hormone,
insulin and glucagon secretion. More particularly, the present
invention is directed to peptides having fewer amino acid moieties
than somatostatin which are effective to inhibit the release of
growth hormone by the pituitary gland or the release of glucagon or
insulin by the pancreas. Various peptides of the invention have
dissociated biological activity in respect to the inhibition of
growth hormone, insulin and glucagon secretion.
A peptide having inhibitory effect on the secretion of growth
hormone has been characterized and is described in U.S. Pat. No.
3,904,594 to Guillemin et al. This peptide has been named
"somatostatin". Somatostatin (also known as somatotropin release
inhibiting factor) is the tetradecapeptide: ##STR1##
Somatostatin, the linear form of somatostatin (dihydrosomatostatin)
and various acylated derivatives of somatostatin and
dihydrosomatostatin are described in the aforementioned U.S.
patent.
Somatostatin and many analogs of somatostatin exhibit activity in
respect to the inhibition of growth hormone (GH) secretion from
cultured, dispersed, rat anterior pituitary cells in vitro and
inhibition of insulin and glucagon secretion in vivo in the rat. It
has been considered highly desirable in the use of somatostatin to
selectively inhibit only the secretion of GH, insulin or glucagon.
Efforts have been made to develop analogs of somatostatin which
possess dissociated biological activity and which inhibit only GH,
insulin or glucagon secretion. Although there have been reports
citing differences in the amounts of somatostatin required for
inhibition of insulin compared to glucagon in the human and the
perfused rat pancreas in vitro, somatostatin and some somatostatin
analogs exhibit similar potencies on the inhibition of these two
hormones in vivo.
The present invention relates to the discovery that certain amino
acids can be removed and/or rearranged in somatostatin and
dihydrosomatostatin peptides to provide novel peptides having fewer
amino acid components and which possess biological activity in
respect to the inhibition of GH, insulin or glucagon secretion.
Some of the novel peptides of the invention have dissociated
activity. The novel peptides of the invention having fewer amino
acid components than somatostatin or dihydrosomatostatin are
considered to be of great value because of the relative simplicity
with which these peptides can be manufactured.
The novel peptides of the invention are defined by the formulae:
##STR2## where R.sub.1 is selected from Asn and des R.sub.1,
R.sub.2 is selected from Trp and D-Trp, R.sub.3 is selected from
Phe and Thr, R.sub.4 is selected from Thr and des R.sub.4, and
R.sub.5 is selected from Ser, Phe, and des R.sub.3 provided that at
least one of R.sub.1, R.sub.4 and R.sub.5 is deleted.
The nomenclature used to describe the peptides of the present
invention is in accordance with the conventional practice of using
the first three letters of the trivial name. Also, in accordance
with such practice, it is the L form of the amino acid that is
intended, unless otherwise expressly indicated. In this connection,
it should be understood that either of the Cys amino acid moieties
can be either D-Cys or L-Cys.
Pharmaceutically acceptable acid addition salts of the peptides are
also within the scope of the present invention. Such acid addition
salts include but are not limited to hydrochloride, hydrobromide,
sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate,
malate, ascorbate, tartrate and the like.
Also considered to be within the scope of the present invention are
intermediates of the formula:
wherein: X is either hydrogen or an .alpha.-amino protecting group.
The .alpha.-amino protecting groups contemplated by X are those
known to be useful in the art in the step-wise synthesis of
polypeptides. Among the classes of .alpha.-amino protecting groups
covered by X are (1) acyl type protecting groups such as formyl,
trifluoroacetyl, phthalyl, toluenesulfonyl (tosyl), benzensulfonyl,
nitrophenylsulfenyl, tritylsulfenyl, o-nitrophenoxyacetyl,
chloroacetyl, acetyl, y-chlorobutyrul, etc.; (2) aromatic urethan
type protecting groups such as benzyloxycarbonyl and substituted
benzyloxycarbonyl such as p-chlorobenzyloxycarbonyl,
p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,
p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups
such as .alpha.-t-butyloxycarbonyl, diisopropylmethoxycarbonyl,
isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; (4)
cycloalkyl urethan type protecting groups such as
cyclopentyloxycarbonyl, adamantyloxycarbonyl,
cyclohexyloxycarbonyl; (5) thiourethan type protecting groups such
as phenylthiocarbonyl; (6) alkyl type protecting groups such as
triphenylmethyl (trityl), benzyl; (7) trialkylsilane groups such as
trimethylsilane. The preferred .alpha.-amino protecting group
defined by R is tertbutyloxycarbonyl.
X.sup.1 and X.sup.6 are each a protecting group for Cys selected
from the group consisting of S-p-methoxybenzyl, S-p-methylbenzyl,
S-acetamidomethyl, S-trityl, S-benzyl, and the like. The preferred
protecting group is S-p-methoxybenzyl. X.sup.1 and/or
.[.X.sup.5`.]. X.sup.6 can be hydrogen which means that there is no
protecting group on the sulfur group.
X.sup.2 is a protecting group for the side chain amino substituent
of lysine or X.sup.2 is hydrogen which means there is no protecting
group on the side chain amino substituent. Illustrative of suitable
side chain amino protecting groups are benzyl,
chlorobenzyloxycarbonyl, benzyloxycarbonyl, tosyl,
t-amyloxycarbonyl, t-butyloxycarbonyl, etc. The selection of such a
side chain amino protecting group is not critical except that it
must be one which is not removed during deprotection of the
.alpha.-amino groups during the synthesis. Hence, the .alpha.-amino
protecting and side chain amino protecting group cannot be the
same.
X.sup.3, X.sup.4 and X.sup.5 are protecting groups for the hydroxyl
group of Thr and Ser and are selected from the group consisting of
acetyl, benzoyl, tert-butyl, trityl, tetrahydropyranyl, benzyl,
.Badd.2,6-dichlorobenzyl and benzyloxycarbonyl. The preferred
protecting group is benzyl. X.sup.3 and/or X.sup.4 and/or X.sup.5
can be hydrogen which means there is no protecting group on the
hydroxyl group.
R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are as previously
defined. R.sub.6 is selected from the class consisting of OH,
OCH.sub.3, esters, amides, hydrazides and benzyl ester or
hydroxymethyl ester anchoring bond used in solid phase synthesis
linked to a solid resin support represented by the formulae:
and
The polymer is preferably a copolymer of styrene with about 0.5 to
2.0% divinyl benzene as a cross-linking agent which causes the
polystyrene polymer to be completely insoluble in certain organic
solvents. In formula III at least one of X, X.sup.1, X.sup.2,
X.sup.3, X.sup.4, X.sup.5 and X.sup.6 is a protecting group.
In selecting a particular side chain protecting group to be used in
synthesis of the peptides of formula I or formula II, the following
rules should be followed: (a) the protecting group must be stable
to the reagent and under the reaction conditions selected for
removing the .alpha.-amino protecting group at each step of the
synthesis, (b) the protecting group must retain its protecting
properties and not be split off under coupling conditions, and (c)
the side chain protecting group must be removable upon the
completion of the synthesis containing the desired amino acid
sequence under reaction conditions that will not alter the peptide
chain.
The peptides of formula I and formula II can be prepared using
solid phase synthesis. The synthesis is commenced from the
C-terminal end of the peptide using an .alpha.-amino protected
resin. Such a starting material can be prepared by attaching an
.alpha.-amino and S-protected Cys to a chloromethylated resin or a
hydroxymethyl resin. The preparation of the hydroxymethyl resin is
described by Bodanszky et al., Chem. Ind. (London) 38, 1597-98
(1966). A chloromethylated resin is commercially available from Bio
Rad Laboratories, Richmond, California and the preparation of such
a resin is described by Stewart et al., "Solid Phase Peptide
Synthesis" (Freeman & Co., San Francisco 1969), Chapter 1, pp.
1-6. The .alpha.-amino and S-protected Cys is coupled to the
chloromethylated resin according to the procedure of Monahan and
Gilon, Biopolymer 12, pp. 2513-19, 1973. Following the coupling of
the .alpha.-amino and S-protected Cys to the resin support, the
.alpha.-amino protecting group is removed such as by using
trifluoroacetic acid in methylene chloride, trifluoroacetic acid
alone or HCl in dioxane. The deprotection is carried out at a
temperature between about 0.degree. C. and room temperature.
Other standard cleaving reagents and conditions for removal of
specific .alpha.-amino protecting groups may be used as described
in Schroder & Lubke, "The Peptides", 1 pp. 72-75 (Academic
Press 1965).
After removal of the .alpha.-amino protecting group of Cys the
remaining .alpha.-amino and side chain protected amino acids are
coupled step-wise in the desired order to obtain a compound of
formula III or as an alternate to adding each amino acid separately
to the synthesis, some of them may be coupled prior to addition to
the solid phase reactor. The selection of an appropriate coupling
reagent is within the skill of the art. Particularly suitable as a
coupling reagent is N,N.sup.1 -dicyclohexyl carbodiimide.
The activating reagents used in the solid phase synthesis of the
peptides are those well known in the peptide art. Examples of
suitable activating reagents are: (1) carbodiimides such as
N,N-diisopropyl carbodiimide, N-ethyl N.sup.1 -(y-dimethylamino
propyl carbodiimide); (2) cyanamides such as N,N-dibenzylcyanamide;
(3) keteimines; (4) isoxazolium salts such as N-ethyl-5-phenyl
isoxazolium-3.sup.1 -sulfonate; (5) monocyclic nitrogen containing
heterocyclic amides of aromatic character containing one through 4
nitrogens in the ring such as imidazolides, pyrazolides,
1,2,4-triazolides. Specific heterocyclic amides that are useful
include N,N.sup.1 -carbonyl diimidazole, N,N.sup.1
-carbonyl-di-1,2,4-triazole; (6) alkoxylated acetylene such as
ethoxyacetylene; (7) reagents which form a mixed anhydride with the
carboxyl moiety of the amino acid such as ethylchloroformate and
isobutylchloroformate and (8) nitrogen-containing heterocyclic
compounds having a hydroxy group on one ring nitrogen such as
N-hydroxyphthalimide, N-hydroxysuccinimide and
1-hydroxybenzotriazole. Other activating reagents and their use in
peptide coupling are described by Schroder & Lubke supra, in
Chapter III and by Kapoor, J. Pharm. Sci., 59, pp. 1-27 (1970).
Each protected amino acid or amino acid sequence is introduced into
the solid phase reactor in about a four-fold excess and the
coupling is carried out in a medium of dimethylformamide: methylene
chloride (1:1) or in dimethylformamide: methylene chloride (1:1) or
in dimethylformamide or methylene chloride alone. In cases where
incomplete coupling occurred the coupling procedure is repeated
before removal of the .alpha.-amino protecting group, prior to the
coupling of the next 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., Analyt.
Biochem. 34, 595 (1970).
After the desired amino acid sequence of formula III has been
synthesized, the peptide is removed from the resin support by
treatment with a reagent such as liquid hydrogen fluoride which not
only cleaves the peptide from the resin but also cleaves all
remaining side chain protecting groups X.sup.1, X.sup.2, X.sup.3,
X.sup.4, X.sup.5 and X.sup.6 and the .alpha.-amino protecting group
X to obtain directly a peptide of formula I. Peptides in accordance
with formula II are obtained by oxidizing formula I peptides in
accordance with known procedures. As an alternate route, the
peptide linked to the resin support may be separated from the resin
by alcoholysis after which the recovered C-terminal methyl ester is
converted to the acid by hydrolysis. Any side chain protecting
group may then be cleaved as previously described or by other
procedures such as catalytic reduction (e.g. Pd on BaSO.sub.4)
using conditions which will keep the Trp moiety intact. When using
hydrogen fluoride for cleaving, anisole is included in the reaction
vessel as a scavenger.
The solid phase synthesis procedure discussed above is well known
in the art and has been essentially described by Merrifield J. Am.
Chem. Soc., 85, p. 2149 (1964).
The peptides of the present invention having dissociated effects in
respect to inhibition of release of growth hormone, insulin and
glucagon are considered to be particularly important in connection
with the treatment of diabetes. The traditional view of diabetes
has been that it is a disease resulting from impaired insulin
production alone. As clinical and research experience has become
more extensive, it has become apparent that some factor in addition
to impairment of insulin secretion is operative in diabetes. It is
known that, while insulin is normally deficient in diabetes,
glucagon is normally present in excess. It is now believed that the
presence of glucagon is at least as important a factor in diabetes
as the absence of insulin.
The fact that a deficiency in insulin is normally accompanied by an
excess of glucagon has made it difficult to study the role of
glucagon in diabetes. While it is easy to add extra quantities of a
hormone such as insulin, it has proved very difficult to lower the
concentration of glucagon. The discovery of somatostatin has
facilitated research in respect to the role of glucagon in
diabetes. Somatostatin inhibits the release of both insulin and
glucagon. The role of somatostatin in diabetes research is detailed
in an article appearing in Science, Vol. 188, pp. 920-923, May 30,
1975. However, there are several problems in respect to the use of
somatostatin as a treatment in diabetes. Somatostatin inhibits the
release of insulin in addition to glucagon. Thus, the need for a
peptide having a dissociated effect on the inhibition of release of
insulin and glucagon has been recognized in connection with
diabetes treatment. The novel peptides of the present invention
provide such dissociative effect. More particularly, certain of the
peptides of the present invention are effective to inhibit
secretion of glucagon while having less effect on the inhibition of
secretion of insulin.
The peptides of the invention provide the benefits of somatostatin
and known somatostatin analogs, but have fewer amino acid
components, e.g. 8 to 10 amino acid moieties as compared to 12 to
14 amino acid moieties for most known somatostatin analogs.
Accordingly, the peptides of the present invention have significant
economic advantage due to the relative ease of manufacture of these
peptides.
The following examples illustrate various features of the present
invention, but are intended to in no way limit the scope of the
invention which is defined in the appended claims.
EXAMPLE I
The peptides of the present invention were synthesized by solid
phase techniques, generally in accordance with the procedure
described in U.S. Pat. No. 3,904,595. The synthesis was conducted
in a stepwise manner on chloromethylated resin. The resin was
composed of fine beads (20-70 microns in diameter) of a synthetic
resin prepared by copolymerization of styrene with one to two
percent divinylbenzene. The benzene rings in the resin were
chloromethylated in a Friedel-Crafts reaction with chloromethyl
methyl ether and stannic chloride. The chlorine thus introduced is
a reactive benzyl chloride type of linkage. The Friedel-Crafts
reaction is continued until the resin contains 0.5 to 2 millimoles
of chlorine per gram of resin. In the further description of the
synthesis of the peptides, the reagents used will be first
described by their chemical name with their common abbreviation in
parenthesis. Thereafter, the reagent will be referred to by the
common abbreviation.
A peptide having the structure:
was synthesized by the following solid phase methodology. Other
peptides, described hereinafter were synthesized by a similar
technique.
The tertiobutyloxycarbonyl-S-paramethoxybenzyl (Boc-SpOMe-Bzl)
derivative of Cys was linked to the resin by any of three known
methods; (1) reflux in ethanol in presence of triethyl amine, (2)
Cesium salt of the Boc protected amino acid is kept at 50.degree.
C. in dimethylformamide (DMF) overnight, (3) the potassium salt of
the Boc-protected amino acid is kept at 80.degree. C. in dimethyl
sulfoxide (DMSO) for 2 hours. Only one milliequivalent of the
protected Cys per milliequivalent of Cl on the resin is used.
Method (3) is described hereinbelow in more detail. To a slurry of
the resin and the dissolved protected Cys in DMSO is added 0.9 mEq
of potassium tertiobutoxide (KOtBut) per mEq of amino acid. The
reaction mixture is exposed to air as little as possible so that no
amber coloration is observed. Reaction at 80.degree. C. for 2 hours
yields a suitable substituted resin for synthesis of the peptides
(approx. 0.2 mEq of amino acid derivative per g of resin). After
deprotection and neutralization, the peptide chain is built on
resin. Deprotection, neutralization and addition of each amino acid
is performed in accordance with schedule I. N.sup..alpha.
-t-butyloxycarbonyl (Boc) derivative of each amino acid is used.
After deprotection of the first residue (i.e., SpOMe.Bzl.Cys)
according to schedule I (steps 3 to 8 included), the N .Iadd.Boc
derivative of Phe is added along with a coupling agent. Thereafter,
the N .Iaddend.Boc derivative of Thr is next added along with a
coupling agent which is dicyclohexylcarbodiimide (DCC) (step 9 of
schedule I). The side chain of Thr is protected with O-benzyl ether
(OBzl). Benzyloxycarbonyl (Z) or benzyloxycarbonyl-2Cl [Z(2-Cl)]
was used as the protecting group for the Lys side chain.
______________________________________ 1. Schedule for coupling of
amino acids in solid phase synthesis (5-10 g resin) Mix times Step
Reagents and operations Min. ______________________________________
1 CH.sub.2 Cl.sub.2 wash 80 ml (2 times) 3 2 Methanol (MeOH) wash
30 Ml (2 times) 3 3 CH.sub.2 Cl.sub.2 wash 80 ml (3 times) 3 4 50
percent trifluoroactetic acid (TFA) 10 containing 5 percent
1,2-ethanedithiol in CH.sub.2 Cl.sub.2 70 ml (2 times) 5 CH.sub.2
Cl.sub.2 wash 80 ml (2 times) 3 6 Triethylamine (Et.sub.2 N) 12.5
percent in 5 CH.sub.2 Cl.sub.2 70 ml (2 times) 7 MeOH wash 40 ml (2
times) 2 8 CH.sub.2 Cl.sub.2 wash 80 ml (3 times) 3 9 Boc-amino
acid (10 mmoles) in 10 ml DMF (1 times) and 30 ml CH.sub.2 Cl.sub.2
plus DCC (10 mmoles) in Ch.sub.2 Cl.sub.2 (2 M) 30 to 120 10 MeOH
wash 40 ml (2 times) 3 11 Et.sub.3 N 12.5 percent in CH.sub.2
Cl.sub.2 70 ml (2 3imes) 12 MeOH wash 30 ml (2 times) 3 13 CH.sub.2
Cl.sub.2 wash 80 ml (2 times) 3
______________________________________
After step 13, an aliquot is taken for a ninhydrin test:
if the test is negative, go back to step 1 for coupling of the next
amino acid; if the test is positive or slightly positive, go back
to steps 9 through 13. Schedule I was used for coupling of each of
the amino acids of the peptide to Cys.
Cleavage of the peptides from the resin (5 grams) and deprotection
of the side chain protecting groups of the peptide was performed in
hydrofluoric acid (75 ml) in the presence of anisole (8 ml). After
elimination of hydrofluoric acid under high vacuum, the
resin-peptide was washed with ether.
The dried resin was immediately extracted with 25% acetic acid (150
ml) and diluted to 3000 ml with degassed H.sub.2 O (N.sub.2). The
pH of the solution was adjusted to 6.6-7.0 with NH.sub.4 OH. The
solution was titrated dropwise under stirring with potassium
ferricyanide solution (1.g/500 ml H.sub.2 O) until a permanent
yellow color was observed. The solution sat for 10 minutes and pH
was adjusted to 5.0 with glacial acetic acid; Bio Rad AG 3-X4A
resin (100-200 mesh, chloride form, 10-15 g) was added to the
turbid solution and stirred for 15 minutes. The solution was
filtered over celite and applied successively onto two columns; (a)
Bio Rad AG 3-X4A resin chloride form (10 ml); (b) Bio Rex-70 resin
(100 ml) cation form. The celite + resin cake was thoroughly washed
with water (500 ml) which was applied onto columns (a) and (b) as a
wash. The peptide material was then eluted from the Bio Rex-70
resin column with pyridine; acetic acid:water (30:4:66) or 50%
acetic acid. Fractions were collected; only the ones containing
peptide (ninhydrin positive) were diluted with water and
immediately lyophilized. 950 mg of crude cream colored material was
obtained. It was applied onto a Sephadex G-25 F gel column
(3.times.200 cm) equilibrated and eluted with 2 N acetic acid.
The elution pattern as observed at 280 nm showed one major
symmetrical peak. After lyophylization the center cut yielded 550
mg which were submitted to counter current distribution (solvent
system n-butanol:acetic acid:water, 4:1:5) 10 ml lower phase per
tube. 100 transfers were performed and the major peak was found in
tubes 57-68. The compound (250 mg) appeared homogeneous on tlc.
The specific optical rotation was [.alpha.].sup.23 =-67.8.+-.2:
(c=1 in 1% acetic acid). Amino acid analysis of this material
showed the expected ratio for the different amino acids.
Active esters can be used in solid phase synthesis and the
classical method of synthesis can also be used to prepare the
peptides of the invention.
In vitro Bioassay: The effects of the various peptides of the
invention were tested in vitro on the secretion of growth hormone
by primary cultures of enzymatically dissociated rat anterior
pituitary cells by the method of Vale et al., Endocrinology 91: p.
562-571 (1972). The assay is made by treating pituitary glands
removed from rats to separate cells therefrom. The cells are placed
in culture dishes in Dulbecco's Modified Eagle Medium (Dulbecco et
al., Virology, Vol. 8, p. 396, 1949). Carbon dioxide gas and oxygen
are supplied to the cell cultures which are maintained at
37.degree. C. for 4-5 days prior to use in the assay. Following
media changes, cell cultures are incubated for a period of 4 hours
and particular somatostatin peptides are added thereto.
Radioimmunoassay analysis is used to determine the rate of growth
hormone secretion which is expressed in nanograms per hour.
An investigation of the effect of somatostatin,
dihydrosomatostatin, (as controls) and the peptides of the
invention to inhibit the release of glucagon and insulin was made
as follows:
In vivo Bioassay: Male Sprague-Dawley-CD rats weighing 180-200 g
housed in temperature and humidity controlled quarters with 14h
light and 10h dark (light 0700-21100) were used in all experiments.
Animals were fed a standard ration and tap water ad libitum.
Experiments were carried out at least 5 days after arrival of rats
from the supplier between the hours 1400 to 1600. After ether
anesthesia, peptides or saline were administered in a volume of 0.2
ml. via the external jugular vein. Animals remained anesthetized
until the time of blood collection from the portal vein. The blood
samples were placed into chilled tubes containing 10 mg EDTA and 50
.mu.l of M Benzamidine per ml of blood.
Plasma was stored at -20.degree. C. for insulin and glucagon
determinations. Insulin levels were determined by the method of
Herbert et al, J. Chem. Endocr. Metab. 25:1375, .[.1956.].
.Iadd.1965.Iaddend., utilizing porcine insulin antisera and (125I)
iodinated insulin tracer. Human insulin standard was obtained from
Schwarz-Mann, Orangeburg, New York. Glucagon was determined by the
method of Faloona and Unger, in Jaffe et al ed., Methods of Hormone
Radioimmunoassay, Academic Press, New York, 1974, p. 317, utilizing
glucagon antisera 30K. Cellulose was determined by the glucose
oxidase method, utilizing a Beckman Glucose Analyzer.
GH determinations were performed on tissue culture media utilizing
the following reagents: NIAMDD rat GH standard (GH-RP-1), NIAMDD
monkey anti-rat GH (GH-Serum-3), and highly purified rat GH for
iodination.
All experiments were carried on in a randomized block design.
Following analysis of variance difference between treatments were
determined by the multiple range tests of Dunnett and Duncan.
Potency values were calculated from four or six point
bioassays.
Various peptides in accordance with the invention were prepared in
accordance with the solid phase methodology described above. The
composition of the peptides is reported hereinbelow in Table I.
Table I also sets forth the percent effectiveness of the peptide
for inhibiting secretion of growth hormone (GH), insulin and
glucagon, with somatostatin taken as the base.
TABLE I
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Somatostatin (control) Growth Peptides of Invention Hormone Insulin
Glucagon R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 100 100 100
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desR.sub. 1 D-Trp .[.Phe.]..Iadd.Thr.Iaddend. desR.sub.4 desR.sub.5
6 80 100 desR.sub.1 D-Trp .[.Phe.]..Iadd.Thr.Iaddend. desR.sub.4
desR.sub.5 (D-Cys).sup.11 14 100 100 desR.sub.1 D-Trp
.[.Phe.]..Iadd.Thr.Iaddend. Thr desR.sub.5 35 10-100 100 Asn D-Trp
.[.Phe.]..Iadd.Thr.Iaddend. desR.sub.4 desR.sub.5 <1 100 <1
Asn Trp .[.Phe.]..Iadd.Thr.Iaddend. Thr desR.sub.5 3 100 <1 Asn
D-Trp .[.Phe.]..Iadd.Thr.Iaddend. Thr desR.sub.5 10 200 10-100
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