Note: Descriptions are shown in the official language in which they were submitted.
3~
The pres~nt invention relates generally to peptides
having dissociated biological ac-tivity in respect to the inhib-
ition of growth hormone, insul;n and glucagon secretion. More
particularly, the present invention is directed to peptides which
are effective to selectively inhibit only the release of growth
hormone ~y the pituitary gland or the release of glucagon or in-
sulin by the pancreas.
A peptide having inhibitory effect on the secretion of
growth hormone has been characterized and is described in United
States Patent No. 3,904,594 to Gulllemin ek al. This peptide
has been named "somatostatin". Somatostatin (also known as som-
atotropin release inhibiting factor) is the tetradecapeptide:
H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH
Somatostatin, the linear form of somatostatin
(dihydroxomatostatin) and various acylated derivatives of soma-
tostatin and dihydrosomatostatin are described in the aforemen-
tioned United States Patent.
Somatostatin and many analogs of somatostatin exhibit ac-
tivity in respect to the inhibition of growth hormone (GH) secre-
tion from cultured, dispersed, rat anterior pituitary cells invitro 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, insu-
lin or glucagon. Efforts have been made to devleop analogs of so-
matostatin which possess dissociated biological activity and whichinhibit only GH, insulin or glucayon secretion. Although there
have been reports citing differences in the amounts of somatos-
tatin 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 inhibi-
tion of these two hormones.
The present invention relates to the discovery that cer-
-1- ~'
3~
tain ami.no acids can be subs~ituted for amino acid substituents
in somatostatin and dihydrosomatostatin to provide peptides which
possess dissociated biological activity in respect to the inhibi-
tion of GH, insulin or glucagon secretion. As a convenient short-
han~ form the novel peptides of the present in~ention are des-
cribed in terms of the amino acid moiety which is substituted,
the position of substitution and whether the substitution is made
in somatostatin (SS) or dihydrosomatostatin (D~ISS). The nomen-
clature used to describe the peptides of the present invention is
in accordance with the conventional practice and in accordance with
such practice, it is the L form of the amino acid that is intended,
unless otherwise expressly indicated.
The novel peptides of the invention are defined by the
formulae herein below, where the individual amino acids of the
peptide are numbered from left to right for ease of identification:
Ala~Gly-Cys-Lys-Xl-Phe-Phe-X2-Lys-Thr-Phe-Thr-X3-X4
1 2 3 4 5 ~ 7 8 9 10 11 12 13 14
and
Ala-Gly-Cys-Lys-Xl-Phe-Phe-X2-Lys-Thr-Phe-Thr-X3-X4
1 2 3 ~ 5 6 7 8 9 10 11 12 13 14
Where X1 is selected from Asn and des-Asn, X2 is selec-
ted from Trp and D-Trp, X3 is selected from Ser and D-Ser and X4
is selected from Cys and D-Cys with the proviso that whenever X2
is D-Trp, X3 is D-Ser and/or X4 is D-Cys; tha-t X3 is D-Ser when-
ever Xl is Asn and X4 is Cys; and that X4 is D-Cys whenever Xl is
Asn and X3 is Ser. [Cys]3 can he either L-Cys or D-Cys without
change of the potency or specific.ity of the peptides.
In one aspect, the invention provides a method for
making a peptlde useful as a pharmaceutical comprising the steps
of preparing an ester of N~ protected Cys amino acid and a chloro-
methylated or a hydroxymethyl resin, deprotecting said Cys, step-
wise coupling N~ and side chain protected amino acids to said Cys
.~ -2-
~ ~r ~
to form a resin-coupled peptide haviny the structure R-Ala-Gly-
Cys(Rl)-Lys(R2)-(Xl)-Phe~Phe-(X2)-Lys(R3)-Thr(R4)-Phe-Thr(R5)-
~X3)(R6)-(X4)(R7)-resin wherein Xl is selected from Asn and des-
Asn; X2 is selected from Trp and D-Trp; X3 is selected from Ser
and D-Ser; and X4 is selected from Cys and D-Cys with the proviso
that whenever X2 is D-Trp, X3 is D-Ser and/or X4 is D~Cys; that
X3 is D-Ser, whenever Xl is Asn and X4 is Cys; and that X4 is
D~Cys whenever Xl is Asn and X3 is Ser; R is selected from the . ~-
class consisting of H and an alpha-amino protecting group; Rl and
R7 are selected from the group consisting of H and a protecting
group for Cys selected from S-p-methoxybenzyl, S-p-methylbenzyl,
S-acetamidomethyl, S-trityl and S-benzyl; R2 and R3 are selected
from the group consisting of H and a side chain amino protecting
group; R4, R5 and R6 are selected from the group consisting of H
and a hydroxyl protecting group selected from the group consist-
ing of acetyl, benzoyl, tert-butyl, benzyl and benzyloxycarbonyl;
with the proviso that at least one of R, Rl, R2, R3/ R4, R5, R6
and R7 is other than hydrogen; cleaving said peptide from the
resin and, optionally, oxidizing said peptide to obtain the corre
sponding cyclic disulfide derivative.
The novel peptides of the present invention having spec-
ific biological activity in respect to release of growth hormone,
insulin and glucagon are: [D-S~13]-SS, [D-Serl3]-DHSS; [D-Trp8]-
[D-Serl3]-SS; [D-Trp8]-[D-Serl3]-DHSS; ~D-Cysl4]-SS; [D-Cysl4]- ~ :
DHSS; lD-Trp8]-
~ -2a-
~a~
[D-Cysl4]SS and [D-Trp83-[D~Cysl4]DHSS.
Pharmaceutically acceptab]e 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, sulfa-te, phosphate, maleate, acetate, citrate,
benzoate, succinate, malate, ascorbate, tartrate and the like.
Also considered to be within the scope o~ the pxesent
invention are intermediates of the formula:
R-Ala-Gly-Cys tR1) -Lys(R2)-(Xl)-Phe-Phe-(X2)-Lys(R3)-Thr(R4)-Phe-
Thr(RS)-(X3) (R6)-(X4) (R7)-(xs). III
wherein: R is either hydrogen or an ~-amino protecting
group The a-amino protecting groups contemplated by R aré those
known to be useful in the art in the step-wise synthesis of poly-
peptides. Among the classes of ~-amino protecting groups covered
by R are (1) acyl type protecting groups such as formyl, triflu-
oroacetyl, phthalyl, toluenesulfonyl ~tosyl~, benzensulfonyl,
nitrophenylsulfenyl, tritylsulfenyl, o-nitrophenoxyacetyl, chlo-
roacetyl, acetyl, y-chlorobutyrul, etc.; (a) aromatic urethan
type protecting groups such as benzyloxycarbonyl and substituted
benzyloxycarbonyl such as p-chlorobenæyloxycarbonyl, p-nitroben-
zyIoxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxy-
carbonyl; (3) aliphatic urethan protecting groups such as ~t-
butyloxycarbonyl, diisopropylmethoxycaxbonyl, isopropyloxycarbonyl,
ethoxycarbonyl, allyloxycarbonyl; (4) cycloalkyl urethan type
protecting groups such as cyclopenthyloxycarbonyl, adamantyloxy-
carbonyl, cyclohexyloxycarbonyl; (5) thiourethan type protecting
groups such as phenylthiocarbonyl; ~6) alkyl type protecting
groups such as triphenylmethyl (trityl), benæyl; (7) trialkyl-
silane groups such as trimethylsilane. The preferred a-amino
protecting group defined by R is tertbutyloxycarbonyl.
Rl and R7 are each a protecting group for Cys or D Cys
selected from the group consisting of S-p-methoxybenzyl, S-p~methyl-
-3
3~
ben~yl, S-acetamidomethyl, S-trityl, S-benzyl, and the like.
The preferred pro~ecting group is S-p-methoxybenzyl. Rl and/or
R7 can be hydrogen which means that there is no protecting group
on the sulfur group.
R2 and R3 are each a protecting group or the side chain
amino substituent of lysine or R2 and/or R3 are hydrogen which
means there is no protecting group on the side chain amino sub-
stituent. 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 protec~ing group is not cri~ical ex-
cept that it must be one which is not removed during deprotection
of the ~-amino groups duriny the synthesisO Hence, the ~-amino
protecting and side chain amino protecting group cannot be the
same;
R4, R5, and R5 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,
2,6-dichlorobenzyl and benzyloxycarbonyl. The preferred pro-
tecting group is benzyl. R4 and/or R5 and/or ~6 can be hydrogen
which means there is no protecting group on the hydroxyl group.
Xl,X2,X3 and X4 are as previously defined. X5 is se-
lected from the class consisting of OH, OCH3, 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:
-O-CH2-polystyrene resin support
and
O-CH2-benzyl-polystyrene resin support
The polymer is preferably a copolymer of styrene with about .5 to
2~ divinyl benzene as a cross linking agent which causes the poly-
~tyrene polymer to be completely insoluble in cextain organic
solve~ts. In formula III at least one of R,Rl,R2,R3,R~,R5,R6
and R7 is a protecting group.
In selecting a particular side chain protecting group
to be used in the synthesis of the peptides of formula I or
formula II, the ~ollowing rules should be followed:
(a) the protecting group must be stable to the reagent and
under the reaction conditions selected for removing the ~-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 pep-
tide 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 ~-amino protected resin.
Such a starting material can be prepared by attaching an ~-amino
and S-protected Cys to a chloromethylated resin or a hydroxy-
methyl resin. The preparation of the hydroxymethyl resin is des-
cxibed by Bodansæky et al., Chem. Ind. (London)38, 1597-98 ~1966).
A chloromethylated resin is commercially available from Bio Rad
Laboratoxies, Richmond, California and the preparation of such a
resin is descrihed by Stewart et al., "Solid Phase Peptide Syn-
2S thesis" (Freeman & Co., San Francisco 1969)l Chapter 1/ pp 1-6.
The ~-amino and S-protected Cys is coupled to the chloromethyl-
ated resin according to the procedure of Monahan and Gilon, Bio-
polymer 12, pp 2513-19, 1973. Following the coupling of the a-
amino and S-protected Cys to the resin support/ the ~-amino pro-
3~ tecting 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 aboutOC
--5--
3~3
and room temperature.
Other st~ndard cleaving reagents and conditions for
removal of specific ~-amino protecting groups may be used as
described in Schroder & Lubke, "The Peptides", 1 pp 72~75
(Academic Press 1965).
After removal of the a-amino protecting group of Cys
the remaining ~amino and ~ide 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 separ-
ately to the synthesis, some of them may be coupled prior toaddition to the solid phase reactor. The selection of an appro-
priate coupling reagent is within the skill o~ the art~ Partic-
ularly suitable as a coupling reagent is N,Nl-dicyclohexyl car- -
bodiimide.
The activating reagents used in the solid phase syn-
thesis of the peptides are those well known in the peptide art.
ExampIes of suitable activating reagents are: (1) carbodiimides
such as N,N-diisopropyl carbodiimide, N-ethyl Nl (y-dimethyl-
amino propyl carbodiimide; (2) cyanamides such as N,N-dibenzyl-
cyanamide; (3) keteimines; (4) isoxazolium salts such as N-
ethyl-5-phenyl isoxazolium-31-sul~onate; (5) monocyclic nitrogen
containing heterocyclic amides of aromatic character containing
one through four nitxogens in the ring such as imidazolides,
pyrazolides, 1,2,4-triazolides. Specific heterocyclic amides thatare
25 -- useful include N,~l-carbonyl diimidazole, N,Nl-carbonyl-di-1,2,4-
triazole; (6) alkoxylated acetylene such as ethoxyacetylene;
(7) reagents which form a mixed anhydride with the carboxylmoiety
of the amino acid such as ethylchloroformate and isobutylchloro-
formate and (8~ nitrogen-containing heterocyclic compounds having
a hydroxy group on one ring nitrogen such as N-hydroxyphthalimide~
N~hydroxysuccinimide and l-hydroxybenzotriazole. Other activating
reagents and their use in peptide coupling are described by Schroder
~lU2~13
& Lubke supra, in Chapter III and by Kapoor, J. Pharm. Sci.~ 59,
pp 1-27.(1970~.
Each protected amino acid or amino acid sequence i5
introduced into the solid phase reactor in about a fourfold ex-
cess and the coupling is carried out in a medium of dimethylform-
amide: 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
a-amino protecting group, prior to the coupling of the next
1~ amiino acid. The success of the coupling reaction at each stage
of the synthesis is monitored by the ninhydrin reaction, as des-
cribed 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 sup-
port by treatment with a reagent such as liquid hydrogen fluoridewhich not only,cleaves the peptide from the resin but also cleaves
all remaining side chain protecting groups Rl, R2, R3, R4, R5, ,~,
- R6 and R7 and the ~a~lino protecting group R to obtain directly
a peptide of formula II. Peptides in accordance with formula I
are obtained by oxidizing formula II pep~ides in accordance with
known procedures. As an alternate route, the peptide linked ~o
the resin support may be separated ~rom the resin by alcoholysis
after which the recovered C-tarminal methyl ester is converted to
the acid by hydrolysis. Any side chain protecting group may then
~5 be cleaved as previously described or by other procedures such as
catalytic reduction (e.g. Pd on BaSO4) using conditions which will
keep the Trp moiety intact. When using hydrogen fluoride for
cleavingr anisole is included in the reaction vessel as a sca~enger.
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
--7--
effects in respect to inhibition of xelease of growth hormone,
insulin and glucagon are considered to be particularly important
in connection with the ~reatmen-t of dlabetes. 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 ac-
companied by an excess of glucagon has made it difficult to study
the role of glucagon in diabetes. While it is eas~ to add extra
quantities of a hormone such as insulin, it has proved ver~ dif-
fidu~i 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
- ins~lin and glucagon. The role of somatostatin in diabetes
research is detailed in an article appearing in Science7 Vol. 1~8,
pp 920-923, 30 May 1975. However, there are several problems in
respec~ to the use of somatostatin as a treatment in diabetes.
Somatostatin inhibits the release of insulin in addition to glu-
cagon. Thus, khe 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 pep-
tides of the present invention provide such dissociative effect.
More particularly, certain of the peptides of the present inven-
tion are effective to inhibit secretion of glucagon while having
less effect on the inhibition of secretion of insulin.
The following examples illustrate various features o
khe present invention but are intended to in no wa~ limit the
3~
scope o the invention which is defined in the appended c]aims.
EXAMPLE I
The peptides of the present invention were synthesized
by solid phase techniques, generally in accordance with the pro-
cedure described in United States Patent No. 3,904,594. ~he syn-
thesis w~s conducted in a stepwise manner on chloromethylated
resin. The resin was composed of fine beads (20-73 microns in
diamet~r) 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
tG 2 millimoles of chlorine per gr~n 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 re-
ferred to hy the common abbreviation.
A peptide having the structure:
Ala-Gly-Cys-Lys-des-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH
1 2 3 4 5 6 7 8 9 10 11 12 13 1~
was synthesized by the following solid phase methodology. Other
peptides, described hereinafter w~re synthesized by a similar
technique.
The tertiobutyloxycarbonyl-S-paramethoxybenzyl (Boc-
SpOMe-Bz]) derivative of Cys was linked to the resin by any of the
three known rnethods: (1) reflux in ethanol in presence of triethyl
amine~ (2) Cesium salt of the Boc protected amino acid is kept at
50 C in dimethylformamide (DMF~ overnight, (3) the potassium salt
of the Boc-protected amino acid is kept at 80 C in dimethyl sul-
foxide (DMSO) fox 2 hours. Only one milliequivalent of the pro-
_g_
~ ~3 r ~
~L~v~
tected Cys per milllequivalent o~ 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 tertiobuto~ide (KOtBut) per mEq of
S amino acid. rrhe reaction mixture is exposed to air as little as
possible so that no amber coloration is observed. Reaction at
80 C for 2 hours yields a suitable substituted resin for synthe-
sis of the peptides (approx. .2 meq of amino acid derivative per
g of resin). After deprotection and neutralization, the peptide
chain is built on the resin. Deprotection, neutralization and
addition of each amino acid is performed in accordance with
schedule I. N~-t-butyloxycarbonyl (Boc) derivative of each amino
acid is used with the exception that any ~-amino protecting group
can be used for the alanine 1 residue provided it i5 cleaved by
HF (benzyloxycarbonyl, Z; Boc and others). After deprotection of
the irst residue (i.2., SpOMe.Bzl.Cys) according to scheduIe I
(steps 3 to 8 incluslve) the N~Boc derivative of Ser is next added
along with a coupling agent which is dicyclohexylcarbodiimide
(DCC).(step 9 of schedule I). The sicle chain of Ser is protected
with benzyl ether (OBzl). The O~Benzyl (OBzl) protecting group is
also used for protection of the threonine side chain. P-nitro-
phenyl ester (ONp) was used to activate the carboxyl end of ASn.
O-nitrophenyl ester can also he used for this purpose. Formyl
groups can be used for the protection of the indole N-H. Benæyl~
oxycarbonyl ~Z) or benæyloxycarbonyl~2Cl [Z ~2-CL)] was used as
the protecting group for the Lys side chain.
--10--
3~3
1. Schedule Eor coupling of amino acids other than Asn ~ :
in sol.id phase synthesis (5-10 g resin)
Step Reagents and operationsMix times
Min.
1 CH2C12 wash 80 ml (2 times) 3
5 2 Methanol (MeOH) wash 30 Ml (2 time-s) 3
3 CH2C12 wash 80 ml (3 times) 3
4 50 percent trifluoroacetic acid (TFA) 10
containing 5 percent 1,2-ethanedithiol
i.n CH2C12 70 ml (2 times)
CH Cl wash 80 ml (2 times) 3
106 Triethylamine tEt N) 12.5 percent in
CH2C1~ 70 ml (2 times) 5
7 MeOH wash 40 ml ~2 times) 2
8 CH2C12 wash 80 ml (3 times) 3
9 Boc-amino acid (10 mmoles) in 10 ml
DMF ~1 times) and 30 ml CH2C12 plus .
DCC (10 mmoles) in ::
CH2C12 (2 M) ; 30 to~ 120
. . _
Step Reagents and operationsMix times
Min.
-
MeOH wash 40 ml (2 times) . 3
11 Et3N 12.5 percent in CH2C12 70 ml (2 times) 3
12 MeOH wash 30 ml (2 times) 3
13 CH2C12 wash 80 ml (2 times) 3
After step 13, an aliquot is taken for a ninhydrin test:
i~ the test is negati~e~ go back to,step 1 for coupling
of the next amino acid; if the test is positive or slightly pos- ;
itive, go back to steps 9 through 13. Schedule I was used for
coupling o~ each of the amino acids of the peptide to Cys with
the exception o~ Asn, when present. For peptides o~ the inven-
tion containing Asn~ steps 1 through 8 are the same and
3~3
schedule II is used for the remiander of the coupling reaction:
II Schedule ~or Boc-Asn-ONp or for any active ester coupl-
ing in solid phase syn-thesis (5-10 g resin)
Step R~agents and operations Mix times
Min.
_
9 D~F wash 60 ml t3 times) 3
Boc~sn-ONp (lt mmoles) in 800
20 ml DMF (1 time)
11 MeOh wash 30 ml (4 times) 3
1012 Et3N 12.5 percent in ~MF 3
30 ml (2 times)
13 MeOH wash 30 ml (2 times? 3
1~ CH2C12 wash 80 ml (3 times) 3
After step 14, an aliquot is taken for a ninhydrin test:
i the test is negative go back to step 1 for coupling
of the next amino acid; if the test is positive or slightly posi-
tive, go back to steps 9 th~ough 14.
Cleavage of the peptides from the resin (5 grams) and
daprotection of the side chain protec~ing groups of the peptide
was performed in hydrofluoric ~cid ~75 ml3 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
H2O(N2). The pH of the solution was adjusted to 6.6-7.0 with
NH40H. The solutionwas titrated dropwise under stirring with
potassium ~erricuanide solution (1 g/500 ml H2O) until a perman-
ent 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 Eiltered over celite and applied successivley onto two
12-
3~3
columns; a) siO Rad AG 3-X4A resin chloride form (10 ml);
b) Bio Rex~70 resin (100 ml) catlon form. ~rhe ce]ite + resin
ca]ce was thoroughly washed with water (500 ml) which was applied
onto columns a) and b) as a wash. The peptide material was then
eluted frorn the Bio Rex-70 resin column with pyridine:acetic
acid:water (30:4:66) or 50% acetic acid. Fractions were col-
lected; only the ones containiny peptide (ninhydrin positive)
were diluted with water and immediately lyophilized. 1.2 g of
crude cream colored ma-terial was obtained. It was applied onto
a Sephadex G-25 F gel column (3 x 200 cm) equilibrated and
eluted with 2 N acetic acid.
The elution pattern as observed at 280 nm showed one
major symmetical peak centered at VO (400 mg). It was subs~-
quently submitted to counter current distribution (solvent
system n-butanol:acetic acid:water, 4:1:5) 10 ml lower phase
per tube~ 237 transfers were performed and the major peak was
found in tubes 48-64. The compound (250 mg) appeared homogen-
eous on tlc.
The specific optical rotation was [~]D23=-38.2~1
~c=l in 1% acetic acid). Amino acid analysis of this material
showed the expected ra~io for the different amino acids.
Active esters can he 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 an-
terior pituitary cells by the method of Vale~et al.,
Endocrinology 91: p 562-571 (1972~. The assay is made by treat-
ing pituitary glands removed from rats to separate cells there-
from. The cells are placed in cultUre dishes in a modificatio~
of Dulbecco's Modified Eagle Medium. ~Valeet ~1, Methods in
-13-
Enzymology). Carbon dioxide gas and oxygen are supplied to
the cell cultures which are maintained at 37C for 4-5 days
priot to use in the assay. Following media changes, cell cul-
tures are incubated for a period of 4 hours and partlcular so-
matostatin peptides are added thereto. Radioimmunoassay analy-
sis is used to determine the rate of growth hormone secretion
which is expressed in nanograms per dish per hour.
An investigation of the efEect of somatostatin,
dihydrosomatostatin, ~as controls) and the peptides of the in-
vention to inhibit the release of glucagon and insulin was madeas follows-
In vo Bioassay: Male Sprague-Dawley-CD rates weighing
180-200g 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 Ollt at least 5 days ater
-
arrival of rats from the supplier between the hours 1400 and
1600. After either anesthesia, peptides or saline ~ere adminis-
terea in a volume of 0.2 ml. via the external jugular vein.
Animals remained anesthetized until the time of blood collection
from the portal vien. The blood samples were placed into chilled
tubes containing 10 mg EDTA and 50 ~1 of 2M Benzamidine per ml
of blood.
Plasma was stored at -20C for insulin and glucagon de~
terminations. Insulin levels were determined by the method of
Herbet et al, J. Chem. Endocr. Metab. 25:1375, 1965, utilizing
_
porcine insulin antisera and (125I) iodinated insulin tracer.
~uman insulin standard was obtained from Schwarz-Mann, Orange-
burg, New York. Glucagon was determined by the method of
Faloonaand Unger, in Jaffe et al ed., Method of Hormone Radio-
immunoassay, Academic Press, New York, 1974, p. 317, utilizing
glucagon antisera 30K. Glucose was detexmined by the glucose
-14-
~2~
oxidase method, utilizing a Beckman Glucose Analyzer.
GH determinations were perormed on tissue culture
media utiliæing the following rea~ents: NIAMDD rat GH standard
(G~ RP-l), NIAMDD monkey anti-rat GH (GH-Serum-3), and highly
purified ra-t GH for iodlnation.
All experiments are carried on in a xandomized block
designO Following analysis of variance difference between
treatments were determined by the multiple range tests of
Dunnett and Dunca. 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 descri-
bed above. The composition of the peptides is reported herein-
below in Table I. Table I also sets foxth the ration of effec-
tiveness of the peptide for inhibiting secretion of growth hor-
- mone (GH), insulin and glucagon, with inhibition of glucagon
taken as the base. Also reported in Table I is the percent po-
tency of the peptide in respect to growth hormone inhibition
with somatostatin considered as being 100 percent efective.
TABLE I
% Potency
Ratio of EffectivenessGrowth
Somatostatin ~I : Insulin : ~71ucagon Hormone
(control) 1 : 1 : 1 Base 100
~ _. .
25 des~sn-[D-~rp8]-SS 12 : 60 : 1 12
[D-Serl3]~SS 10 : 10 . : 1 10
[D-l~p8]-[D-Serl3]-SS 18 : 261 r
[D-C~Sl4]-SS 2.7 ~ 1 270
[D-Trp~]~SS-[D-Cysl~]-SS .7 : .13 : 1 ' 650
30 [~{~s ]-[D-Serl3]-SS : 7 : <1
-15-
