Dodecapeptides preventing glucose and triglyceride assimilation

Polypeptides of the formula: ##STR1## where X.sub.1 is either D- or L- Lys, Arg, His or Orn; PA0 X.sub.2 is either D- or L- Asn, His, Glu, Asp, Tyr, Trp or Phe; PA0 X.sub.3 is either D- or L- Trp; the linear precursor intermediates thereof and pharmaceutically acceptable salts and amides thereof are inhibitors of growth hormone release, active for periods of two hour and more. In addition, the compounds of this invention normalize post-prandial glucose and triglyceride levels in diabetics. The compounds are useful for treatment of patients suffering from diabetes mellitus and excessive growth hormone secretion (acromegaly).

SUMMARY OF THE INVENTION 
Dodecapeptides of the formula: 
##STR2## 
where 
X.sub.1 is either D- or L- Lys, Arg, His or Orn; 
X.sub.2 is either D- or L- Asn, His, Glu, Asp, Tyr or Trp; 
X.sub.3 is either D- or L- Trp; 
the linear precursor intermediates thereof and pharmaceutically acceptable 
salts and amides thereof are inhibitors of growth hormone release, active 
for periods of two hours and more. In addition, the polypeptides of this 
invention normalize post-prandial glucose and triglyceride levels in 
diabeties. The compounds are therefore useful in treatment of patients 
suffering from diabetes mellitus and excessive growth hormone secretion 
(acromegaly). 
DETAILED DESCRIPTION OF THE INVENTION 
In accordance with this invention there is provided a group of 
dodecapeptides of the formula: 
##STR3## 
where 
X.sub.1 is either D- or L- Lys, Arg, His or Orn; 
X.sub.2 is either D- or L- Asn, His, Glu, Asp, Tyr, Trp or Phe; 
X.sub.3 is either D or L-Trp; 
the linear precursor intermediates thereof and pharmaceutically acceptable 
salts and amides thereof. These polypeptides reduce growth hormone release 
for periods up to and in excess of two hours post dosing at the very 
unusual level of 98 percent reduction. In addition, these polypeptides 
normalize post-prandial glucose and triglyceride levels in patients 
suffering from diabetes mellitus. 
The pharmaceutically acceptable salts of the compounds of this invention 
are those non-toxic addition salts produced by known methods from acids 
conventionally employed with pharmaceuticals such as hydrochloric, 
hydrobromic, sulfuric, phosphoric, polyphosphoric, maleic, acetic, citric, 
benzoic, succinic, malonic or ascorbic acid and the like. Similarly the C- 
terminal Glu and Asp carboxylic acid salts of alkali metals and ammonia 
are produced by careful neutralization of the acid. By amides of the 
compounds disclosed herein, the applicant intends to embrace alkyl amides 
containing from 1 to 4 carbon atoms, which amides are produced 
conventionally. 
The linear precursor intermediates of the cyclic dodecapeptides of this 
invention may be depicted as follows: 
##STR4## 
in which 
X.sub.1, X.sub.2 and X.sub.3 are defined above and 
R is hydrogen or an alpha amino protecting group; 
R.sub.1 is hyrogen or an amino protecting group; 
R.sub.2 is hydrogen or an imino protecting group for D- or or L- His or a 
phenolic hydroxyl protecting group for D- or L- Tyr; 
R.sub.3 is hydrogen or an amino protecting group; 
R.sub.4, R.sub.5 and R.sub.6 are hydrogen or hydroxy protecting groups; 
R.sub.7 is --NHR.sub.10, in which R.sub.10 is alkyl of 2 to 4 carbon atoms, 
or --OR.sub.11, in which R.sub.11 is hydrogen or alkyl of 1 to 6 carbon 
atoms or --OCH.sub.2 (resin support); 
R.sub.8 and R.sub.9 are hydrogen or sulfhydryl protecting groups. 
These intermediates comprise the fully protected and partially protected 
dodecapeptides bound to a resin support employed in solid phase synthesis 
of the polypeptide as well as the fully deprotected linear polypeptide 
removed from the resin support. 
The protecting groups employed during preparation of the linear 
intermediates are conventional in solid phase polypeptide synthesis. Thus, 
in the above formula, the protecting group embraced in the definition of R 
may be formyl, trifluoroacetyl, phthalyl, p-chlorobenzyloxycarbonyl, 
p-nitrobenzyloxycarbonyl, tert-butyloxycarbonyl (BOC), 
2,2,2-trichloroethoxycarbonyl, amyloxycarbonyl, cyclopentyloxycarbonyl, 
cyclohexyloxycarbonyl, trityl, etc., the preferred group being 
tert-butyloxycarbonyl. 
Examples of the sulfhydryl protecting groups R.sub.8 and R.sub.9 and the 
hydroxyl protecting groups R.sub.4-6 of tyrosyl, seryl or threonyl are 
benzyl, p-methoxybenzyl, p-chlorobenzyl, p-nitrobenzyl, p-methyl-benzyl, 
trityl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl and the like. The 
p-methoxybenzyl group is preferred for protection of cysteinyl sulfur 
while the benzyl group is preferred for the tyrosyl, seryl and threonyl 
moieties. 
Protecting groups for the nitrogen atom of lysine and ornithine include 
tosyl, benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, and 
tert-butyloxycarbonyl, preferably the 2-chlorobenzyloxycarbonyl group. 
Protecting groups for arginine and the imino group of histidine include 
nitro, tosyl, benzyloxycarbonyl, adamantyloxycarbonyl and 
tert-butyloxycarbonyl. Where the protecting group is nitro or tosyl, the 
protection is on either one of the N.omega., N.omega.' nitrogens and in 
the case of benzyloxycarbonyl, or adamantyloxycarbonyl, the protection is 
on the N.delta. nitrogen and either one of the N.omega., N.omega.' 
nitrogen atoms of arginine. The preferred protecting group is tosyl. 
The criterion for selecting protecting groups for R-R.sub.9 are (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 (i.e. not be split off under coupling conditions), 
and (c) the protecting group must be readily removable upon conclusion of 
the polypeptide synthesis, under conditions that do not otherwise effect 
the polypeptide structure. 
The dodecapeptides of this invention may be prepared by either solid phase 
or liquid phase methodology, well known to the art. The support employed 
in the solid phase synthesis of the compounds of this invention is a 
chloromethylated or hydroxymethylated polystyrene resin cross-linked with 
divinylbenzene. Such resin supports are prepared by known methods and are 
commercially available. 
The following examples illustrate the preparation of 
H-Phe-Arg-Glu-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-Ser-Phe (4,9-disulfide) which 
is representative in its solid phase preparation and biological activity, 
of the other compounds of the invention generically described, supra.

EXAMPLE 1 
N-tert-Butyloxycarbonyl-L-phenylalanyl-N.sup.g 
-tosyl-L-arginylbenzylL-glutamyl-S-p-methoxybenzyl-L-cysteinyl-L-phenylala 
nyl-D-tryptophyl-N-2-chloro-benzyloxycarbonyl-L-lysyl-O-benzyl-L-threonyl-S 
-pmethoxybenzyl-L-cysteinyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-L-phenylal 
anylhydroxymethyl polystyrene ester 
Chloromethylated polystyrene resin (Lab Systems Inc.) 1% cross-linked with 
divinylbenzene was esterified with BOC-L-Phe-OH according to Gisin Helv. 
Chim. Acta, 56, 1476 (1973). The polystyrene resin ester was placed into a 
reaction vessel of an Automatic Peptide Synthesizer Beckman and treated 
according to Program No. 1 and 2 for the incorporation of BOC-Ser(BZL)OH, 
BOC-Thr(BZL)OH, BOC-Cys(SMBZL)OH, BOC-Thr(BZL)OH, BOC-Lys(CLZ)OH, 
BOC-D-Trp-OH, BOC-Phe-OH, BOC-Cys(SMBZL)OH, BOC-Glu(OBZL)OH, 
BOC-Arg(TOS)OH and finally BOC-Phe-OH to afford the title peptide resin. 
PROGRAM NO. 1 
Peptide Synthesizer-Beckman 990 
1. Wash with CH.sub.2 Cl.sub.2 X 3. 
2. Treat with TFA-CH.sub.2 Cl.sub.2 -EDT, 1:1.5% for 5 minutes. 
3. Repeat (2) for 25 minutes. 
4. Wash with CH.sub.2 Cl.sub.2 X 4. 
5. Treat with TEA 12% in DMF for 1 minute. 
6. Repeat (5) for 5 minutes. 
7. Wash with CH.sub.2 Cl.sub.2 X 3. 
8. Add 4 equivalents of BOC-protected amino acid and stir for 5 minutes. 
9. Add 2 equivalents of 1M-DIC solution in DMF and stir for 25 minutes. 
10. Add 2 equivalents of 1M-DIC solution in DMF and stir for 180 minutes. 
11. Wash with CH.sub.2 Cl.sub.2 X 3. 
12. Wash with methanol X 3. 
13. Wash with CH.sub.2 Cl X 3. 
PROGRAM NO. 2 
Peptide Synthesizer, Beckman 990 
1. Wash with CH.sub.2 Cl.sub.2 X 3. 
2. Add 2 equivalents of BOC-protected amino acid and stir for 5 minutes. 
3. Add 2 equivalents of 1M-DIC solution in DMF and stir for 180 minutes. 
4. Wash with DMF X 3. 
5. Wash with CH.sub.2 Cl.sub.2 X 3. 
6. Wash with methanol X 3. 
7. Wash with CH.sub.2 Cl.sub.2 X 3. 
EXAMPLE 2 
L-Phenylalanyl-L-arginyl-L-glutamyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl 
-L-lysyl-L-threonyl-L-cysteinyl-L-threonyl-L-seryl-L-phenyl-alanine cyclic 
(4-9) disulfide 
The peptidoresin of the previous example (12 g) was mixed with anisole (20 
ml) and treated with liquid HF, in the absence of air, in an ice bath for 
60 minutes. The excess HF was removed under vacuo as fast as possible and 
the residue was taken in 50% aq. acetic acid (100 ml). The filtrate was 
poured into 6 liters of deaerated water and the pH was brought to 6.8 with 
NH.sub.4 OH. A solution of K.sub.3 Fe(CN).sub.6 (3 g in 500 ml H.sub.2 O) 
was added dropwise over a period of 10 minutes until the yellow color 
persisted for 15 minutes and the solution was brought to pH5 with gl. 
acetic acid. Bio-Rad AG3-X4A (Cl.sup.- forms) was added and then 
filtered. The filtrate was passed through Bio-Rex 70 (H.sup.+ form) and 
the absorbed peptidic material was eluted with a mixture of 
water-pyridine-acetic acid, 66-30-4, v/v, then lyophilized to yield 2 g of 
crude material. 
The above material was chromatographed through a column (2.5.times.150 cm) 
of Sephadex G25 and eluted with 50% a.q. acetic acid. Fractions (5 ml 
each) No. 141-165 were pooled and lyophilized to yield 900 mg of the title 
compound as the acetate salt. 
TLC: Avicel precoated glass plates R.sub.f (n-BuOH-H.sub.2 O-AcOH, 4:5:1, 
v/v) 0.78, Silica gel 60, 254F, precoated glass plates; R.sub.f 
(n-BuOH-Pyridine-AcOH-H.sub.2 O, 15:10:3:6, v/v) 0.50; R.sub.f 
(EtOAc-Pyridine-AcOH-H.sub.2 O, 5:5:1:3, v/v) 0.80. 
HPLC: .mu.Bondapak C.sub.18 -column (4 mm.times.30 cm) 71%, 72.5% 
0.1M-NH.sub.4 OAc solution pH4, 27.5% CH.sub.3 CN. 
Aminoacid Analysis: Thr(2) 2.05, Ser(1) 1.09, Glu(1) 1, Cys(2) 1.55, Phe(3) 
3, Lys(1) 1.02, Trp(1) 0.55, Arg (1) 0.84. 
The product of the preceding examples illustrate the activity of the 
compounds of this invention for growth hormone suppression in the 
following standard procedure: 
Albino male rats are administered Nembutal intraperitoneally at a dose of 
50 milligrams per kilogram. Fifteen minutes later a subcutaneous injection 
of the compound of Example 2 or physiological saline (control) is 
administered. 
Ten minutes later 0.5 milliliters of arginine (300 milligrams per 
milliliter, pH 7.2) is injected into the heart. Five minutes after receipt 
of the arginine the rats are decapitated and blood is collected into 
trasylol-EDTA. An appropriate aliquot is assayed for growth hormone (GH) 
by radioimmunoassay. The results of the assay are as follows: 
______________________________________ 
Dose Time GH 
Compound .mu.g/kg minutes ng/ml 
______________________________________ 
Saline -- 30 150 .+-. 43 
Example 2 1 30 121 .+-. 28 
Example 2 5 30 62 .+-. 15 
Example 2 20 30 12 .+-. 6 
Example 2 100 30 1 .+-. 0.2 
______________________________________ 
The duration of activity of the product of Example 2 was as follows: 
______________________________________ 
Dose Time GH 
Compound .mu.g/ml minutes ng/ml 
______________________________________ 
Saline -- 60 161 .+-. 37 
Example 2 1000 60 3 .+-. 1 
Saline -- 120 129 .+-. 23 
Example 2 1000 120 3 .+-. 1 
______________________________________ 
The product of Example 2 has been studied in two diabetic dogs in a meal 
study. The dogs were fasted for 18 hours and then received a standard 
meal, a normal dose of insulin and either saline or the product of Example 
2. Blood samples were obtained for the next eight hours. The results, 
demonstrate that the product of Example 2 normalizes post-prandial glucose 
and triglyceride levels. 
______________________________________ 
+ Example 2 + Saline 
Time (hrs) 
Dog 731 Dog 765 Dog 731 
Dog 765 
______________________________________ 
Plasma Glucose, % of zero time.sup.a 
0 100 100 100 100 
1/2 105 110 134 100 
1 56 94 185 116 
2 41 75 178 137 
3 39 43 149 137 
4 62 29 137 131 
5 85 32 133 121 
6 118 44 118 138 
7 109 48 127 128 
8 116 51 123 127 
Plasma Triglycerides, mg/dl 
0 37 48 77 41 
1/2 31 34 32 35 
1 29 25 137 69 
2 23 28 166 173 
3 31 19 87 150 
4 46 21 72 49 
5 74 21 57 63 
6 113 43 54 47 
7 71 30 43 62 
8 32 32 65 55 
______________________________________ 
.sup.a Zero time glucose values: dogs received compound of Example 2, Dog 
731: 87 mg/dl, Dog 765: 180 mg/dl; dogs receiving saline, Dog 731: 260 
mg/dl, Dog 765: 208 mg/dl 
From this work it is clear that the product of Example 2 retards absorption 
of the meal as reflected in the reduced triglyceride absorption at and 
around two hours. The increase peaking at 6 hours in dog 731 indicates 
that the meal is now crossing the gut in that animal. It is apparent, 
however, that glucose and triglycerides did not cross the gut in the 
initial post-prandial stage in such quantities as to overwhelm the amount 
of insulin present. 
As with administration of any therapeutic agent used in the treatment of 
diabetes mellitus, the compounds of this invention must be individualized 
for the patient under guidance and close control of the attending 
physician to reach optimum blood levels of growth hormone, insulin and 
glucagon. Doses for achieving the desired state vary with the condition of 
the patient, such as age, amount of endogenous insulin produced, the 
presence of glucagon secreting tumors, the route of administration, the 
duration of treatment, severity of the condition being treated etc. 
Thus, the compounds of this invention may be administered alone or in 
combination with insulin with or without carriers or excipients 
conventional to the route of administration selected, which may be oral, 
intravenous, subcutaneous, intramuscular, intranasal, intrarectally, etc. 
Suitable pharmaceutical composition for application are apparent to those 
skilled in the art.