NPY peptide analogs

This invention is generally directed to the field of Neuropeptide Y(NPY) 
and to methods for pharmaceutical treatment of mammals using analogs of 
such a peptide. More specifically, the invention relates to NPY analogs, 
to pharmaceutical compositions containing such peptide analogs and to 
methods of treatment of mammals using such peptide analogs to lower blood 
pressure. 
BACKGROUND OF THE INVENTION 
Experimental and clinical observations have supported the concept that 
neuropeptides play central roles in neurotransmission as well as the 
regulation of secretory functions of adenohypophysial, pancreatic, 
adrenalcortical and gut cells. Among the thirty or so neuropeptides that 
have been implicated in neuronal function in the mammalian central nervous 
system, several have also been suggested to function as neurotransmitters 
or neuromodulators primarily in afferent neurons. 
NPY is a 36-residue, amidated peptide hormone which was first isolated from 
porcine brain and characterized. NPY is anatomically co-distributed and 
co-released with norepinephrine in and from sympathetic postganglionic 
neurons. Stimulation of the sympathetic nervous system under physiologic 
circumstances, e.g. exercise, cold exposure, surgical stress, etc., 
promotes an elevation of plasma concentrations of NPY. NPY is believed to 
participate along with norepinephrine in the regulation of vascular smooth 
muscle tone and maintenance of blood pressure. In addition to the 
post-synaptic action to increase vascular smooth muscle tone, NPY may also 
act presynaptically to inhibit both its own release and that of 
norepinephrine. This mechanism is similar to the presynaptic actions of 
norepinephrine that, acting through certain receptors, facilitates a local 
feedback regulation of the neurohumoral regulation of blood pressure. Over 
the past 30 years, knowledge of norepinephrine's role in regulation of 
blood pressure has resulted in increasing our understanding of 
cardiovascular regulation and the successful development of a variety of 
pharmacotherapeutic substances used clinically to treat disorders of 
cardiovascular function. These compounds are, in general, structural 
analogs of norepinephrine and serve as either agonists or antagonists of 
norepinephrine to treat hypotension or hypertension, respectively. These 
drugs, however useful, have not solved the problems of management of a 
variety of cardiovascular disorders. 
An additional action of NPY is to decrease cardiac contractility 
(inotropy). This is an extremely important action of NPY, because it is 
known that, under many circumstances in which inotropy is decreased, 
diseases of life-threatening importance, e.g. congestive heart failure and 
cardiogenic shock, are associated with probable increased release of NPY 
into the blood. Prevention of NPY release, using a presynaptic NPY 
agonist, or NPY's action, using a postsynaptic NPY antagonist, may be 
beneficial in these disease states. 
NPY has also been reported to produce coronary artery vasoconstriction and 
thereby may decrease myocardial blood flow resulting in myocardial 
ischemia. Such a circumstance can result in angina pectoris or, under more 
severe circumstances, may result in myocardial infarction and death. In 
recent years, several classes of drugs have proven effective in dilating 
coronary arteries to prevent such events. The use of analogs of NPY are 
expected to prove useful in treatment of such problems. 
Porcine NPY has the formula (SEQ ID NO:1): 
Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Ar 
g-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg -Tyr, 
wherein the C-terminus is amidated. The formula of Human NPY has been 
deduced from clones obtained from total RNA by preparing cDNA and then 
employing DNA sequencing technologies and is accepted to be (SEQ ID NO:2): 
Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-Ar 
g-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg -Tyr, 
wherein the C-terminus is amidated. 
Peptide YY (porcine) (PYY) has the formula (SEQ ID NO:3): 
Tyr-Pro-Ala-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Ser-Ar 
g-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val -Thr-Arg-Gln-Arg-Tyr, 
wherein the C-terminus is amidated. 
SUMMARY OF THE INVENTION 
NPY has a wide range of biological actions including cardiovascular effects 
such as increasing mean arterial pressure (MAP) when injected into 
conscious rats. Administration of NPY analogs which are shortened at the 
N-terminus and which may have a D-isomer substitution at at least one 
position in the chain substantially decrease the MAP. The following 
peptides, hereinafter termed NPY analogs, are preferred, which peptides 
have one of the following formulae: 
(a) H-Ala-Arg-Tyr-Xaa.sub.21 -Xaa.sub.22 -Ala-Leu-Arg-His-Xaa.sub.27 
-Ile-Xaa.sub.29 -Xaa30-Xaa.sub.31 -Xaa.sub.32 -Arg-Xaa.sub.34 -Xaa.sub.35 
-Xaa.sub.36 -NH.sub.2 wherein Xaa.sub.21 is Tyr or D-Tyr; Xaa.sub.22 is 
Ser or D-Ser; Xaa.sub.27 is Tyr or D-Tyr; Xaa.sub.29 is Asn or D-Asn; 
Xaa.sub.30 is Leu or D-Leu; Xaa.sub.31 is Ile or D-Ile; Xaa.sub.32 is Thr 
or D-Thr; Xaa.sub.34 is Gln or D-Gln; Xaa.sub.35 is Arg or D-Arg; and 
Xaa.sub.36 is Tyr or D-Tyr; and wherein at least one D-isomer is present; 
and 
(b) H-Ser-Arg-Tyr-Xaa.sub.21 -Xaa.sub.22 -Ser-Leu-Arg-His-Xaa.sub.27 
-Leu-Xaa.sub.29 -Xaa.sub.30 -Xaa.sub.31 -Xaa.sub.32 -Arg-Xaa.sub.34 
-Xaa.sub.35 -Xaa.sub.36 -NH.sub.2, wherein Xaa.sub.21 is Tyr or D-Tyr; 
Xaa.sub.22 is Ala or D-Ala; Xaa.sub.27 is Tyr or D-Tyr; Xaa.sub.29 is Asn 
or D-Asn; Xaa.sub.30 is Leu or D-Leu; Xaa.sub.31 is Val or D-Val; 
Xaa.sub.32 is Thr or D-Thr; Xaa.sub.34 is Gln or D-Gln; Xaa.sub.35 is Arg 
or D-Arg; and Xaa.sub.36 is Tyr or D-Tyr; and wherein at least one 
D-isomer is present. 
Such analogs of NPY, which preferably include one or two D-isomers, have 
the following applications: potent post-synaptic treatment of hypertension 
and cardiogenic shock, the treatment of acute cardiovascular circulatory 
failure, and the elevation of intracellular calcium. 
These synthetic peptide NPY analogs substantially lower blood pressure for 
an extended time period. Pharmaceutical compositions in accordance with 
the invention include NPY analogs, or nontoxic addition salts thereof, 
dispersed in a pharmaceutically acceptable liquid or solid carrier. Such 
peptides or pharmaceutically acceptable addition salts thereof may be 
administered to mammals in accordance with the invention for lowering 
blood pressure or other regulation as described above. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The nomenclature used to define the peptides is that specified by Schroder 
& Lubke, "The Peptides", Academic Press (1965) wherein, in accordance with 
conventional representation, the N-terminus appears to the left and the 
C-terminus to the right. Where the amino acid residue has isomeric forms, 
it is the L-form of the amino acid that is represented herein unless 
otherwise expressly indicated. By Nva is meant norvaline, and by Nle is 
meant norleucine. 
Very broadly, NPY analogs are provided having the following formula (SEQ ID 
NO:14), with the Xaa groups being defined using subscripts beginning with 
Xaa.sub.19 from left to right by position: 
Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Arg-Xaa-Arg-Xaa 
wherein X-Q is present at the N-terminus; X is H or C.sup..alpha. Me or 
N.sup..alpha. Me or desamino or an acyl group having 7 carbon atoms or 
less; Q is Xaa.sub.17 -Xaa.sub.18, Xaa.sub.18 or desQ; Xaa.sub.17 is Met, 
Arg, Nle, Nva, Leu, Ala or D-Ala; Xaa.sub.18 is Ala, Ser, Ile, D-Ala, 
D-Ser or D-Ile; Xaa.sub.19 is Arg, Lys, Gln or des Xaa.sub.19 ; Xaa.sub.20 
is Tyr, Phe or des Xaa.sub.20 ; Xaa.sub.21 is Tyr, Glu, His, Ala or des 
Xaa.sub.21 ; Xaa.sub.22 is Ser, Ala, Thr, Asn or Asp; Xaa.sub.23 is Ala, 
Asp, Glu, Gln, Asn or Ser; Xaa.sub.25 is Arg or Gln; Xaa.sub.26 is His, 
Arg or Gln; Xaa.sub.27 is Phe or Tyr; Xaa.sub.28 is Ile, Leu, Val or Arg; 
Xaa.sub.29 is Asn or Ile; Xaa.sub.30 is Leu, Met, Thr or Val; Xaa.sub.31 
is Ile, Val or Leu; Xaa.sub.32 is Thr or Phe; Xaa.sub.34 is Gln, Pro or 
His; Xaa.sub.36 is Phe or Tyr; and the C-terminus may be amidated; 
provided however that one or more of the residues in positions 21-23 
and/or positions 26-36 is preferably substituted by the D-isomer thereof. 
When X is not H, C.sup..alpha. Me N.sup..alpha. Me , or desamino, X is 
preferably acetyl (Ac) , acrylyl (Acr) , formyl (For) or benzoyl (Bz); 
however, X is most preferably H. 
Certain preferred NPY analogs have the formula: H-Xaa.sub.18 
-Arg-Tyr-Tyr-Xaa.sub.22 -Xaa.sub.23 -Leu-Arg-His-Xaa.sub.27 -Xaa.sub.28 
-Asn-Leu-Xaa.sub.31 -Thr-Arg-Gln-Arg-Xaa.sub.36 -NH.sub.2, wherein 
Xaa.sub.18 is Ala or Ser; Xaa.sub.22 is Ser or Ala; Xaa.sub.23 is Ala or 
Ser; Xaa.sub.27 is Phe or Tyr; Xaa.sub.28 is Ile or Leu; Xaa.sub.31 is Ile 
or Val; and Xaa.sub.36 is Phe or Tyr; provided that at least one of the 
residues in positions 21-23 and 26-36 is a D-isomer of the residue 
indicated. 
Other preferred NPY analogs have the formula: H-Xaa.sub.17 -Xaa.sub.18 
-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile 
-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH.sub.2, wherein Xaa.sub.17 is Arg, Leu 
or des Xaa.sub.17 ; Xaa.sub.18 is Ser or Ala or Ile; and wherein at least 
one of the residues in positions 21-23 and 26-36 is a D-isomer of the 
residue indicated. 
Still other preferred NPY analogs have the formula: H-A1 
a-Arg-Tyr-Xaa.sub.21 -Xaa.sub.22 -Ala-Leu-Arg-His-Xaa.sub.27 
-Ile-Xaa.sub.29 -Xaa.sub.30 -Xaa.sub.31 -Xaa.sub.32 -Arg-Xaa.sub.34 
-Xaa.sub.35 -Xaa.sub.36 -NH.sub.2, wherein Xaa.sub.21 is Tyr or D-Tyr, 
Xaa.sub.22 is Ser or D-Ser, Xaa.sub.27 is Tyr or D-Tyr, Xaa.sub.29 is Asn 
or D-Asn, Xaa.sub.30 is Leu or D-Leu, Xaa.sub.31 is Ile or D-Ile, 
Xaa.sub.31 is Thr or D-Thr, Xaa.sub.34 is Gln or D-Gln, Xaa.sub.35 is Arg 
or D-Arg, and Xaa.sub.36 is Tyr or D-Tyr, wherein at least one D-isomer is 
present. 
Another preferred NPY analog group has the formula: 
H-Ser-Arg-Tyr-Xaa.sub.21 -Haa.sub.22 -Ser-Leu-Arg-His-Xaa.sub.27 
-Leu-Xaa.sub.29 -Xaa.sub.30 -Xaa.sub.31 -Xaa.sub.32 -Arg-Xaa.sub.34 
-Xaa.sub.35 -Xaa.sub.36 -NH.sub.2, where in Xaa.sub.21 is Tyr or D-Tyr, 
Xaa.sub.22 is Ala or D-Ala, Xaa.sub.27 is Tyr or D-Tyr, Xaa.sub.29 is Asn 
or D-Asn, Xaa.sub.30 is Leu or D-Leu, Xaa.sub.31 is Val or D-Val, 
Xaa.sub.32 is Thr or D-Thr, Xaa.sub.34 is Gln or D-Gln, Xaa.sub.35 is Arg 
or D-Arg, and Xaa.sub.36 is Tyr or D-Tyr; and N-terminally shortened 
fragments thereof, wherein either 1 or 2 D-isomers are present. 
Still other preferred NPY analogs have the formula: H-Xaa.sub.18 
-Arg-Tyr-Xaa.sub.21 -Xaa.sub.22 -Xaa.sub.23 -Leu-Arg-His-Xaa.sub.27 
-Xaa.sub.28 -Asn-Leu-Xaa.sub.31 -Thr-Arg-Gln-Xaa.sub.35 -Tyr-NH.sub.2, 
wherein Xaa.sub.18 is Ala or Ser; Xaa.sub.21 is Tyr or D-Tyr, Xaa.sub.22 
is Ser or Ala, Xaa.sub.23 is Ala or Ser, Xaa.sub.27 is Tyr or D-Tyr, Xaa28 
is Ile or Leu, Xaa.sub.31 is Ile or Val, and Xaa.sub.35 is Arg or D-Arg; 
wherein one D-isomer is present. 
There is also provided a method for lowering the blood pressure of a 
mammal, which method comprises administering an effective amount of a 
synthetic peptide, or a nontoxic salt thereof, having the formula: 
H-Ala-Arg-Tyr-Xaa.sub.21 -Xaa.sub.22 -Ala-Leu-Arg-His-Xaa.sub.27 
-Ile-Xaa.sub.29 -Xaa.sub.30 -Xaa.sub.31 - Xaa.sub.32 -Arg-Xaa.sub.34 
-Xaa.sub.35 -Xaa.sub.36 -NH.sub.2, wherein Xaa.sub.21 is Tyr or D-Tyr, 
Xaa.sub.22 is Ser or D-Ser, Xaa.sub.27 is Tyr or D-Tyr, Xaa.sub.29 is Asn 
or D-Asn, Xaa.sub.30 is Leu or D-Leu, Xaa.sub.31 is Ile or D-Ile, 
Xaa.sub.32 is Thr or D-Thr, Xaa.sub.34 is Gln or D-Gln, Xaa.sub.35 is Arg 
or D-Arg, and Xaa.sub.36 is Tyr or D-Tyr; provided that at least one 
D-isomer is present. 
In addition, there is also provided a method for lowering the blood 
pressure of a mammal, which method comprises administering an effective 
amount of a synthetic peptide, or a nontoxic salt thereof, having the 
formula: H-Xaa.sub.18 -Arg-Tyr-Xaa.sub.21 -Xaa.sub.22 -Xaa.sub.23 
-Leu-Arg-His-Xaa.sub.27 -Xaa.sub.28 -Xaa.sub.29 -Xaa.sub.30 -Xaa.sub.31 
-Xaa.sub.32 -Arg-Xaa.sub.34 -Xaa.sub.35 -Xaa.sub.36 -NH.sub.2, wherein 
Xaa.sub.18 is Ala or Ser; Xaa.sub.21 is Tyr or D-Tyr; Xaa.sub.22 is Ser, 
D-Ser, Ala or D-Ala; Xaa.sub.23 is Ala or Ser; Xaa.sub.27 is Tyr or D-Tyr; 
Xaa.sub.28 is Ile or Leu; Xaa.sub.29 is Asn or D-Asn; Xaa.sub.30 is Leu or 
D-Leu; Xaa.sub.31 is Ile, D-Ile, Val or D-Val; Xaa.sub.32 is Thr or D-Thr; 
Xaa.sub.34 is Gln or D-Gln; Xaa.sub.35 is Arg or D-Arg; and Xaa.sub.36 is 
Tyr or D-Tyr; provided that at least one D-isomer is present. 
The peptides can be synthesized by any suitable method, such as by 
exclusively solid-phase techniques, by partial solid-phase techniques, by 
fragment condensation or by classical solution addition. Synthetic NPY 
analogs may also be entirely or partially synthesized by recently 
developed recombinant DNA techniques, which may likely be used for 
large-scale production. 
For example, the techniques of exclusively solid-phase synthesis are set 
forth in the textbook "Solid-Phase Peptide Synthesis" Stewart & Young, 
Freeman & Co., San Francisco, 1969, and are exemplified by the disclosure 
of U.S. Pat. No. 4,105,603, issued Aug. 8, 1978 to Vale et al. The 
fragment condensation method of synthesis is exemplified in U.S. Pat. No. 
3,972,859 (Aug. 3, 1976). Other available syntheses are exemplified by 
U.S. Pat. No. 3,842,067 (Oct. 15, 1974) and U.S. Pat. No. 3,862,925 (Jan. 
28, 1975). 
Synthesis by the use of recombinant DNA techniques, for purposes of this 
application, should be understood to include the suitable employment of a 
structural gene coding for all or an appropriate section of the NPY analog 
to transform a microorganism, using an expression vector including a 
promoter and operator together with such structural gene, and causing such 
transformed microorganism to express the peptide or such a synthetic 
peptide fragment. A non-human animal may also be used to produce the 
peptide by gene-farming using such a structural gene in the microinjection 
of embryos as described in U.S. Pat. No. 4,870,009 issued Sep. 26, 1989. 
Common to coupling-type chemical syntheses of peptides is the protection of 
the labile side chain groups of the various amino acid moieties with 
suitable protecting groups which prevent a chemical reaction from 
occurring at that site until the group is ultimately removed. Usually also 
common is the protection of an alpha-amino group on an amino acid or a 
fragment while that entity reacts at the carboxyl group, followed by the 
selective removal of the alpha-amino protecting group to allow subsequent 
reaction to take place at that location. Accordingly, it is common that, 
as a step in the synthesis, an intermediate compound is produced which 
includes each of the amino acid residues located in its desired sequence 
in the peptide chain with various of these residues having side-chain 
protecting groups. 
In preparing the peptides of the present invention, intermediates may be 
created such as those having the formula (II): X.sub.1 -Xaa.sub.17 
(X.sup.7)-Xaa.sub.18 (X.sup.3)-Xaa.sub.19 (X.sup.4 or X.sup.6 or 
X.sup.7)-Xaa.sub.20 (X.sup.2)-Xaa.sub.21 (X.sup.2 or X.sup.5 or 
Xs)-Xaa.sub.22 (X.sup.3 or X.sup.5 or X.sub.6)-Xaa.sub.23 (X.sup.3 or 
X.sup.5 or X.sup.6)-Leu-Xaa.sub.25 (X.sup.6 or X7)-Xaa.sub.26 (X.sup.6 or 
X.sup.7 or X.sup.8) -Xaa.sub.27 ( X.sup.2) -Xaa.sub.28 
(X.sup.7)-Xaa.sub.29 (X.sup.6)-Xaa.sub.30 (X.sup.3)-Xaa-.sub.31 
-Xaa.sub.32 (X.sup.3)-Arg (X.sup.7)-Xaa.sub.34 (X.sup.6 or 
X.sup.8)-Arg(X.sup.7)-Xaa.sub.36 (X.sub.2)-X.sup.9 wherein the R-groups 
are as hereinbefore defined. 
X.sup.1 is either hydrogen or an alpha-amino protecting group and when X in 
the desired peptide is a particular acyl group, that group can be used as 
the protecting group. The alpha-amino protecting groups contemplated by 
X.sup.1 are those known to be useful in the art in the step-wise synthesis 
of polypeptides. Among the classes of alpha-amino protecting groups which 
may be urethane X used as X.sup.1 are: (1) aromatic urethane-type 
protecting groups, such as fluorenylmethyloxycarbonyl (FMOC), 
benzyloxycarbonyl(Z) and substituted Z, such as p-chlorobenzyloxycarbonyl, 
p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 
p-methoxybenzyloxycarbonyl; (2) aliphatic urethan protecting groups, such 
as t-butyloxycarbonyl(BOC), diisopropylmethoxycarbonyl, 
isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; and (3) cycloalkyl 
urethan-type protecting groups, such as and cyclopentyloxycarbonyl, 
adamantyloxycarbonyl,and cyclohexyloxycarbonyl. The preferred alpha-amino 
protecting group is BOC. 
X.sup.2 is a protecting group for the phenolic hydroxyl group of Tyr, 
selected from the group consisting of tetrahydropyranyl, tert-butyl, 
trityl, benzyl, Z, 4-bromobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl 
(2BrZ) and 2,6-dichlorobenzyl(DCB). 2BrZ is preferred. 
X.sup.3 is a protecting group for the hydroxyl group of Thr and Ser and is 
preferably selected from the class consisting of acetyl(Ac), benzoyl(Bz), 
tert-butyl, triphenylmethyl(trityl), tetrahydropyranyl, benzyl ether(Bzl) 
and 2,6-dichlorobenzyl(DCB). The most preferred protecting group is Bzl. 
X.sup.3 can be hydrogen, which means there is no protecting group on the 
hydroxyl group. 
X.sup.4 is hydrogen or a protecting group for the side chain amino group of 
Lys, such as 2-chlorobenzyloxycarbonyl(2-Cl-Z), Tos, CBZ, 
t-amyloxycarbonyl and BOC. 
X.sup.5 is hydrogen or an ester-forming protecting group for the side chain 
carboxyl group of Asp and Glu, preferably selected from the class 
consisting of benzyl, 2,6-dichlorobenzyl, .beta. or .gamma.-cyclohexyl 
(CyHx) , methyl, ethyl and t-butyl ester. CyHx is most preferred. 
X.sup.6 is hydrogen or a protecting group for the amino group of Asn and 
Gln such as xanthyl(Xan). It is preferably left unprotected and coupled in 
the presence of 2 eg. of HOBt. 
X.sup.7 is a protecting group for the guanidino group of Arg preferably 
selected from the class consisting of nitro, p-toluenesulfonyl(Tos), Z, 
adamantyloxycarbonyl and BOC, or is hydrogen. Tos is most preferred. 
X.sup.8 is hydrogen or a protecting group for the imidazole nitrogen of 
His, such as Tos or 2,4-dinitrophenyl (DNP). 
Although the side chain methylthiol group of Met can be protected by oxygen 
or the like, preferably Met is left unprotected. 
The selection of a side chain amino protecting group is not critical except 
that it should be one which is not removed during deprotection of the 
alpha-amino groups during the synthesis. Hence, the alpha-amino protecting 
group and the side chain amino protecting group cannot be the same. 
X.sup.9 may be NH.sub.2, or an ester or amide anchoring bond used in solid 
phase synthesis for linking to a solid resin support, represented by the 
formula: 
--O--CH.sub.2 -polystyrene resin support, 
--O--CH.sub.2 -benzyl-polyamide resin support, 
--NH-benzhydrylamine (BHA) resin support, and 
--NH-paramethylbenzhydrylamine (MBHA) resin support. The polyamide polymer 
is commercially available and is discussed in detail in Bioorganic 
Chemistry, 8, 351-370 (1979) where a preferred version of it is discussed 
in FIG. 6 therein. Use of BHA or MBHA resin is preferred, and cleavage 
directly gives the NPY analog amide. 
When X is acetyl(Ac), for example, in the final formula, it may be possible 
to employ it as the X.sup.1 protecting group for the alpha-amino group of, 
for example, Ala by adding it to such amino acid before the coupling of 
this last amino acid to the peptide chain. However, a reaction is 
preferably carried out with the peptide on the resin (after deblocking the 
alpha-amino group while the side-chain groups remain protected), e.g. by 
reacting with acetic acid in the presence of dicyclohexyl 
carbodiimide(DCC) or preferably with acetic anhydride or by another 
suitable reaction as known in the art. Other examples of X include acrylyl 
and benzoyl which can be similarly incorporated. 
In the Formula (II) for the intermediate, at least one of X.sup.1, X.sup.2, 
X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8 and X.sup.9 is a 
protecting group or an anchoring bond. Thus, there are also provided 
methods for manufacturing an NPY peptide analog of interest by (a) first 
forming a peptide of Formula (II) wherein: X, X.sup.1, X.sup.2, X.sup.3, 
X.sup.4, X.sup.5, X.sup.6, X.sup.7 and X.sup.8 are each either hydrogen or 
a protective group and X.sup.9 is either a protective group or an 
anchoring bond to resin support or NH.sub.2, with at least one X-group 
being either a protecting group or an anchoring bond; (b) splitting off 
the protective group or groups or anchoring bond from said peptide of 
Formula (II); and (c) if desired, converting the resulting peptide of 
interest into a nontoxic salt thereof. 
In selecting a particular side chain protecting group to be used in the 
synthesis of the peptides, the following rules are followed: (a) the 
protecting group should 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 should 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. 
When the peptides are not prepared using recombinant DNA technology, they 
are preferably prepared using solid phase synthesis, such as that 
described by Merrifield, J. Am. Chem. Soc., 85, p 2149 (1964), although 
other equivalent chemical syntheses known in the art can also be used as 
previously mentioned. Solid-phase synthesis is commenced from the 
C-terminus of the peptide by coupling a protected alpha-amino acid to a 
suitable resin as generally set forth in U.S. Pat. No. 4,244,946 issued 
Jan. 21, 1981 to Rivier et al., the disclosure of which is incorporated 
herein by reference. Such a starting material for NPY analogs can, for 
example, be prepared by attaching alpha-amino- and side-chain-protected 
Tyr to a BHA resin. 
Tyr protected by BOC and DCB is coupled to the BHA resin using methylene 
chloride or dimethylformamide (DMF) as solvent with a suitable coupling 
reagent. The selection of an appropriate coupling reagent is within the 
skill of the art. Particularly suitable as a coupling reagent is 
N,N'-dicyclohexyl carbodiimide (DCC). The activating reagents used in the 
solid phase synthesis of the peptides are well known in the peptide art. 
Examples of suitable activating reagents are carbodiimides, such as 
N,N'-diisopropyl carbodiimide and 
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. Other activating reagents 
and their use in peptide coupling are described by Schroder & Lubke, 
supra, in Chapter III and by Kapoor, J. Phar. Sci., 59, pp 1-27 (1970). 
Following the coupling of BOC-Tyr(DCB) to the resin support, the 
alpha-amino protecting group is removed, as by using trifluoroacetic 
acid(TFA) in methylene chloride, TFA alone or with HC1 in dioxane. 
Preferably 50 % TFA in methylene chloride is used with 0-5 % 1,2 
ethanedithiol. 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, the remaining 
alpha-amino- and side-chain-protected amino acids are coupled step-wise in 
the desired order to obtain the intermediate compound defined 
hereinbefore. As an alternative to adding each amino acid separately in 
the synthesis, some of them may be coupled to one another prior to 
addition to the solid phase reactor. Each protected amino acid or amino 
acid sequence is introduced into the solid phase reactor in about a two to 
fourfold excess, and the coupling is carried out in a medium of 
dimethylformamide(DMF):CH.sub.2 Cl.sub.2 (1:1) or in DMF or CH.sub.2 
Cl.sub.2 alone. In instances where the coupling is carried out manually, 
the success of the coupling reaction at each stage of the synthesis can be 
monitored by the ninhydrin reaction, as described by E. Kaiser et al., 
Anal. Biochem. 34, 595 (1970). In cases where incomplete coupling occurs, 
the coupling procedure is repeated before removal of the alpha-amino 
protecting group prior to the coupling of the next amino acid. The 
coupling reactions can be performed automatically, as on a Beckman 990 
automatic synthesizer, using a program such as that reported in Rivier et 
al., Biopolymers, 1978, 17, pp.1927-1938. 
After the desired amino acid sequence has been completed, the intermediate 
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 the alpha-amino protecting group X.sup.1 and 
all remaining side chain protecting groups X.sup.2, X.sup.3, X.sup.4, 
X.sup.5, X.sup.6, X.sup.7 and X.sup.8 to obtain the peptide.

The following Examples set forth preferred methods for synthesizing NPY 
analogs by the solid-phase technique and generally is in accordance with 
the procedure set forth in U.S. Pat. No. 4,415,558 to Vale, et al, issued 
Nov. 15, 1983, the disclosure of which is incorporated herein by 
reference. 
EXAMPLE I 
The synthesis of pNPY(17-36), see SEQ ID NO:1, is conducted in a stepwise 
manner on a methylbenzhydrylamine hydrochloride resin, such as available 
from Bachem, Inc., having a substitution range of about 0.1 to 0.5 
mmoles/gm. resin. The synthesis is performed on an automatic Beckman 990B 
peptide synthesizer under an N.sub.2 atmosphere. Coupling of BOC-Tyr(2BrZ) 
results in the substitution of about 0.35 mmol. Tyr per gram of resin. The 
program used is generally that reported in Marki et al., J. Am. Chem. 
Soc., 103, 3178-3185 (1981). 
After deprotection and neutralization, the peptide chain is built 
step-by-step on the resin. Generally, one to two mmol. of BOC-protected 
amino acid in methylene chloride is used per gram of resin, plus one 
equivalent of 2 molar DCCI in methylene chloride, for two hours. When 
BOC-Arg(Tos) is being coupled, a mixture of 50% DMF and methylene chloride 
is used. Bzl is used as the hydroxyl side-chain protecting group for Ser 
and Thr. P-nitrophenyl ester(ONp) is used to activate the carboxyl end of 
Asn and Gln, and for example, BOC-Asn(ONp) is coupled overnight using one 
equivalent of HOBt in a 50% mixture of DMF and methylene chloride. 
Although the amido group of Asn or Gln may be protected by Xan when DCCI 
coupling is used instead of the active ester method, BOC-Asn or BOC-Gln is 
preferably used in the presence of 2 eq. N-hydroxybenzotriazole per 
equivalent of BOC-AA. Tos is used to protect the guanidino group of Arg 
and the imidazole group of His, and the side chain carboxyl group of Asp 
and Glu is protected by OBzl. At the end of the synthesis, the following 
composition is obtained: X.sub.1 -Leu-Ala-Arg (X.sup.7) -Tyr (X.sup.2) 
-Tyr (X.sup.2) -Ser (X.sup.3) -Ala-Leu-Arg (X.sup.2) -His(X.sup.8) 
-Tyr(X.sup.2)-Ile-Asn-Leu-Ile-Thr(X.sup.3)-Arg(X.sup.7) -Gln-Arg 
(X.sup.7)-Tyr(X.sup.2)-MBHA resin; wherein X.sub.1 is BOC, X.sub.2 is 
2BrZ, X.sub.3 is Bzl, X.sub.7 is Tos and X.sub.8 is DNP. Xan may have been 
partially or totally removed by TFA treatment used to deblock the 
alpha-amino protecting group. 
In order to cleave and substantially deprotect the resulting protected 
peptide-resin, it is treated with 1.5 ml. anisole, and 30 ml. hydrogen 
fluoride(HF) per gram of peptide-resin, first at -20.degree. C. for 20 
min. and then at 0.degree. C. for 40 minutes. After elimination of the HF 
under high vacuum, the resin-peptide is washed with dry diethyl ether, and 
the peptides are then extracted with water and separated from the resin by 
filtration. 
The cleaved peptide is then purified by reverse-phase HPLC and then 
rechromatographed for final purification using preparative HPLC as 
described in Rivier et al., Peptides: Structure and Biological Function 
(1979) pp. 125-128. The chromatographic fractions are carefully monitored 
by HPLC, and only the fractions showing substantial purity are pooled. 
EXAMPLE II 
The peptide hNPY(17-36), see SEQ ID NO:2, synthesized using the method as 
described in Example I. 
EXAMPLE III 
The peptides NPY(18-36) and [Phe.sup.27 ]-NPY(18-36) are synthesized using 
the method as described in Example I. 
EXAMPLE IV 
The peptide [Ac-D-Ala.sup.17 ]-NPY(17-36) having the formula: 
Ac-D-Ala-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-G 
ln-Arg-Tyr-NH.sub.2 is synthesized using the general method as described in 
Example I, and then reacting with acetic acid in the presence of DCC, or 
reacting with acetic anhydride, after deblocking the alpha-amino group on 
the final D-Ala residue. 
EXAMPLE V 
The peptide NPY(19-36) is synthesized using the method as described in 
Example I. 
EXAMPLE VI 
The peptide [Nle.sup.17 ]-NPY(17-36) is synthesized using the method as 
described in Example I. 
EXAMPLE VII 
The peptide [D-Ser.sup.18 ]-NPY(18-36) having the formula: 
H-D-Ser-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Ar 
g-Tyr-NH.sub.2 is synthesized using the method as described in Example I. 
EXAMPLE VIII 
The peptide [Ala.sup.17, His.sup.21 ]-NPY(17-36) is synthesized using the 
method as described in Example I. 
EXAMPLE IX 
The peptide [D-Ile.sup.18 ]-NPY(18-36) having the formula: 
D-Ile-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg- 
Tyr-NH.sub.2 is synthesized using the method as described in Example I. 
EXAMPLE X 
The peptide [Ac-Arg.sub.17 ]-NPY(17-36) is synthesized using the general 
method as described in Example I, and then reacting with acetic acid in 
the presence of DCC, or reacting with acetic anhydride, after deblocking 
the alpha-amino group on the final residue. 
EXAMPLE XI 
The peptide [Gln.sup.19 ]-NPY(19-36) is synthesized using the method as 
described in Example I. 
EXAMPLE XII 
The peptide [Phe.sup.20 ]-NPY(18-36) is synthesized using the method as 
described in Example I. 
EXAMPLE XIII 
The peptide [C.sup..alpha. MeLeu.sub.17 ]-pNPY(17-36) is synthesized using 
the method as described in Example I. 
EXAMPLE XIV 
The peptide [N.sup..alpha. MeLeu.sup.17 ]-pNPY(17-36) is synthesized using 
the method as described in Example I. 
EXAMPLE XV 
The peptide [desaminoAla.sup.18 ]-NPY (18-36) is synthesized using the 
method as described in Example I. 
EXAMPLE XVI 
The peptide [For-Ala.sup.18, Glu.sup..sub.23, Arg.sub.26 ]-NPY(18-36) is 
synthesized using the general method as described in Example I to create 
the peptide chain, and the BOC protecting group on the final Ala residue 
is removed. The peptide-resin is then treated with 98% formic acid at 
5.degree.-15.degree. C. and acetic anhydride is added dropwise, and the 
reaction mixture is stirred for 1 hour. After completion of the reaction, 
cleavage from the resin and of the protecting groups takes place as set 
forth in Example I. 
EXAMPLE XVII 
The peptide [Nva.sup.17, Ala.sup.21, Leu.sup.28 ]-NPY(17-36) is synthesized 
using the method as described in Example I. 
EXAMPLE XVIII 
The peptide [Thr.sup.22, Gln.sup.23 ]-NPY(18-36) having the formula (SEQ ID 
NO:4): 
Ala-Arg-Tyr-Tyr-Thr-Gln-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Ty 
r, wherein the C-terminus is amidated, is synthesized using the method as 
described in Example I. 
EXAMPLE XIX 
The peptide [desamino Leu.sup.17, Asn.sup.23, Val.sub.30 ]-NPY(17-36) is 
synthesized using the general method as described in Example I. 
EXAMPLE XX 
The peptide [Asp.sup.22, Ser.sub.23, Thr.sup.30 ]-NPY(18-36) having the 
formula (SEQ ID NO:5): 
Ala-Arg-Tyr-Tyr-Asp-Ser-Leu-Arg-His-Tyr-Ile-Asn-Thr-Ile-Thr-Arg-Gln-Arg-TY 
r, wherein the C-terminus is amidated, is synthesized using the method as 
described in Example I. 
EXAMPLE XXI 
The peptide [Gln.sup.25, Leu.sup.31 , Pro.sup.34 ]-NPY(18-36) having the 
formula (SEQ ID NO: 6): 
Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Gln-His-Tyr-Ile-Asn-Leu-Leu-Thr-Arg-Pro-Arg-Ty 
r, wherein the C-terminus is amidated, is synthesized using the method as 
described in Example I. 
EXAMPLE XXII 
The peptide [Gln.sup.26, Arg.sup.28,Phe.sup.36 ]-NPY (17-36) having the 
formula (SEQ ID NO:7): 
Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-Gln-Tyr-Arg-Asn-Leu-Ile-Thr-Arg-Gln-Ar 
g-Phe, wherein the C-terminus is amidated, is synthesized using the method 
as described in Example I. 
EXAMPLE XXIII 
The peptide [Phe.sub.36 ]-pPYY(19-36) is synthesized using the method as 
described in Example I. 
EXAMPLE XXIV 
The peptide pPYY(18-36) is synthesized using the method as described in 
Example I. 
EXAMPLE XXV 
The peptide [Ac-Ser.sup.18,Phe.sup.27 ]-pPYY(18-36) having is synthesized 
using the general method as described in Example I, and then reacting with 
acetic anhydride, after deblocking the alpha-amino group on the final Ser 
residue. 
EXAMPLE XXVI 
The peptide [Nle.sup.17,Asn.sup.22,Phe.sup.27 ]-NPY (17-36) is synthesized 
using the method as described in Example I. 
EXAMPLE XXVII 
The peptide [D-Ala.sup.18, Glu.sup.21, His.sup.34 ]-NPY(18-36) having the 
formula: 
H-D-Ala-Arg-Tyr-Glu-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-His-Ar 
g-Tyr-NH.sub.2 is synthesized using the method as described in Example I. 
EXAMPLE XXVIII 
The peptide [Bz-Leu.sup.7, Pro.sub.34,Phe.sub.36 ]-pNPY(17-36) having the 
formula (SEQ ID NO:8): 
Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Pro-Ar 
g-Phe, wherein the N-terminal is acylated with benzoyl and the C-terminus 
is admidated, is synthesized using the general method as described in 
Example I, and then reacting with benzoic acid in the presence of DCC 
after removing the BOC group on the final Leu residue. 
EXAMPLE XXIX 
The peptide [Lys.sup.19, Phe.sup.27, Val.sup.28 ]-NPY(18-36) having the 
formula (SEQ ID NO:9): 
Ala-Lys-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Phe-Val-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Ty 
r, wherein the C-terminus is amidated, is synthesized using the method as 
described in Example I. 
EXAMPLE XXX 
The peptide [D-Ala.sup.17, Val.sup.M, Phe.sub.32 ]-NPY(17-36) having the 
formula: 
D-Ala-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Val-Asn-Leu-Ile-Phe-Arg-Gln- 
Arg-Tyr-NH.sub.2 is synthesized using the general method as described in 
Example I, and then reacting with acetic acid in the presence of DCC, or 
reacting with acetic anhydride, after deblocking the alpha-amino group on 
the final D-Ala residue. 
EXAMPLE XXXI 
The peptide [C.sup..alpha. MeSer.sup.18, Met.sup.30, Phe.sup.36 
]-Npy(18-36) is synthesized using the method as described in Example I. 
EXAMPLE XXXII 
The peptide [Arg.sup.17, Ile.sup.18, Phe.sub.27,36 ]-Npy(17-36) having the 
formula (SEQ ID NO:10): 
Arg-Ile-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Phe-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Ar 
g-Phe, wherein the C-terminus is amidated, is synthesized using the method 
as described in Example I. 
EXAMPLE XXXIII 
The peptide [Ser.sup.18, Phe.sup.27 ]-pNPY(17-36) having the formula (SEQ 
ID NO:11): 
Leu-Ser-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Phe-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Ar 
g-Tyr, wherein the C-terminus is amidated, is synthesized using the method 
as described in Example I. 
EXAMPLE XXXIV 
The peptide [N.sup..alpha. MeIle.sup.18,Gln.sup.25, Phe.sup.27 ]-Npy(18-36) 
is synthesized using the general method as described in Example I. 
EXAMPLE XXXV 
The peptide [D-Ser.sup.18, Phe.sup.36 ]-Npy(18-36) having the formula: 
H-D-Ser-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Ar 
g-Phe-NH.sub.2 is synthesized using the method as described in Example I. 
EXAMPLE XXXVI 
The peptide [Asp.sup.23, Arg.sup.26 ]-hNPy(17-36) having the formula (SEQ 
ID NO:12): 
Met-Ala-Arg-Tyr-Tyr-Ser-Asp-Leu-Arg-Arg-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Ar 
g-Tyr, wherein the C-terminus is amidated, is synthesized using the method 
as described in Example I. 
EXAMPLE XXXVII 
The peptide [Glu.sup.23, Ile.sub.29 ]-NPY(18-36) having the formula (SEQ ID 
NO:13): 
Ala-Arg-Tyr-Tyr-Ser-Glu-Leu-Arg-His-Tyr-Ile-Ile-Leu-Ile-Thr-Arg-Gln-Arg-Ty 
r, wherein the C-terminus is amidated, is synthesized using the method as 
described in Example I. 
EXAMPLE XXXVIII 
The peptide [D-Ala.sup.17 ]-NPY(17-36)-OH having the formula: 
D-Ala-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln- 
Arg-Tyr-OH is synthesized on a chloromethylated resin using the general 
method as described in Chemistry Letters, K. Horiki et al., 165-168 
(1978). 
EXAMPLE XXXIX 
The peptide pPYY(17-36) is synthesized using the method as described in 
Example I. 
EXAMPLE XL 
The peptide pPYY(19-36) is synthesized using the method as described in 
Example I. 
EXAMPLE XLI 
The peptide pPYY(20-36) is synthesized using the method as described in 
Example I. 
EXAMPLE XLII 
The peptide pPYY(21-36) is synthesized using the method as described in 
Example I. 
EXAMPLE XLIII 
The peptides listed below are synthesized using the method of Example I. 
______________________________________ 
Peptide [.alpha.].sub.D 
______________________________________ 
[D-Tyr.sup.21 ]-NPY(18-36) 
-48.0.degree. 
[D-Ser.sup.22 ]-NPY(18-36) 
-36.0.degree. 
[D-Ala.sup.23 ]-NPY(18-36) 
-48.0.degree. 
[D-His.sup.26 ]-NPY(18-36) 
-38.0.degree. 
[D-Tyr.sup.27 ]-NPY(18-36) 
-40.0.degree. 
[D-Ile.sup.28 ]-NPY(18-36) 
-43.0.degree. 
[D-Asn.sup.29 ]-NPY(18-36) 
-38.0.degree. 
[D-Leu.sup.30 ]-NPY(18-36) 
-35.0.degree. 
[D-Ile.sup.31 ]-NPY(18-36) 
-38.0.degree. 
[D-Thr.sup.32 ]-NPY(18-36) 
-35.0.degree. 
[D-Arg.sup.33 ]-NPY(18-36) 
-39.4.degree. 
[D-Gln.sup.34 ]-NPY(18-36) 
-40.3.degree. 
[D-Arg.sup.35 ]-NPY(18-36) 
-41.0.degree. 
[D-Tyr.sup.36 ]-NPY(18-36) 
-48.9.degree. 
______________________________________ 
These peptides are judged to be substantially pure using the TLC and HPLC. 
The optical rotations [.alpha.].sub.D of these peptides are measured in 
1.0 M acetic acid (c=0.5) at about 25.degree. C. and are reported above. 
Synthetic NPY analogs are tested for their effect on mean arterial blood 
pressure (MAP) in conscious rats. Saline solutions of the peptides are 
injected intra-arterially into conscious rats, and arterial pressure is 
monitored via an indwelling femoral cannula directly coupled to a pressure 
transducer. MAP is calculated as [(systolic-diastolic/3)+diastolic] and is 
generally determined 1 minute after administration of the peptide and then 
at varying periods thereafter. Control animals receive saline vehicle 
alone. 
pNPY(17-36), NPY(18-36) and NPY(19-36) significantly decrease MAP at five 
and ten minutes after administration. The maximal response is seen with 
NPY(18-36), which produces a fall in MAP from 102.+-.2 mmHg to 50.+-.6 
mmHg after five minutes. All fragments increase heart rate (HR) after 
injection when compared to the control group. There is no significant 
difference in the maximal HR obtained between any of the fragments. 
PYY(17-36), PYY(19-36), PYY(20-36) and PYY(21-36) also significantly 
decrease MAP at 5 and 15 minute intervals. Because of the homology between 
the NPY and PYY, these fragments of PYY are broadly considered as NPY 
analogs. 
NPY(18-36) decreases MAP in a dose-dependent manner. At five minutes after 
injection, there is no significant change in MAP in the group that 
received 10 .mu.g, however, there is a significant decrease in MAP in the 
30 .mu.g group and a more pronounced effect in the groups that receive 100 
and 300 .mu.g. There is no significant difference in the decrease in MAP 
obtained after administration of 100 or 300 .mu.g. All doses produce 
maximal decrease in MAP five minutes after administration, except for the 
10 .mu.g dose which has a nadir MAP at t=15 min. Heart rates are only 
significantly increased in the 30 .mu.g group. 
NPY is given to a group of animals that are pretreated with NPY(18-36) to 
determine if the hypotension seen after NPY(18-36) administration is due 
to antagonism of the hypertensive actions of NPY. The increase in MAP (a 
MAP) obtained by NPY administration (1 .mu.g, intra-arterially) to animals 
pretreated with 
NPY(18-36) (300 .mu.g) is not significantly different from that obtained 
after NPY administration to control animals (28.+-.5 mmHg vs 32.+-.5 mmHg, 
respectively, with 3 animals for each test). 
The peptides disclosed in Example XLIII in which singular D-isomer 
substitutions were made in a C-terminal fragment of the NPY peptide, 
namely NPY(18-36), along with certain other NPY peptide analogs were 
tested for their effect on mean arterial blood pressure (MAP) using the 
protocol set forth above. Saline solutions of the peptides were 
administered intraarterially in a 100-.mu.L bolus in an amount equal to 
about 0.4 mg/Kg body weight. The change in MAP was measured by measuring 
the pressure just prior to administration and at intervals of minute for 
the first 5 minutes after administration, and then at intervals of 5 
minutes each for 1 hour; the results set forth hereinafter were calculated 
as an average using the data obtained during the first 10 minutes 
following administration (with the indicated standard deviation of error 
based upon the variance in the number of animals actually treated, which 
in most cases was 6 rats). The results for these tests are set forth in 
Table 1 which follows: 
TABLE 1 
______________________________________ 
Peptide .DELTA. MAP (mmHg) 
______________________________________ 
[D-Tyr.sup.20 ]-NPY(18-36) 
-4.4 .+-. 7 
[D-Tyr.sup.21 ]-NPY(18-36) 
-53 .+-. 9 
[D-Ser.sup.22 ]-NPY(18-36) 
-37 .+-. 8 
[D-Ala.sup.23 ]-NPY(18-36) 
-25 .+-. 9 
[D-Leu.sup.24 ]-NPY(18-36) 
0.1 .+-. 2 
[D-Arg.sup.25 ]-NPY(18-36) 
6 .+-. 7 
[D-His.sup.26 ]-NPY(18-36) 
-13 .+-. 7 
[D-Tyr.sup.27 ]-NPY(18-36) 
-38 .+-. 10 
[D-Ile.sup.28 ]-NPY(18-36) 
-14 .+-. 10 
[D-Asn.sup.29 ]-NPY(18-36) 
-30 .+-. 15 
[D-Leu.sup.30 ]-NPY(18-36) 
-40 .+-. 6 
[D-Ile.sup.31 ]-NPY(18-36) 
-41 .+-. 5 
[D-Thr.sup.32 ]-NPY(18-36) 
-32 .+-. 9 
[D-Arg.sup.33 ]-NPY(18-36) 
-25 .+-. 8 
[D-Gln.sup.34 ]-NPY(18-36) 
-32 .+-. 11 
[D-Arg.sup.35 ]-NPY(18-36) 
-45 .+-. 12 
[D-Tyr.sup.36 ]-NPY(18-36) 
-36 .+-. 10 
______________________________________ 
pNPY also transiently elevates intracellular Ca++concentrations with an 
EC.sub.50 of 2.0 nM. Maximal Ca++ increases are 200-550 nM above basal 
levels (40-70 nM). NPY(18-36) (100 nM) increases Ca++less than 10% as much 
as does an equal concentration of NPY, indicating that NPY(18-36) is only 
a very weak agonist in this system. Moreover, NPY(18-36) does not 
antagonize the Ca++mobilization by NPY; after addition of 100 nM NPY 
(18-36), 100 nM NPY still elevates intracellular Ca++to the same extent as 
under control conditions. Furthermore, pNPY(18-36) neither inhibits cAMP 
formation nor antagonizes NPY-mediated inhibition. 
NPY analogs or nontoxic addition salts thereof, combined with a 
pharmaceutically acceptable carrier to form a pharmaceutical composition, 
may be administered to mammals, including humans, either intravenously, 
subcutaneously, intramuscularly, percutaneously, e.g.intranasally, 
intracerebrospinally, orally or by suppository. The peptide should be at 
least about 90% pure and preferably should have a purity of at least about 
98% when administered to humans. This purity means that the intended 
peptide constitutes the stated weight % of all like peptides and peptide 
fragments present. Administration may be employed by a physician to lower 
blood pressure to counteract hypertension; the required dosage will vary 
with the particular condition being treated, with the severity of the 
condition and with the duration of desired treatment. 
Such peptides are often administered in the form of pharmaceutically 
acceptable nontoxic salts, such as acid addition salts or metal complexes, 
e.g., with zinc, iron, calcium, barium, magnesium, aluminum or the like 
(which are considered as addition salts for purposes of this application). 
Illustrative of such acid addition salts are hydrochloride, hydrobromide, 
sulphate, phosphate, tannate, oxalate, fumarate, gluconate, alginate, 
maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, 
tartrate and the like. If the active ingredient is to be administered in 
tablet form, the tablet may contain a binder, such as tragacanth, corn 
starch or gelatin; a disintegrating agent, such as alginic acid; and a 
lubricant, such as magnesium stearate. If administration in liquid form is 
desired, sweetening and/or flavoring may be used, and intravenous 
administration in isotonic saline, phosphate buffer solutions or the like 
may be effected. 
The peptides should be administered under the guidance of a physician, and 
pharmaceutical compositions will usually contain the peptide in 
conjunction with a conventional, pharmaceutically-acceptable carrier. 
Usually, the dosage will be from about 1 to about 200 micrograms of the 
peptide per kilogram of the body weight of the host. As used herein all 
temperatures are .degree. C. and all ratios are by volume. Percentages of 
liquid materials are also by volume. 
Although the invention has been described with regard to its preferred 
embodiments, which constitute the best mode presently known to the 
inventors, it should be understood that various changes and modifications 
as would be obvious to one having the ordinary skill in this art may be 
made without departing from the scope of the invention which is set forth 
in the claims appended hereto. For example, fragments of PYY that are 
further N-terminally shortened by one or several residues, e.g. PYY(22-36) 
are also expected to have a similar effect upon MAP. In many instances the 
C-terminal free acid has substantially the same biological effect as the 
C-terminal amide, and thus they are considered to be equivalents. 
Various features of the invention are emphasized in the claims which 
follow. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 14 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TyrProSerLysPro AspAsnProGlyGluAspAlaProAlaGluAsp 
151015 
LeuAlaArgTyrTyrSerAlaLeuArgHisTyrIleAsnLeuIleThr 
20 2530 
ArgGlnArgTyr 
35 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
Tyr ProSerLysProAspAsnProGlyGluAspAlaProAlaGluAsp 
151015 
MetAlaArgTyrTyrSerAlaLeuArgHisTyrIleAsnLeuIleThr 
202530 
ArgGlnArgTyr 
35 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
TyrProAlaLysProGluAlaProGlyGluAspAlaSerProGluGlu 
151015 
LeuSerArgTyrTyrAlaSerLeuArgHisTyrLeuAs nLeuValThr 
202530 
ArgGlnArgTyr 
35 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
AlaArgTyrTyrThrGlnLeuArgHisTyrIleAsnLeuIleThrArg 
151015 
GlnArgTyr 
(2) INFORMATION FOR SEQ ID NO:5: 
(i ) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
AlaArgTyrTyrAspSerLeuArgHisTyrIleAsnThrIleThrArg 
15 1015 
GlnArgTyr 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
AlaArgTyrTyrSerAlaLeuGlnHisTyrI leAsnLeuLeuThrArg 
151015 
ProArgTyr 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
LeuAlaArgTyrTyrSerAlaLeuArgGlnTyrArgAsnLeuIleThr 
151015 
ArgGlnArgPhe 
20 
( 2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
LeuAlaArgTyrTyrSerAlaLeuArgHisTyrIleAsnLeuIleThr 
15 1015 
ArgProArgPhe 
20 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
AlaLysTyrTyrSerAlaLeuArgHisPheValAsnLeuIleThrArg 
151015 
GlnArgTyr 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
ArgIleArgTyrTyrSerAlaLeuArgHisPheIleAsnLeuIleThr 
151015 
ArgGlnArgPhe 
20 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
LeuSerArgTyrTyrSerAlaLeuArgHisPheIl eAsnLeuIleThr 
151015 
ArgGlnArgTyr 
20 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
MetAlaArgTyrTyrSerAspLeuArgArgTyrIleAsnLeuIleThr 
151015 
ArgGlnArgTyr 
20 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
AlaArgTyrTyrSerGluLeuArgHisTyrIleIleLeuIleThrArg 
151015 
GlnArgTyr 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
Xaa XaaXaaXaaXaaLeuXaaXaaXaaXaaXaaXaaXaaXaaArgXaa 
151015 
ArgXaa