Vasonatrin peptide and analogs thereof

Synthetic analogs of C-type natriuretic peptide are provided, together with methods for their production and use as natriuretics, diuretics, and/or vasodilators, or as intermediates for or modulators of such useful compounds or of native natriuretic peptides.

Atrial natriuretic peptide (ANP) is the first described peptide in a family 
of hormones known to have important roles in the regulation of body fluid 
homeostasis. See, B. M. Brenner et al., Physiol. Rev., 70, 665 (1990). The 
description of the potent diuretic and natriuretic properties of atrial 
extracts by A. J. de Bola et al., Life Sci., 28, 89 (1981) was the first 
evidence that the heart could be an endocrine organ. The subsequent 
isolation and characterization of this activity by T. G. Flynn et al., 
Biochem. Biophys. Res. Commun., 117, 859 (1981) and other groups 
characterized ANP as the first secreted cardiac hormone. It is secreted by 
atrial myocytes in response to increased intravascular volume. Once it is 
in the circulation, its effects are primarily on the kidney, vascular 
tissue, and adrenal gland, in which its actions lead to the excretion of 
sodium (natriuresis) and water (diuresis) by the kidneys and a decrease in 
intravascular volume and blood pressure. (S. A. Atlas et al., in Atrial 
Hormones and Other Natriuretic Factors, P. J. Mulrow et al, ed., Am. 
Physiol. Soc., Bethesday, Md. (1987) at pages 53-76.) 
More recently, Matsuo and his coworkers have isolated two new natriuretic 
peptides. Brain natriuretic peptide (BNP) and C-type natriuretic peptide 
(CNP) were both isolated from porcine brain extracts on the basis of their 
potent relaxant effects on chick rectum. (See T. Sudeh et al., Nature, 
332, 78 (1988); Biochem. Biophys. Res. Commun., 168, 863 (1990)). Like 
ANP, these hormones are synthesized from large precursor proteins, and the 
mature, active peptides have a 17-amino-acid loop formed by an 
intramolecular disulfide linkage. In the human peptides (FIG. 1), eleven 
of these amino acids are identical in ANP, BNP, and CNP, whereas the N- 
and C-terminal tails vary in both length and composition. See, Y. 
Kambayashi et al., FEBS Lett., 259, 341 (1990); Y. Tawaragi et al., 
Biochem. Biophys. Res. Commun., 175, 645 (1991). CNP has no C-terminal 
tail, and studies of the structure of the gene for CNP demonstrated that 
translation is terminated by a stop codon immediately after the final 
cysteine codon in the mRNA. 
Among species, the amino acid sequence of both ANP and CNP are highly 
conserved, whereas the structure of BNP varies greatly. For example, the 
mature 28-amino-acid human and porcine ANPs are identical, and there is 
only one substitution in the rat peptide. This existence of this 
structural variation, coupled with the presence of at least three types of 
receptors specific for the natriuretic peptides, suggests that the 
physiological control of body fluid homeostasis is complex. ANP and CNP 
both decrease cardiac preload. However, unlike ANP, CNP is not 
natriuretic. See, A. J. Stingo et al., Am. J. Physiol., 262, H308 (1992). 
The diverse actions of ANP, BNP and CNP on both the cardiovascular system 
and the kidney as well as their roles in pathophysiological states such as 
heart failure, hypertension, and renal disease have made the native 
peptides and their analog molecules of great interest to both clinical and 
basic scientists. See, for example, J. A. Lewicki et al. (U.S. Pat. Nos. 
5,114,923, 4,804,650 and U.S. Pat. No. 4,757,048), L. K. Johnson et al. 
(U.S. Pat. No. 5,047,397) and L. K. Johnson et al. (U.S. Pat. No. 
4,935,492). Therefore, a continuing need exists to identify and 
characterize bioactive peptides belonging, or related to, this class of 
materials. Such peptides may be useful both to elucidate the mechanism of 
action of these compounds, as well as to provide new pharmaceutical agents 
with useful profiles of bioactivity. 
SUMMARY OF THE INVENTION 
The present invention provides a peptide compound having natriuretic, 
diuretic and/or vasodilator activity in mammals, which has the formula 
(I): 
EQU X.sub.1 --A.sub.y --X.sub.2 --A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 
--A.sub.5 --X.sub.3 --A.sub.z --X.sub.y (I) 
wherein A.sub.1 is Lys, Arg, Orn, Ala, Thr, Asn or Gln; A.sub.2 is Ile, 
Met, Val or Leu; A.sub.3 is Glu or Asp; A.sub.4 is Lys, Arg, Orn, Ala, 
Thr, Asn or Gln; A.sub.5 is Val, Leu or Ileu; X.sub.1 is a peptide of from 
1-125 amino acid residues, preferably 1-5 amino acid residues comprising 
Gly at the carboxyl terminus, and preferably Gly at the N-terminus, with 
the remainder being individually selected from the group consisting of 
Leu, Ser and Lys. Most preferably, X.sub.1 is (H)-Gly-Leu-Ser-Lys-Gly (SEQ 
ID NO; 5), or an N-terminal deleted form such as (H)Leu-Ser-Lys-Gly (SEQ 
ID NO: 5), (H)Ser-Lys-Gly, (H)Lys-Gly or (H)Gly; A.sub.y and A.sub.z are 
amino acid residues which together form a bridging bond, wherein said bond 
is selected from the group consisting of disulfide, methylene, 
sulfide/methylene, amide and ester; preferably, disulfide; X.sub.2 is a 
peptide of 1-10 amino acid residues, preferably of about 1-3 residues, 
individually selected from the group consisting of Phe, Trp, Val, Leu, 
Ile, Gly, Ser and Cys; X.sub.3 is a peptide of 1-10 amino acid residues, 
preferably about 1-7 residues and X.sub.y is a peptide of about 1-5 
residues selected from the group consisting of R-Asn-, R-Ser-Asn, 
R-Phe-Ser-Asn, R-Arg-Phe-Ser-Asn-(SEQ ID NO: 7) and 
R-Tyr-Arg-Phe-Ser-Asn-(SEQ ID NO: 8) wherein R is OH, NH.sub.2, NHR.sup.3 
or NR.sup.3 R.sup.4, wherein R.sup.3 and R.sup.4 are independently 
(C.sub.1 -C.sub.4)alkyl, (C.sub.3 -C.sub.5)cycloalkyl, phenyl or benzyl. 
Preferably, X.sub.3 is a heptapeptide comprising peptidyl residues 
individually selected from the group consisting of Gly, Ser, Met and Leu. 
Preferably, when A.sub.2 is Ile, A.sub.1 is not Arg, and when A.sub.2 is 
Arg, A.sub.2 is not Ile. 
Preferably, the total ring size is equivalent to that obtained by disulfide 
bridge formation between cysteine residues separated by 7-15 amino acid 
(peptidyl) residues. Optionally, one or more of the amide linkages between 
adjacent amino acid residues can be replaced by a linkage such as 
--CH.sub.2 --NH--, --CH.sub.2 --S--, --CH.sub.2 CH.sub.2 --, --CH=CH--, 
--COCH.sub.2 --, --CH(OH)CH.sub.2 -- or --CH.sub.2 SO--. X.sub.1 can also 
be an amino acid protecting group such as fluorenylmethyloxycarbonyl, 
benzyloxycarbonyl, (2-(2',6'-methoxynaphthyl)propionyl, diphenylpropionyl, 
cyclohexylacetyl, 3-indolepropionyl, 2-naphthoxy and the like. Other such 
groups are disclosed in U.S. Pat. No. 4,935,492. 
In the peptides of the invention, the L-form of any amino acid residue 
having an optical isomer is intended unless otherwise indicated. In some 
cases, A.sub.2 and/or A.sub.5 can be replaced by the D-forms. Bioactive 
peptides of formula (I) which do not comprise the disulfide bond are also 
within the scope of the invention. 
In a preferred embodiment, the present invention provides a peptide having 
natriuretic, diuretic and/or vasodilator activity in mammals, of the 
formula (II): 
##STR1## 
(SEQ ID NO: 9), wherein A' is Lys or Arg, A" is Leu, Ile or Met and 
R.sup.2 is R-Asn-, R-Ser-Asn-, R-Phe-Ser-Asn-, R-Arg-Phe-Ser-Asn-(SEQ ID 
NO: 7) and R-Tyr-Arg-Phe-Ser-Asn-(SEQ ID NO: 8); wherein R is OH, 
NH.sub.2, NHR.sup.3 or NR.sup.3 R.sup.4 wherein R.sup.3 and R.sup.4 are 
independently phenyl or (C.sub.1 -C.sub.4)alkyl; and wherein the bracket 
connecting the two cysteinyl moieties of formula (I) indicates that they 
are connected by a disulfide bond as depicted on FIG. 2. Preferably, 
R.sup.2 is R-Tyr-Arg-Phe-Ser-Asn-(SEQ ID NO: 8), and most preferably, R is 
OH, i.e., H in formula (I) represents the amino terminus of the peptide 
and (OH) represents the carboxyl terminus of the peptide. Preferably, A' 
is Lys (K) and A" is Leu (L). Thus, using single letter amino acid code, 
this preferred peptide, or "VNP", can be represented as: 
##STR2## 
(SEQ ID NO: 4), wherein the sequence is read conventionally, from the 
left, amino terminus to the right, carboxy terminus. The bracket 
represents a disulfide bond between the cysteinyl residues. 
VNP is a more potent, endothelium independent, vasorelaxing peptide in both 
arteries and veins than ANP or CNP. VNP also has potent natriuretic 
effects in vivo. Thus, the present invention also provides a composition 
useful as a natriuretic, diuretic and/or vasodilator comprising a 
therapeutically effective amount of the present peptide in combination 
with a pharmaceutically acceptable carrier, and a method for _inducing 
natriuresis, diuresis or vasodilation in a mammal, preferably a human, 
comprising administering to said mammal a pharmaceutically effective 
amount of said composition. The present compositions can also be employed 
to modify the renin-angiotensis-aldosterone system. 
Thus, the present peptides may be useful, either singly or in combination, 
to treat (ameliorate or prevent) a number of pathological conditions, 
including acute or chronic kidney failure, hypertension, congestive heart 
failure, cirrhosis of the liver, nephrotic syndrome, and other "edematous 
states".

DETAILED DESCRIPTION OF THE INVENTION 
1. Synthesis 
Peptides of this invention can be synthesized by the solid phase peptide 
synthesis (or Merrifield) method. This established and widely used method, 
including the experimental procedures, is described in the following 
references: Stewart et al., Solid Phase Peptide Synthesis, W. H. Freeman 
Co., San Francisco (1969); Merrfield, J. Am. Chem. Soc., 85, 2149 (1963); 
Meienhofer in "Hormonal Proteins and Peptides," ed.; C. H. Li, Vol. 2 
(Academic Press, 1973), pp. 48-267; and Barany and Merrifield in "The 
Peptides," eds. E. Gross and F. Meinenhofer, Vol. 2 (Academic Press, 
1980), pp. 3-285. The synthesis is commenced from the carboxy-terminal end 
of the peptide using an alphaamino protected amino acid. 
Fluorenylmethyloxy-carbonyl (Fmoc) or t-butyloxycarbonyl (Boc) protective 
groups can be used for all amino groups even through other protective 
groups are suitable. For example, Boc-Asn-OH, Boc-Ser-OH, Boc-Phe-OH, 
Boc-Arg-OH or Boc-Tyr-OH (i.e., selected ANP analog carboxy-terminal amino 
acids) can be esterified to chloromethylated polystyrene resin supports. 
The polystyrene resin support is preferably a copolymer of styrene with 
about 0.5 to 2% divinyl benzene as a cross-linking agent which causes the 
polystyrene polymer to be insoluble in certain organic solvents. See 
Carpino et al., J. Org. Chem., 37, 3404 (1972); Meinhofer, Int. J. Pept. 
Pro. Res., 11, 246 (1978); and Merrifield, J. Am. Chem. Soc., 85, 2149 
(1963). These and other methods of peptide synthesis are also exemplified 
by U.S. Pat. Nos. 3,862,925; 3,842,067; 3,972,859, 4,105,602 and U.S. Pat. 
No. 4,757,048. 
The immobilized peptide is then N-deprotected and other amino acids having 
protected amino groups are added in a stepwise manner to the immobilized 
peptide. At the end of the procedure, the final peptide is cleaved from 
the resin, and any remaining protecting groups are removed, by treatment 
under acidic conditions such as, for example, with a mixture of 
hydrobromic acid and trifluoroacetic acid or with hydrofluoric acid, or 
the cleavage from the resin may be effected under basic conditions, for 
example, with triethylamine, the protecting groups then being removed 
under acid conditions. 
The cleaved peptides are isolated and purified by means well known in the 
art such as, for example, lyophilization followed by either exclusion or 
partition chromatography on polysaccharide gel media such as Sephadex 
G-25, or countercurrent distribution. The composition of the final peptide 
may be confirmed by amino acid analysis after degradation of the peptide 
by standard means. 
Salts of carboxyl groups of the peptide may be prepared in the usual manner 
by contacting the peptide with one or more equivalents of a desired base 
such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a 
metal carbonate or bicarbonate base such as, for example, sodium carbonate 
or sodium bicarbonate; or an amine base such as, for example, 
triethylamine, triethanolamine, and the like. 
Acid addition salts of the polypeptides may be prepared by contacting the 
polypeptide with one or more equivalents of the desired inorganic or 
organic acid, such as, for example, hydrochloric acid. 
Esters of carboxyl groups of the polypeptides may be prepared by any of the 
usual means known in the art for converting a carboxylic acid or precursor 
to an ester. One preferred method for preparing esters of the present 
polypeptides, when using the Merrifield synthesis technique described 
above, is to cleave the completed polypeptide from the resin in the 
presence of the desired alcohol either under basic or acidic conditions, 
depending upon the resin. Thus, the C-terminal end of the peptide when 
freed from the resin is directly esterified without isolation of the free 
acid. 
Amides of the polypeptides of the present invention may also be prepared by 
techniques well known in the art for converting a carboxylic acid group or 
precursor, to an amide. A preferred method for amide formation at the 
C-terminal carboxyl group is to cleave the polypeptide from a solid 
support with an appropriate amine, or to cleave in the presence of an 
alcohol, yielding an ester, followed by aminolysis with the desired amine. 
N-acyl derivatives of an amino group of the present polypeptides may be 
prepared by utilizing an N-acyl protected amino acid for the final 
condensation, or by acylating a protected or unprotected peptide. O-acyl 
derivatives may be prepared, for example, by acylation of a free hydroxy 
peptide or peptide resin. Either acylation may be carried out using 
standard acylating reagents such as acyl halides, anhydrides, acyl 
imidazoles, and the like. Both N- and O-acylation may be carried out 
together, if desired. 
The synthesis may use manual techniques or be completely automated, 
employing, for example, an Applied BioSystems 431A Peptide Synthesizer 
(Foster City, Calif.) or a Biosearch SAM II automatic peptide synthesizer 
(Biosearch, Inc., San Rafael, Calif.), following the instructions provided 
in the instruction manual and reagents supplied by the manufacturer. 
Disulfide bonds between Cys residues can be introduced by mild oxidation 
of the linear peptide by KCN as taught in U.S. Pat. No. 4,757,048 at Col. 
20. 
Alternatively, selected compounds of the present invention can be produced 
by expression of recombinant DNA constructs prepared in accordance with 
well-known methods such as those described in Seihamer et al. (U.S. Pat. 
No. 5,114,923), entitled "Recombinant Techniques for Production of Novel 
Natriuretic and Vasodilator Peptides." Such production can be desirable to 
provide large quantities or alternative embodiments of such compounds. 
2. Structural Features 
The compounds of the invention contain the pentapeptide core sequence 
A.sub.3 -A.sub.4 -A.sub.5, which, in preferred embodiments, corresponds to 
the conserved region DRI present in ANP, CNP and BNP. Preferably, the 
sequence A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 --A.sub.5 is RMDRI (SEQ ID 
NO: 10), KMDRI (SEQ ID NO: 11), KLDRI (SEQ ID NO: 12), KIDRI (SEQ ID NO: 
18) or RLDRI (SEQ ID NO: 14), most preferably, is the KLDRI (SEQ ID NO: 
12) sequence of rat, porcine or human CNP. 
Preferably, X.sub.1 is (H)GLSKG-(SEQ ID NO: 15) wherein (H) indicates the 
amino terminus of the peptide; and X.sub.4 is --NSFRY(R) (SEQ ID NO: 16), 
wherein (R) is preferably (OH), indicating the carboxy terminus of the 
peptide. Preferably, X.sub.2 is a tripeptide, most preferably -FGL-, or a 
dipeptide such as -FG- or -GL-, and X.sub.3 is a heptapeptide, most 
preferably -GSMSGLG- (SEQ ID NO: 17). X.sub.3 can also be a truncated form 
of -GSMSGLG- (SEQ ID NO: 17) wherein one or more of the Gly or Ser 
residues may be replaced by another of Gly (G), Ser (S) or Cys (C) or by 
Ala, and Leu may be replaced by Val or Ile. 
The cyclic disulfides included within the invention are directly analogous 
to the naturally occurring CNPs, which contain 17 amino acid 
residue-membered disulfide rings, inclusive of the two cysteine residues 
which provide the sulfhydryl groups for the formation of the disulfide 
bond. However, those embodiments of the compounds of the invention which 
contain the cyclic disulfide may contain either more, or less, than 17 
amino acid residues in the cyclic structure. 
As indicated, the cyclic compounds of the present invention can be provided 
by bonding cysteine residues, or alternate amino acid residues A.sub.y and 
A.sub.z with an equivalent bond or linking group such as, for example, 
--CH.sub.2 --CH.sub.2 --. The replacement of a sulfhydryl group on the 
cysteine residue with an alternative group will effectively replace the 
cysteine residue with an alternative amino acid. For example, to replace 
one sulfhydryl group with a --CH.sub.2 -- group, the cysteine residues 
will be replaced by the analogous alpha-aminobutyric acid. These cyclic 
analog peptides can be formed, for example, in accordance with the 
methodology of M. Lebl and V. J. Hruby, Tetrahedron Lett., 25, 2067 
(1984), or by employing the procedure disclosed in U.S. Pat. No. 
4,161,521. 
Ester or amide bridges may also be formed by reacting the OH or serine or 
threonine and the carboxyl of aspartic acid or glutamic acid, to yield a 
bridge of the structure --CH.sub.2 --CO.sub.2 CH.sub.2 --. Similarly, an 
amide can be obtained by reacting the side-chain of lysine and aspartic or 
glutamic acid to yield a bridge of the structure --CH.sub.2 
C(O)NH--(CH.sub.2).sub.4 --. Methods for synthesis of these bridges are 
found in P. W. Schiller et al., Biochem. Biophy. Res. Comm., 127 558 
(1985); Int. J. Peptide and Protein Res., 25, 171 (1985). Other 
bridge-forming amino acid residues and reactions are provided in U.S. Pat. 
No. 4,935,492. 
The following references describe preparation of peptide analogs which 
include non-peptidyl bonds to link amino acid residues. A. F. Spatola, 
Vega Data, Vol. 1, Issue 3 (March 1983), "Peptide Backbone Modifications" 
(general review); A. F. Spatola in "Chemistry and Biochemistry of Amino 
Acids Peptides and Proteins", B. Weinstein, eds., Marcel Dekker, New York, 
p. 267 (1983) (general review); J. S. Morley, Trends Pharm. Sci. (1980) at 
pp. 463-468 (general review); D. Hudson et al., Int. J. Pept. Prot. Res., 
14, 177 (1979); (--CH.sub.2 NH--, --CH.sub.2 C--H.sub.2 --); A. F. Spatola 
et al., Life Sci., 38, 1243 (1986) (--CH.sub.2 --S); M. M. Hann, J. Chem. 
Soc. Perkin Trans. I, 307 (1982) (--CH--CH--), cis and trans); R. G. 
Almquist et al., J. Med. Chem., 23, 1392 (1980) (--COCH.sub.2 --); C. 
Jennings-White et al., Tetrahedron Lett., 23, 2533 (1982) (--COCH.sub.2 
--); M. Szelke et al., European Patent Appln EP 45665 (1982) 
(--C(OH)CH.sub.2 --); M. W. Holladay et al., Tetrahedron Lett., 24, 4401 
(1983) (--C(OH)CH.sub.2 --); and V. J. Hruby, Life Sci., 31, 189 (1982) 
(--CH.sub.2 --S--). 
3. Doses and Dosage Forms 
The compounds of the present invention have natriuretic, diuretic and 
hypotensive activity in the intact mammal, and may possess vasorelaxant 
activity and/or inhibit the release of aldosterone and renin. 
Thus, these compounds, and compositions containing them, can find use as 
therapeutic agents in the treatment of various edematous states such as, 
for example, congestive heart failure, nephrotic syndrome and hepatic 
cirrhosis, in addition to hypertension and renal failure due to 
ineffective renal perfusion or reduced glomerular filtration rate. 
The present invention also provides compositions containing an effective 
amount of compounds of the present invention, including the nontoxic 
addition salts, amides and esters thereof, together with physiologically 
acceptable liquid, gel or solid diluents, adjuvants and excipients. 
These compounds and compositions can be administered to mammals for 
veterinary use, such as with domestic animals, and for clinical use in 
humans in a manner similar to other therapeutic agents. In general, the 
dosage required for therapeutic efficacy will range from about 0.01 to 
1000 mcg/kg, more usually 0.1 to 1000 mcg/kg of the host body weight. 
Dosages within these ranges can be administered via bolus doses, via a 
plurality of unit dosage forms, or by constant infusion over an extended 
period of time, usually exceeding 24 hours, until the desired therapeutic 
benefits have been obtained. 
Typically, such compositions are prepared for injection or infusion, either 
as liquid solutions or suspensions. Solid forms suitable for solution in, 
or suspension in, liquid vehicles prior to injection or infusion may also 
be prepared. The preparation may also be emulsified. The active ingredient 
can be mixed with diluents or excipients which are physiologically 
acceptable and compatible with the active ingredient(s). Suitable diluents 
and excipients are, for example, water, saline, PBS, glycerol, or the 
like, and combinations thereof. In addition, if desired, the compositions 
may contain minor amounts of auxiliary substances such as wetting or 
emulsifying agents, stabilizing or pH-buffering agents, and the like. 
Such compositions are conventionally administered parenterally, by 
injection, for example, either subcutaneously or intravenously. 
Formulations which are suitable for other modes of administration include 
suppositories, insufflated powders or solutions, eyedrops, nosedrops, 
intranasal aerosols, and, in some cases, oral formulations. Oral 
formulations include such normally employed excipients as, for example, 
pharmaceutical grades of alkylcelluloses, mannitol, dextrose, lactose, 
starch, magnesium stearate, sodium saccharin, cellulose, magnesium 
carbonate, and the like. Thus, these compositions can take the form of 
solutions, suspensions, tablets, pills, hard or soft gelatin capsules, 
sustained-release formulations such as liposomes, gels or hydrogels; or 
powders, and can contain 10%-95% of active ingredient, preferably 25%-70%. 
The peptide compounds may be formulated into the compositions as neutral or 
salt forms. Pharmaceutically acceptable nontoxic salts include the acid 
addition salts (formed with the free amino groups) and which are formed by 
reaction with inorganic acids such as, for example, hydrochloric, sulfuric 
or phosphoric acids, or organic acids such as, for example, acetic, 
oxalic, tartaric, mandelic, citric, malic, and the like. Salts formed with 
the free carboxyl groups may be derived from inorganic bases such as, for 
example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and 
such organic bases such as amines, i.e., isopropylamine, trimethylamine, 
2-ethylamino ethanol, histidine, procaine, and the like. 
4. Other Utility 
In addition to the compounds of the present invention which display 
natriuretic, diuretic or vasorelaxant activity, compounds of the present 
invention can also be employed as intermediates in the synthesis of such 
useful compounds. Alternatively, by appropriate selection, compounds of 
the present invention whose activity levels are reduced or eliminated 
entirely can serve to modulate the activity of other diuretic, natriuretic 
or vasorelaxant compounds, including compounds outside the scope of the 
present invention by, for example, binding to alternate receptors, 
stimulating receptor turnover, or providing alternate substrates for 
degradative enzyme or receptor activity and thus inhibiting these enzymes 
or receptors. When employed in this manner as antagonists, such compounds 
can be delivered as admixtures with other active compounds or can be 
delivered separately, for example, in their own carriers. 
Compounds of the present invention can also be used to prepare antisera for 
use in immunoassays employing labeled reagents, usually antibodies. 
Conveniently, the peptides can be conjugated to an antigenicity-conferring 
carrier, if necessary, by means of dialdehydes, carbodiimide or by using 
commercially available linkers. These compounds and immunologic reagents 
may be labeled with a variety of labels such as chromophores, fluorophores 
such as, e.g., fluorescein or rhodamine, radioisotopes such as .sup.125 I, 
.sup.35 S, .sup.14 C, or .sup.3 H, or magnetized particles, by means known 
to the art. 
These labeled compounds and reagents, or labeled reagents capable of 
recognizing and specifically binding to them, can find use as, e.g., 
diagnostic reagents. Samples derived from biological specimens can be 
assayed for the presence or amount of substances having a common antigenic 
determinant with compounds of the present invention. In addition, 
monoclonal antibodies can be prepared by methods known in the art, which 
antibodies can find therapeutic use, e.g., to neutralize overproduction of 
immunologically related compounds in vivo. 
The invention will be further described by reference to the following 
detailed examples wherein VNP was synthesized in the Mayo Protein Core 
Facility using fluorenylmethoxy-carbonyl (FMOC) chemistry on an ABI 431A 
peptide synthesizer (Applied Biosystems Inc., Foster City, Calif.) with 
the protocols and reagents supplied by the manufacturer. The peptide was 
purified by reverse phase high performance liquid chromatography (HPLC) 
using a Vydac C8 column (The Separations Group, Hesperia, Calif.). The 
synthesis was confirmed by amino acid analysis and plasma absorption mass 
spectrometry. 
The samples from the plasma and urine were analyzed using reverse phase 
HPLC with a Vydac C18 column (4.6 mm.times.250 mm) (The Separations 
Groups, Hesperia, Calif.). The components of the HPLC system were two 
Beckman 114 pumps (Beckman Instruments, San Ramon, Calif.), ABI 759A 
absorbance detector (Applied Biosystems, Inc., Foster City, Calif.), and 
an IBM PS2 50Z computer with Beckman System Gold Chromatography software. 
The A buffer was 0.1% trifluoracetic acid and the B buffer was 80% 
acetonitrile/20% water/0.1% trifluoroacetic acid. The separation was 
performed with a gradient of 5% to 70% B buffer in 60 minutes. 
The results obtained are expressed as the means .+-.SEM. In organ chamber 
studies, n equals the number of dogs from which rings were taken. Rings 
with and without endothelium were studied in parallel, and Student's 
t-test for unpaired observations was used to determine statistical 
significance among the responses of rings with and without endothelium and 
between responses of arteries and veins. In rat studies, the data were 
analyzed using ANOVA for repeated measures followed by Fisher's least 
significant difference test when appropriate within the group. Data 
between groups were analyzed by Student's unpaired t-test. Statistical 
significance was determined at p&lt;0.05. 
Example 1. In Vitro Studies 
Rings cut from femoral, saphenous, renal and pulmonary arteries and veins 
obtained from random source mongrel dogs (anesthetized with 30 mg/kg 
pentobarbital sodium intravenously) were suspended for the measurement of 
isometric force in organ chambers filled with aerated (95% O.sub.2 and 5% 
CO.sub.2) modified Krebs-Ringer bicarbonate solution (composition in mM: 
118.3 NaCl, 4.7 KCl, 2.5 CaCl.sub.2, 1.2 MgSO.sub.4, 1.2 KH.sub.2 
PO.sub.4, 25.0 NaHCO.sub.3, 0.026 calcium sodium EDTA, and 11.1 dextrose; 
control solution) at 37.degree. C. In one-half rings, the endothelium was 
removed by gently rubbing the intimal surface with a cotton swab wetted 
with control solution. Each ring was stretched to the optimal point on its 
length-tension curve as determined by the tension developed to 
norepinephrine (3.times.10.sup.-7 M) at each level of stretch. The 
presence of endothelium was determined at the beginning of the experiment 
by a relaxation to acetylcholine (10.sup.-6 M) during a contraction to 
norepinephrine at optimal length. The maximal tension of each ring was 
determined by norepinephrine (10.sup.-4 M). To study responses to ANP, CNP 
and VNP, the rings were contracted with phenylephrine (10.sup.-6 M). These 
contractions averaged 30-40% of the maximal contraction to norepinephrine. 
The peptides were added cumulatively once the contraction had stabilized. 
The following commercially available drugs were used: acetylcholine 
chloride (Sigma Chemical, St. Louis, Mo.), human ANP and human CNP 
(Peninsula Laboratories, Belmont, Calif.), L-norepinephrine bitartrate 
(Sigma), phenylephrine bitartrate (Sigma). All drugs were dissolved in 
distilled water immediately prior to study, and the concentrations are 
reported as the final molar concentration (M) in the organ chamber. 
At maximal dose (3.times.10.sup.-6 log M), ANP and CNP had no effects on 
femoral and saphenous arteries and veins, but VNP exposure resulted in a 
potent relaxation (90-100%) in femoral and saphenous arteries. In femoral 
and saphenous veins, ANP had no effect. CNP relaxes femoral veins by 90% 
and saphenous veins by 40%. VNP caused a 90% relaxation in both femoral 
and saphenous veins. 
Table 1 summarizes the maximal relaxations (3.times.10.sup.-6 M) of ANP, 
CNP and VNP in isolated normal canine blood vessels. 
TABLE 1 
______________________________________ 
The maximal responses of ANP, CNP and VNP (3 .times. 10.sup.-6 M) 
in isolated normal canine blood vessels. 
ANP CNP VNP 
(n = 6-9) 
(n = 6-9) (n = 4-6) 
______________________________________ 
Systemic blood vessels 
Arteries 
Endothelium 
Femoral with 10 .+-. 5.sctn. 
4 .+-. 3 
91 .+-. 4*+ 
without 19 .+-. 12 
10 .+-. 6 
89 .+-. 5*+ 
Saphenous 
with 0 .+-. 0 11 .+-. 7 
73 .+-. 3*+ 
without 3 .+-. 3 14 .+-. 8 
81 .+-. 5*+ 
Renal with 27 .+-. 6+ 
8 .+-. 2 
52 .+-. 4*+ 
without 30 .+-. 6+ 
8 .+-. 2 
62 .+-. 12*+ 
Veins 
Femoral with 6 .+-. 3 37 .+-. 8* 
81 .+-. 6*+ 
without 4 .+-. 4 73 .+-. 8*.paragraph. 
86 .+-. 6* 
Saphenous 
with 0 .+-. 0 16 .+-. 1* 
60 .+-. 11*+ 
without 0 .+-. 0 43 .+-. 10* 
89 .+-. 6*+ 
Renal with 12 .+-. 3 43 .+-. 4* 
65 .+-. 6*+ 
without 11 .+-. 4 71 .+-. 5*.paragraph. 
90 .+-. 4*+.paragraph. 
Pulmonary blood vessels 
Artery with 60 .+-. 7+ 
12 .+-. 3 
57 .+-. 5+ 
without 75 .+-. 8+.paragraph. 
33 .+-. 5.paragraph. 
63 .+-. 7+ 
Vein with 49 .+-. 5 31 .+-. 6* 
71 .+-. 2*+ 
without 16 .+-. 3.paragraph. 
66 .+-. 4*.paragraph. 
84 .+-. 3*+.paragraph. 
______________________________________ 
.sctn. Data are shown as Mean .+-. SEM of a percent change in tension fro 
a contraction to phenylephrine (10.sup.-6 M). 
*p &lt; .05 vs ANP 
+ p &lt; .05 vs CNP 
.paragraph. p &lt; .05 vs with endothelium 
ANP atrial natriuretic peptide 
CNP Ctype natriuretic peptide 
VNP vasonatrin peptide 
In systemic arteries (femoral, saphenous and renal), ANP and CNP produced 
modest relaxations with the maximum being less than 30% of the contraction 
to phenylephrine. VNP, on the other hand, produced relaxations which 
ranged between 50-90% of the contraction; these relaxations were not 
modified significantly by the endothelium. In the comparable systemic 
veins, relaxations to CNP were significantly greater than relaxations 
evoked by ANP. Relaxations to CNP were increased significantly by removal 
of the endothelium. Relaxations induced by VNP were significantly greater 
than those induced by CNP in all systemic veins tested. Only in the renal 
veins were the relaxations enhanced by removal of the endothelium. The 
potency for relaxation in the systemic blood vessels for the peptides can 
be summarized as: VNP&gt;ANP.gtoreq.CNP in arteries; VNP&gt;CNP&gt;ANP in veins. 
In the pulmonary blood vessels, all three peptides produced significant 
relaxation. The order of potency in pulmonary arteries is: ANP=VNP&gt;CNP. 
Relaxations induced by ANP and CNP were increased by removal of the 
endothelium. The order of potency in the pulmonary veins without 
endothelium is: VNP&gt;CNP&gt;ANP. Relaxations induced by ANP were greater in 
the presence of endothelium while those to CNP and VNP were greater in the 
absence of the endothelium. 
Example 2. In Vivo Studies 
Experiments were conducted in accordance with the Animal Welfare Act. 
Wistar rats and spontaneously hypertensive rats (SHR) (400 g; Harlan 
Sprague-Dawley, Indianapolis, Ind.) were anesthetized with Inactin (100 
mg/kg; intraperitoneal; BYK Gulden, Konstanz, Germany). The body 
temperature was maintained between 36.degree. and 38.degree. C. by a 
heating pad. Tracheostomy was performed; however, the animals were not 
artificially ventilated. Polyethylene catheters (PE-50; Becton Dickinson 
Co., Parsippany, N.J.) were placed in the left Jugular vein for infusions 
of saline and drugs, in the right jugular vein to right atrium to monitor 
right atrial pressure, and in the carotid artery for the collection of 
blood samples and to monitor mean arterial pressure. A PE-90 catheter was 
placed in the bladder for urine collection. 
Experiments were conducted in three groups in normal rats: ANP group (n=4), 
CNP group (n=4), and VNP group (n=4). VNP also was studied in SHR rats 
(n=4). Intravenous infusions of saline solution (0.9% NaCl) were performed 
(1 ml/100 gm body weight/hr) through the left jugular vein catheter. After 
completion of surgery, rats were allowed to stabilize for 30 minutes. In 
each group, a 15-min baseline period followed. After the baseline period, 
saline solution (0.9% NaCl) was administered in a bolus fashion (0.1 ml) 
and was followed by a 15-min period. After the saline period, the peptide 
(ANP, CNP or VNP) was administered in a bolus fashion (0.1 ml) at 5 
.mu.g/kg which was followed by a 15-min period. This was followed by a 
second bolus (0.1 ml) at 50 .mu.g/kg and a 15-min period. After a 30-min 
washout, a 15-min recovery period followed. During each experimental 
period, mean arterial pressure (MAP), heart rate (HR), and right atrial 
pressure (RAP) were measured. At the midpoint of baseline, second bolus 
fashion (50 .mu.g/kg) and recovery periods, blood was sampled for plasma 
cGMP. At the end of each period, urine was measured for volume (UV), and 
samples were stored for electrolytes and cGMP analysis. 
Blood for plasma cGMP analysis was collected into EDTA tubes, immediately 
placed on ice, and centrifuged at 2,5000 rpm at 4.degree. C. Plasma was 
separated and stored at -20.degree. C. until assay. Urine for cGMP 
determination was heated to &gt;90.degree. C. before storage. Plasma and 
urine cGMP were determined by a specific RIA as previously described by A. 
L. Steiner et al., J. Hypertension, 5 (Suppl. 5) 551-553 (1987). 
Table 2 summarizes the cardiovascular and renal actions of ANP, CNP and VNP 
administration in normal rats. 
TABLE 2 
__________________________________________________________________________ 
The cardiorenal actions of ANP, CNP and VNP in normal rats 
Baseline 
Saline 5 .mu.g/kg 
50 .mu.g/kg 
Recovery 
__________________________________________________________________________ 
ANP group (n = 4) 
MAP (mmHg) 119 .+-. 14 
120 .+-. 14 
97 .+-. 12* 
69 .+-. 7*+ 
92 .+-. 11*.paragraph. 
HR (beats/min) 
375 .+-. 23 
365 .+-. 17 
353 .+-. 14 
355 .+-. 19 
368 .+-. 23 
RAP (mmHg) 0.8 .+-. 1.6 
0.5 .+-. 1.8 
-2.3 .+-. 0.8* 
-3.8 .+-. 0.8*+ 
-0.8 .+-. 0.9.paragraph. 
UV (.mu.l/min) 
6.7 .+-. 1.2 
7.2 .+-. 1.8 
79 .+-. 10* 
341 .+-. 41*+ 
48 .+-. 14.paragraph. 
UNaV (.mu.mol/min) 
0.4 .+-. 0.1 
0.4 .+-. 0.1 
13.2 .+-. 3.5* 
68.2 .+-. 15.7*+ 
8.9 .+-. 3.5.paragraph. 
UKV (.mu.mol/min) 
1.8 .+-. 0.1 
0.7 .+-. 0.1 
10.3 .+-. 3.5 
24.4 .+-. 8.7 
2.4 .+-. 0.1 
PcGMP (pmol/ml) 
4.1 .+-. 0.7 
12 .+-. 2 
-- 25.7 .+-. 7.1* 
3.2 .+-. 0.3.paragraph. 
UcGMPV 
(pmol/min) 
11 .+-. 2 
12 .+-. 2 
605 .+-. 151* 
2580 .+-. 505*+ 
105 .+-. 63.paragraph. 
CNP group (n = 4) 
MAP (mmHg) 116 .+-. 11 
115 .+-. 12 
100 .+-. 12* 
87 .+-. 9*+ 
104 .+-. 11*.paragraph. 
HR (beats/min) 
350 .+-. 9 
360 .+-. 14 
363 .+-. 19 
365 .+-. 19 
363 .+-. 16 
RAP (mmHg) 1.2 .+-. 0.9 
0.2 .+-. 1.4 
0 .+-. 1.5 
-2.3 .+-. 0.6* 
-0.1 .+-. 0.9* 
UV (.mu.l/min) 
5.7 .+-. 0.5 
5.7 .+-. 0.6 
27.6 .+-. 2.0*.sctn. 
66.4 .+-. 7.4*+.sctn. 
21.2 .+-. 6.3.paragraph. 
UNaV (.mu.mol/min) 
0.6 .+-. 0.2 
0.6 .+-. 0.2 
4.1 .+-. 1.4.sctn. 
12.1 .+-. 3.2*+.sctn. 
2.9 .+-. 0.3 
UKV (.mu.mol/min) 
1.2 .+-. 0.3 
1.2 .+-. 0.3 
5.1 .+-. 1.6 
7.6 .+-. 2.5 
2.4 .+-. 1.1 
PcGMP (pmol/ml) 
2.2 .+-. 0.1 
-- -- 19.4 .+-. 2.7* 
3.6 .+-. 0.3.paragraph. 
UcGMPV 
(pmol/min) 
23 .+-. 7 
22 .+-. 6 
97 .+-. 32.sctn. 
323 .+-. 81*.sctn. 
70 .+-. 39.paragraph. 
VNP group (n = 4) 
MAP (mmHg) 112 .+-. 14 
109 .+-. 12 
93 .+-. 15* 
79 .+-. 15*+ 
93 .+-. 15*.paragraph. 
HR (beats/min) 
390 .+-. 24 
398 .+-. 22 
393 .+-. 20 
408 .+-. 25 
390 .+-. 20 
RAP (mmHg) -2.5 .+-. 0.6 
-2.1 .+-. 0.7 
-3.5 .+-. 0.9 
-4.3 .+-. 0.8* 
-2.9 .+-. 0.9.paragraph. 
UV (.mu.l/min) 
6.5 .+-. 1.1 
6.4 .+-. 0.3 
32.2 .+-. 7.5*.sctn. 
136 .+-. 5.5*+.sctn.# 
8.4 .+-. 2.7.paragraph. 
UNaV (.mu.mol/min) 
0.9 .+-. 0.3 
0.7 .+-. 0.2 
5.3 .+-. 1.9 
22.5 .+-. 2.2*+.sctn.# 
0.9 .+-. 0.3.paragraph. 
UKV (.mu.mol/min) 
0.9 .+-. 0.1 
1.0 .+-. 0.2 
3.3 .+-. 1.4 
11.9 .+-. 4.7 
1.3 .+-. 0.6 
PcGMP (pmol/ml) 
3.7 .+-. 1.2 
-- -- 33.3 .+-. 8.7* 
4.4 .+-. 1.4.paragraph. 
UcGMPV 
(pmol/ml) 
13 .+-. 7 
12 .+-. 6 
113 .+-. 27*.sctn. 
961 .+-. 82*+.sctn.# 
88 .+-. 48.paragraph. 
__________________________________________________________________________ 
Mean .+-. SEM 
*p &lt; .05 vs Baseline 
+ p &lt; .05 vs 5 .mu.g/kg 
.paragraph. p &lt; .05 vs 50 .mu.g/kg 
.sctn. p &lt; .05 vs ANP group 
# p &lt; .05 vs CNP group 
ANP atrial natriuretic peptide 
CNP Ctype natriuretic peptide 
VNP vasonatrin peptide 
MAP mean arterial pressure 
HR heart rate 
RAP right atrial pressure 
UV urine volume 
UNaV urine sodium excretion 
UKV urine potassium excretion 
PcGMP plasma cGMP 
UcGMPV urine cGMP volume 
As demonstrated by the data in Table 2, bolus administration (0.1 ml) of 
saline solution had no cardio-vascular or renal actions. Bolus 
administration (0.1 ml) of high dose (50 .mu.g/kg) ANP, CNP and VNP 
resulted in a significant decrease in MAP and RAP, and increased urine 
flow, sodium excretion, plasma cGMP and urinary cGMP volume. The increase 
in urine flow, sodium excretion and urinary cGMP volume were significantly 
higher with VNP than those of CNP, but were less than those of ANP. 
Table 3 reports the cardiovascular and renal effects of VNP in normal and 
SHR rats. 
TABLE 3 
__________________________________________________________________________ 
The cardiorenal actions of VNP in normal and SHR rats 
Baseline 
Saline 5 .mu.g/kg 
50 .mu.g/kg 
Recovery 
__________________________________________________________________________ 
Normal rats (n = 4) 
MAP (mmHg) 112 .+-. 14 
109 .+-. 12 
93 .+-. 15* 
79 .+-. 15*+ 
93 .+-. 15*.paragraph. 
HR (beats/min) 
390 .+-. 24 
398 .+-. 22 
393 .+-. 20 
408 .+-. 25 
390 .+-. 20 
RAP (mmHg) -2.5 .+-. 0.6 
-2.1 .+-. 0.7 
-3.5 .+-. 0.9 
-4.3 .+-. 0.8* 
-2.9 .+-. 0.9.paragraph. 
UV (.mu.l/min) 
6.5 .+-. 1.1 
6.4 .+-. 0.3 
32.2 .+-. 7.5* 
136 .+-. 5.5*+ 
8.4 .+-. 2.7.paragraph. 
UNaV (.mu.mol/min) 
0.9 .+-. 0.3 
0.7 .+-. 0.2 
5.3 .+-. 1.9 
22.5 .+-. 2.2*+ 
0.9 .+-. 0.3.paragraph. 
UKV (.mu.mol/min) 
0.9 .+-. 0.1 
1.0 .+-. 0.2 
3.3 .+-. 1.4 
11.9 .+-. 4.7 
1.3 .+-. 0.6 
PcGMP (pmol/ml) 
3.7 .+-. 1.2 
-- -- 33.3 .+-. 8.7* 
4.4 .+-. 1.4.paragraph. 
UcGMPV 
(pmol/min) 
13 .+-. 7 
12 .+-. 6 
113 .+-. 27* 
961 .+-. 82*+ 
88 .+-. 48.paragraph. 
SHR rats (n = 4) 
MAP (mmHg) 169 .+-. 17.sctn. 
168 .+-. 17.sctn. 
132 .+-. 23* 
119 .+-. 20*+ 
138 .+-. 19*.paragraph. 
HR (beats/min) 
388 .+-. 13 
390 .+-. 11 
393 .+-. 13 
405 .+-. 10 
405 .+-. 17 
RAP (mmHG) 0.5 .+-. 1.2 
0.5 .+-. 1.3 
-1.1 .+-. 0.8 
-2.5 .+-. 0.6*+ 
-0.9 .+-. 1.0.paragraph. 
UV (.mu.l/min) 
3.7 .+-. 0.3.sctn. 
3.8 .+-. 0.7.sctn. 
26.8 .+-. 3.5* 
48.2 .+-. 7.4*+.sctn. 
9.1 .+-. 3.7.paragraph. 
UNaV (.mu.mol/min) 
0.5 .+-. 0.2 
0.6 .+-. 0.2 
5.2 .+-. 1.6* 
9.2 .+-. 2.1*+.sctn. 
1.7 .+-. 0.7.paragraph. 
UKV (.mu.mol/min) 
0.6 .+-. 0.2 
0.6 .+-. 0.2 
3.9 .+-. 1.5 
6.6 .+-. 2.6 
1.1 .+-. 0.4 
PcGMP (pmol/ml) 
3.1 .+-. 0.7 
-- -- 17.7 .+-. 3.6* 
3.1 .+-. 0.5.paragraph. 
UcGMPV 
(pmol/min) 
9 .+-. 1 
8 .+-. 2 
115 .+-. 27* 
234 .+-. 42*+.sctn. 
26 .+-. 12.paragraph. 
__________________________________________________________________________ 
Mean .+-. SEM 
*p &lt; .05 vs Baseline 
+ p &lt; .05 vs 5 .mu.g/kg 
.paragraph. p &lt; .05 vs 50 .mu.g/kg 
.sctn. p &lt; .05 vs normal rats 
SHR spontaneous hypertensive rat 
VNP vasonatrin peptide 
MAP mean arterial pressure 
HR heart rate 
RAP right arterial pressure 
UV urine volume 
UNaV urine sodium excretion 
UKV urine potassium excretion 
PcGMP plasma cGMP 
UcGMPV urine cGMP volume 
As shown by the data in Table 3, the baseline MAP of SHR was significantly 
higher than that of normal rats, and the baseline urine volume of SHR was 
markedly lower than that of normal rats. During high dose bolus infusion 
of VNP, MAP and RAP significantly decreased, urine flow, sodium excretion, 
plasma cGMP and urinary cGMP volume were significantly increased in both 
normal and SHR groups. While the lowering of MAP to VNP was similar in 
both groups, the renal actions and urinary cGMP effects of VNP were 
attenuated in SHR rats as compared to normal rats. VNP is a more potent, 
endothelium independent vasorelaxing peptide in both arteries and veins as 
compared to ANP and CNP. VNP also has a potent natriuretic effect in vivo. 
Examples 1 and 2 confirm that ANP is a potent natriuretic and vasoactive 
peptide, which has vasorelaxing actions in renal and pulmonary arteries. 
As shown by K. J. Koller et al., Science, 251, 120 (1991), these 
biological actions of ANP are mediated through the ANPR-A receptor. CNP 
has potent vasodilator actions in veins with less actions in arteries, 
while not exhibiting natriuretic effects. See C. M. Wei et al., Am. J. 
Physiol., 264, H71 (1993); A. J. Stingo et al., Am. J. Physiol., 262, H308 
(1992). The biological actions of CNP are mediated through ANPR-B 
receptor. (See K. J. Koller, cited above.) In the present study, CNP was 
also confirmed to exhibit hemodynamic actions similar to those previously 
reported. However, CNP in the rat, unlike the dog, has modest but 
significant natriuretic actions. 
The structural difference between ANP and CNP is a difference in amino acid 
sequences and CNP lacks a COOH-terminus. Thus, preferred peptides of the 
invention share properties of both ANP and CNP, suggesting that 
COOH-terminus may play an important role for biological activity, possibly 
by leading to binding to both the ANPR-A and ANPR-B receptors. 
Furthermore, in femoral vein and pulmonary artery, CNP has more potent 
effects in vessels without endothelium, but the actions of VNP are similar 
with and without the presence of endothelium. This may be due to 
endothelial mechanism(s) which inhibit the actions of CNP but not VNP. 
This may also be related to the presence of the VNP COOH-terminus. 
Because VNP has potent arterial and venous vasodilating actions and 
natriuretic effects, the peptides of the invention may be useful in the 
treatment of cardiovascular and renal diseases, such as hypertension, 
congestive heart failure, and eclampsia. In preliminary studies, VNP also 
exhibits potent vasorelaxing actions in isolated vessels in congestive 
heart failure dogs. Moreover, this peptide is also able to relax vessels 
pre-contracted with endothelin, which is a potent endothelium-derived 
vasoconstrictor. 
All publications and patents cited herein are herein incorporated by 
reference to the same extent as if each individual publication or patent 
was specifically and individually indicated to be incorporated by 
reference. 
It will be apparent to one of ordinary skill in the art that many changes 
and modifications can be made in the invention without departing from the 
spirit or scope of the appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 17 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Mature human atrial natriuretic peptide 
(ANP) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
SerLeuArgArgSerSerCysPheGlyGlyArgMetAspArgIleGly 
151015 
AlaGlnSerGlyLeuGlyCysAsnSerPheArgTyr 
2025 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Mature human brain natriuretic peptide 
(BNP) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
SerProLysMetValGlnGlySerGlyCysPheGlyArgLysMetAsp 
151015 
ArgIleSerSerSerSerGlyLeuGlyCysLysValLeuArgArgHis 
202530 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Mature human C-type natriuretic peptide 
(CNP) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GlyLeuSerLysGlyCysPheGlyLeuLysLeuAspArgIleGlySer 
151015 
MetSerGlyLeuGlyCys 
20 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Vasonatrin peptide (VNP) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GlyLeuSerLysGlyCysPheGlyLeuLysLeuAspArgIleGlySer 
151015 
MetSerGlyLeuGlyCysAsnSerPheArgTyr 
2025 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Fragment of X1 of Formula I 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GlyLeuSerLysGly 
15 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Fragment of X1 of Formula I 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
LeuSerLysGly 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Amino acid residue of Xy of Formula I 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
ArgPheSerAsn 
1 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Amino acid residue of Xy of Formula I 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TyrArgPheSerAsn 
15 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Formula II 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GlyLeuSerLysGlyCysPheGlyLeuXaaXaaAspArgIleGlySer 
151015 
MetSerGlyLeuGlyCys 
20 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
ArgMetAspArgIle 
15 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
LysMetAspArgIle 
15 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
LysLeuAspArgIle 
15 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
LysIleAspArgIle 
15 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
ArgLeuAspArgIle 
15 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
GlyLeuSerLysGly 
15 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
AsnSerPheArgTyr 
15 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
GlySerMetSerGlyLeuCys 
15 
__________________________________________________________________________