# Patent

## Title
NPR-B agonists

## Abstract

Disclosed are novel compounds having NPR-B agonistic activity. Preferred compounds are linear peptides containing 8-13 conventional or non-conventional L- or D-amino acid residues connected to one another via peptide bonds.

## Background
The present application is a divisional application of U.S. patent application Ser. No. 14/011,855, filed Aug. 28, 2013, and which issued as U.S. Pat. No. 9,169,293, issued on Oct. 27, 2015, which is a divisional application of U.S. patent application Ser. No. 12/888,556 filed Sep. 23, 2010, and which issued as U.S. Pat. No. 8,546,523, issued on Oct. 1, 2013, which claims priority to U.S. provisional application Ser. No. 61/245,960 filed Sep. 25, 2009, each of which is incorporated by reference in its entirety.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 16, 2016, is named 2006685_1199_ST25 and is 392,326 bytes in size.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to novel compounds which are useful in the treatment and prevention of disorders mediated by natriuretic peptides or proteins. More particularly, the present invention relates to novel peptides, pharmaceutical compositions comprising one or more novel peptides described herein, and their use in methods of treating or preventing ocular disorders, such as glaucoma, ocular hypertension, and optic neuropathies, cardiovascular disease, kidney disease, lung disease, and other disorders mediated by natriuretic peptides or proteins.
2. Description of Related Art
The natriuretic peptides (NP's) are a family of cyclic peptide hormones that have first been described by their involvement in the regulation of natriuresis, diuresis and blood pressure control. To date, four natriuretic peptides have been discovered in man, i.e. atrial natriuretic peptide (ANP; SEQ ID NO:1), B-type or brain natriuretic peptide (BNP; SEQ ID NO:2), C-type natriuretic peptide (CNP; SEQ ID NO:3) and urodilatin (SEQ ID NO:4) (see FIG. 1; and Cho et al., 1999, Heart Dis. 1:305-328). All NP's are synthesized as prepro-hormones which are activated by proteolytic cleavage before their release into the circulation. The NP's bind to natriuretic peptide receptors (NPR), a group of 3 different membrane bound receptors with guanylyl cyclase activity (Pandey 2005, Peptides 26:901-932).
ANP was first discovered as a blood pressure decreasing factor in rat atrial homogenates in 1981 (de Bold 1981, Life Sci 28:89-94). Human pre-pro-ANP (SEQ ID NO: 5) contains 151 amino acids and is stored after N-terminal cleavage as 126 amino acid pro-ANP (SEQ ID NO:6), predominantly in atrial granules. Cardiac stretch, due to systemic volume overload induces the rapid release of ANP from these stores. Upon secretion into the circulation, the C-terminal part of pro-ANP is cleaved by the atrial peptidase corin to the biologically active 28 amino acid form of ANP (SEQ ID NO:1) (Yan 2000, Proc Natl Acad Sci 97:8525-8529). The remaining N-terminal part can be further cleaved into 3 different hormones. i.e. Long Acting Natriuretic Peptide (LANP, amino acids 1-30; SEQ ID NO:7), Vessel Dilator (VSDL, amino acids 31-67; SEQ ID NO:8) and Kaliuretic Peptide (KP, amino acids 79-98; SEQ ID NO:9) (Vesely 2004, Eur J Clin Invest 34:674-682).
After BNP was discovered in porcine brain as a factor that showed smooth muscle relaxing activity (Sudoh T, 1988, Nature 332:78), a much greater tissue expression was found in preparations of cardiac ventricles (Mukoyama 1991, J Clin Invest 87:1402-1412), which led to the conclusion that BNP is, similarly to ANP, a cardiac peptide hormone. Although BNP can be found in storage granules in the atria, the expression in ventricles is transcriptionally regulated (Tamura 2000, Proc Natl Acad Sci 93:4239-4244). Synthesis of pre-pro-BNP is induced through cardiac wall stretch and leads to a 134 amino acid long peptide (SEQ ID NO:10) which is further cleaved by an unknown protease to yield the 108 amino acid long pro-BNP (SEQ ID NO:11). Additional cleavage liberates the active 32 amino acid C-terminal fragment of BNP (SEQ ID NO:2) and the inactive 76 amino acid N-terminal fragment also referred to as NT-pro-BNP (SEQ ID NO:12). To date, no known splice variants of human BNP exists.
CNP was first isolated from porcine brain almost 10 years after the discovery of ANP (Sudoh 1990, Biochem Biophys Res Comm 168:863-870). It is primarily expressed in the central nervous system and endothelial cells. Unlike other NP's, CNP is nearly not present in cardiac tissue, which suggest a more paracrine function on vascular tone and muscle cell growth. The 126 amino acid precursor molecule pro-CNP (SEQ ID NO: 13) is processed by the intracellular endoprotease furin into the mature 53 amino acid peptide CNP-53 (SEQ ID NO:14), which is the most abundant form in the brain (Totsune 1994, Peptides 15:37-40), endothelial cells (Stingo, 1992, Am J Phys 263:H1318-H1321) and the heart (Minamino 1991, Biochem Biophys Res Comm 179:535-542). In both, cerebral spinal fluid (Togashi 1992, Clin Chem 38:2136-2139) and human plasma (Stingo 1992, Am J Phys 263:H1318-H1321) the most common form is CNP-22 (SEQ ID NO:3), which is generated from CNP-53 by an unknown extracellular protease. Unlike the other NP's CNP-22 lacks the C-terminal extension of the 17 amino acid ring (see FIG. 1).
ANP (SEQ ID NO:1), BNP (SEQ ID NO:2) and CNP (SEQ ID NO:3) show a highly conserved amino acid sequence among different vertebrate species (see FIG. 1; and Cho 1999, Heart Dis. 1:305-328). The NP's are inactivated by two distinct mechanisms, i.e. enzymatic cleavage through neutral endopeptidases and binding to the NP clearance receptor (NPR-C; SEQ ID NO:15), which is followed by internalization and intracellular degradation of the NP (Stoupakis 2003, Heart Dis. 5:215-223).
The discovery of the natriuretic peptides ANP, BNP and CNP was followed by the description and cloning of their specific receptors, natriuretic peptide receptor-A, -B and -C (NPR-A, -B, -C) (Fuller 1988, J Biol Chem. 263:9395-9401; Chang 1989 Nature 341:68-72; Chinkers 1989, Nature 338:78-83). NPR-A (SEQ ID NO:16) preferentially binds ANP and BNP, while NPR-B (SEQ ID NO:17) is most specific for CNP and NPR-C(SEQ ID NO:15) binds all natriuretic peptides (Koller 1991, Science 252:120-123).
The primary structure of NPR-A and NPR-B contain an extracellular ligand binding domain, transmembrane domain, intracellular kinase homology domain containing phosphorylation sites and a C-terminal guanylate cyclase domain (reviewed in Misono 2005, Peptides 26:957-68). The latter classifies NPR-A and NPR-B as particulate guanylate cyclases, also known as GC-A and GC-B (E.C.4.6.1.2). In contrast, NPR-C is lacking intracellular homology domains, but evidence is increasing for NPR-C's role not only as a scavenger receptor for natriuretic peptides, but for its' functional coupling to inhibitory G-proteins and phosphoinositide turnover (Maack 1987, Science 238:675-678; Murthy and Makhlouf 1999, J Biol Chem 274:17587-17592; Anand-Srivastava 2005, Peptides 26:1044-1059). Reflecting the grade of sequence homology in natriuretic peptides, natriuretic peptide receptors show a high degree of homology in their extracellular ligand binding domains, with the calculated similarities being 41% between NPR-A and NPR-B and 29% between NPR-A and NPR-C (van den Akker 2001, J Mol Biol. 311:923-937).
Ligand binding to NPRs requires a dimer of glycosylated receptor subunits (Fenrick et al. 1994, Mol Cell Biochem. 137:173-182; Kuhn 2003, Circ Res. 93:700-709) and is followed by a conformational change leading to activation of the guanylate cyclase domains. Subsequently, activity of particulate guanylate cyclases is regulated through phosphorylation (reviewed in Kuhn 2003, Circ Res. 93:700-709). Phosphorylation of NPRs is maximal in the basal state, while ligand binding is followed by dephosphorylation and subsequent desensitization of the receptor.
Natriuretic receptors are expressed in many tissues throughout the organism. NPR-A, NPR-B and NPR-C are present in the cardiovascular system and the kidney, with NPR-C being the most abundant receptor subtype accounting for 80% of NPR-expression in some tissues. NPR-B is present in a particularly high level in rat pineal gland, testis and ovaries. NPR-A and NPR-B ligands both induce endothelium-independent vasorelaxation, where ANP and BNP mainly act on arterial vasculature. In contrast, CNP mainly targets the venous system; with the exception of coronary arteries, that relax in response to CNP stimulation (Marton et al. 2005, Vascul Pharmacol 43:207-212). Importantly, induction of hypotension via NPR-B activation requires 10-fold higher concentrations of ligand compared to blood pressure reduction in response to NPR-A activation (Wei et al. 1993, Am J Physiol. 264:H71-H73; Woods and Jones 1999, Am J Physiol. 276:R1443-R1452). Relaxation of smooth muscle by activation of NPR-B has been shown in a variety of tissues, including blood vessels, seminiferous tubules and uterus. Also contraction of the ocular trabecular meshwork tissue is reduced by activation of natriuretic peptide receptors, confirming functional similarities of trabecular meshwork and smooth muscle cells (Stumpff and Wiederholt 2000, Ophthalmologica 214:33-53).
Another main target organ of natriuretic peptides is the kidney. Ligands of NPR-A induce natriuresis and diuresis by a dual mechanism (reviewed in Beltowski and Wojcicka 2002, Med Sci Monit. 8:RA39-RA52): (1) increased excretion of sodium by a reduced re-uptake of sodium ions in the distal tubulus, subsequently leading also to higher retention of water in the final urine: and (2) dilation of the affluent and concomitant contraction of the effluent glomerular capillary, increasing glomerular filtration rate, at the cost of reduction of renal perfusion (Endlich and Steinhausen 1997, Kidney Int. 52:202-207). In contrast to NPR-A-specific ligands, NPR-B-specific ligands do not induce significant natri- and diuresis, and in addition, show a peculiarity regarding glomerular flow regulation: CNP was shown to dilate both affluent and effluent capillaries in the glomerulus, thus increasing renal blood flow, but not glomerular filtration (Endlich and Steinhausen 1997, Kidney Int. 52:202-207).
In addition to effects of NP-receptor (NPR) activation on blood pressure and kidney function, powerful effects of natriuretic peptides on proliferative processes in a variety of cell types have been documented in the literature. Antiproliferative properties of NPR activation are documented for vascular smooth muscle cells, fibroblasts of different origins, mesangial cells, cancer cells and chondrocytes (reviewed in Schulz 2005, Peptides 26:1024-1034). At least for VSMC, evidence for the involvement of the transcription factor GAX in the regulation of proliferation has given an indication as to which intracellular mechanisms might be involved in growth regulation through NPR (Yamashita et al. 1997, Hypertension 29:381-387). Though tissue growth is mainly regulated by proliferative activity, some organs feature variations in cell size to influence tissue mass. This might be a physiological process, as during endochondral ossification, when chondrocytes mature by undergoing hypertrophy, or a pathological event, as in cardiac hypertrophy, which often precedes chronic heart failure. Both of the above-mentioned events of hypertrophy are regulated by NPR-B. NPR-B deficiency causes dwarfism due to abnormal endochondral ossification, characterized by size reduction of the hypertrophic zone of the epiphyseal growth plate (Bartels et al. 2004, Am J Hum Genet. 75:27-34; Tamura et al. 2004, Proc Natl Acad Sci. 101:17300-17305).
Quite different, a partial knock out of NPR-B in rats promoted cardiac hypertrophy, i.e. hypertrophy of cardiomyocytes (Langenickel et al. 2006, Proc Natl Acad Sci. 103:4735-4740).
Natriuretic peptides, having activity at the natriuretic receptors, were later discovered in various tissues, as well. For example, ANP was discovered in the early 1980s as an endogenous diuretic and vasorelaxant peptide, whose principle circulating form consists of 28 amino acids (SEQ ID NO:1). Subsequently, other natriuretic peptides, such as BNP (SEQ ID NO:2) and CNP (SEQ ID NO:3), were discovered. The presence of natriuretic peptides and their receptors in ocular tissues, especially those involved in the regulation of IOP, have been demonstrated. For example, in rat and rabbit eyes, ANP, BNP, and CNP, as well as NPR-A, NPR-B, and NPR-C mRNA were found in the ciliary processes, retina, and choroid (Mittag et al. 1987, Curr Eye Res. 6:1189-1196; Nathanson 1987, Invest Ophthalmol Vis Sci. 28:1357-1364; Fernandez-Durango et al. 1995, Exp Eye Res. 61:723-729). Similar results were found in bovine ciliary processes and cultured bovine ciliary epithelial cells. (Millar et al. 1997, J Ocul Pharmacol Ther. 13:1-11; Shahidullah and Wilson 1999, Br J Pharmacol. 127:1438-1446). The presence of the peptides and their receptors in the ciliary epithelium suggests that they may play a role in the production of aqueous humor.
In addition to the ciliary processes, natriuretic peptide receptors were also found in tissues associated with the outflow of aqueous humor. ANP binding sites were localized in the longitudinal ciliary muscle of the guinea pig. (Mantyh et al. 1986, Hypertension. 8:712-721). In cultured human TM and ciliary muscle cells, CNP is the most potent and efficacious in stimulating the production of cyclic GMP, indicating the presence of functional NPR-B. Activation of this receptor reduces carbachol-induced calcium influx. (Pang et al. 1996, Invest Ophthalmol Vis Sci. 37:1724-1731). This result predicts that activation of NPR-B should cause relaxation of these tissues. Indeed, CNP significantly decreases the carbachol-induced contraction of monkey and human ciliary muscles. (Ding and Abdel-Latif, 1997, Invest Ophthalmol Vis Sci. 38:2629-2638). Change in contractility in TM and ciliary muscle may affect the outflow facility of aqueous humor.
Cyclic GMP and compounds that increase cyclic GMP in ocular tissues, such as nitric oxide donors, have been shown to lower IOP. (Nathanson 1988, Eur J Pharmacol. 147:155-156; Becker 1990, Invest Ophthalmol Vis Sci. 31:1647-1649; Nathanson 1992, J Pharmacol Exp Ther. 260:956-965; Stein and Clack 1994, Invest Ophthalmol Vis Sci. 35:2765-2768). Since natriuretic peptides potently increase cyclic GMP production, they were predicted to lower IOP, too. In the past 20 years, the natriuretic peptides have been shown to be highly effective as IOP-lowering agents. For example, various researchers have independently shown that intravitreal injection of ANP in rabbits consistently and significantly lowers IOP. This effect lasts for many hours. (Sugrue and Viader, 1986, Eur J Pharmacol. 130:349-350; Mittag et al. 1987, Curr Eye Res. 6:1189-1196; Nathanson 1987 Invest Ophthalmol Vis Sci. 28:1357-1364; Korenfeld and Becker 1989, Invest Ophthalmol Vis Sci. 30:2385-2392; Takashima et al. 1996, Invest Ophthalmol Vis Sci. 37:2671-2677). The IOP effect of ANP correlates with an increase in cyclic GMP production in the iris-ciliary body. (Korenfeld and Becker 1989, Invest Ophthalmol Vis Sci. 30:2385-2392). Intravitreal injection of BNP (Takashima et al. 1996, Invest Ophthalmol Vis Sci. 37:2671-2677) or CNP (Takashima et al. 1998, Exp Eye Res. 66:89-96) is also highly efficacious in lowering IOP. In addition to intravitreal injection, subconjunctival (Yang et al. 1997, Chin J Ophthalmol. 33:149-151) or intracameral (Sugrue and Viader 1986, Eur J Pharmacol. 130:349-350; Fernandez-Durango et al. 1999, Eur J Pharmacol. 364:107-113) injection of the natriuretic peptides have been shown to be ocular hypotensive as well. Systemic administration of ANP in the rabbit, (Tsukahara et al. 1988, Ophthalmologica. 197:104-109) or human (Diestelhorst and Krieglstein 1989, Int Ophthalmol. 13:99-101) also lowers IOP. Unfortunately, it has not been possible to deliver these peptides topically due to their inability to penetrate the cornea. Therefore, these potent and efficacious IOP-lowering compounds have not been developed for such use.
There is a need for novel NPR-B agonists having improved bioavailability, as compared to isolated or synthesized natriuretic peptides, that can be used in the treatment of natriuretic peptide-mediated disorders, such as ocular disorders, diabetes-related disorders, vascular disorders, cardiac and cardiovascular pathologies, inflammation and other disorders described herein. The novel NPR-B agonists, compositions and methods of the present invention meet these needs.
SUMMARY OF THE INVENTION
The present invention provides novel NPR-B agonists, also referred to herein as natriuretic peptide mimics or similars, that are therapeutically useful for lowering intraocular pressure (IOP) and treating other disorders where activation of the type B natriuretic peptide receptor will be beneficial. Specifically, the invention provides novel NPR-B agonists that activate the type B natriuretic peptide receptor (NPR-B). The invention further provides compositions containing such novel NPR-B agonists. The compositions provided herein may be ophthalmic compositions for use in methods of treating or preventing particular ophthalmic diseases such as glaucoma, preferably by lowering intraocular pressure, using such novel NPR-B agonists. Alternatively, the compositions provided herein may be used in methods of treating or preventing cardiovascular disorders, kidney disease, lung disease, skeletal disorders, infertility, and other disorders mediated by natriuretic peptides or proteins.
The invention is in part based on the inventors' finding that the novel NPR-B agonists described herein can provide improved bioavailability, increased chemical stability, and increased metabolic stability in body fluids or tissues, due to their significantly reduced molecular size as compared to the known natriuretic peptides. Certain embodiments of the present application generally pertain to novel peptides containing modified amino acids and that bind to and activate NPR-B with high specificity, as described in more detail herein.
It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
As used herein, the term “NPR-B agonist” refers to the novel molecules described herein that activate the NPR-B with high potency.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device and/or method being employed to determine the value.
As used herein the specification, “a” or “an” may mean one or more, unless clearly indicated otherwise. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE FIGURES
The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1. Illustrates the amino acid sequence of ANP (SEQ ID NO:1), BNP (SEQ ID NO:2) and CNP (SEQ ID NO:3).
FIG. 2. Illustrates the effects of CNP, ANP, BNP and mini-ANP (SEQ ID NO:18) on cyclic GMP production in GTM-3 cells. GTM-3 cells have been shown to express NPR-B (Pang et al. 1996, Invest Ophthalmol Vis Sci. 37:1724-1731). The cells were treated with CNP (triangles), ANP (squares), BNP (diamonds) and mini-ANP (circles). The symbols represent mean values and standard deviations. The highest concentration of compounds used was 45 μM for ANP, BNP and mini-ANP and 5 μM for CNP. EC50 values were determined using the 4-Parameter Logistic Equation. CNP EC50=38.8 nM, ANP EC50=1.63 μM, BNP EC50=1.18 μM, mini-ANP EC50>45 μM. The Emax (maximum activation) of each compound was determined relative to the maximum activation of CNP, i.e. CNP Emax=100%, ANP Emax=15%, BNP Emax=20% and mini-ANP Emax=0%.
FIG. 3. Illustrates the effects of CNP, ANP, BNP and mini-ANP on cyclic GMP production in NPR-A transfected 293-T cells. NPR-A transfected 293-T cells were treated with CNP (triangles), ANP (squares), BNP (diamonds), and mini-ANP (circles). The symbols represent mean values and standard deviations. EC50 was determined using the 4-Parameter Logistic Equation. EC50 of ANP=73.0 nM, EC50 of CNP=1.60 μM, EC50 of BNP=1.85 μM, EC50 of mini-ANP=1.54 μM.



DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention is in part based on the finding that novel NPR-B agonists having improved bioavailability as compared to known natriuretic peptides are useful for lowering elevated intraocular pressure and treating glaucoma. Thus, the present invention is generally directed to novel NPR-B agonists and their use in methods of treating or preventing disorders mediated by natriuretic peptides or proteins. In one particularly preferred embodiment, the novel NPR-B agonists described herein are formulated for the treatment of ophthalmic diseases such as glaucoma, preferably by lowering the elevated intraocular pressure often associated with glaucoma, using a pharmaceutical composition that comprises one or more novel NPR-B agonists, as described herein. In other preferred embodiments, the novel NPR-B agonists described herein are formulated for the treatment of other natriuretic peptide- or protein-mediated disorders such as cardiovascular disorders, kidney disorders, lung disorders, skeletal disorders, fertility disorders, and fibrosis.
The hallmark feature of all known NP's is the 17 amino acid ring which is formed by an intramolecular cysteine bridge (see FIG. 1). The integrity of the cyclic structure of NP's is believed to be critical for the functional activity, i.e. NP receptor transduced cGMP production. The present inventors have discovered that certain linear peptides, such as the novel peptides described herein, having increased chemical and metabolic stability and the improved bioavailability as compared to known NP's, are useful in the treatment of natriuretic peptide- or protein-mediated disorders.
A. Novel Peptides
The present invention provides novel NPR-B agonists having biological activity that is improved in certain aspects as compared to that of the known natriuretic peptides. The novel peptides of the invention include conventional and non-conventional amino acids. Conventional amino acids are identified according to their standard, three-letter codes, as set forth in Table 1, below.







TABLE 1







For conventional amino acids the 3-letter codes were used:










3-letter codes
Amino acids
3-letter codes
Amino acids





Ala
Alanine
Met
Methionine


Cys
Cysteine
Asn
Asparagine


Asp
Aspartic acid
Pro
Proline


Glu
Glutamic acid
Gln
Glutamine


Phe
Phenylalanine
Arg
Arginine


Gly
Glycine
Ser
Serine


His
Histidine
Thr
Threonine


Ile
Isoleucine
Val
Valine


Lys
Lysine
Trp
Tryptophane


Leu
Leucine
Tyr
Tyrosine









Non-conventional amino acids are identified according to a three-letter code, or other abbreviation, when present in the novel NPR-B agonists of the invention. Table 2, below, provides the full name, three-letter code or abbreviation, and structure of each non-conventional amino acid appearing in the sequences of the novel peptides described herein.







TABLE 2







List of abbreviations of non-conventional amino acids and other chemical structures.









Name
Abbr
Structure





(S)-2-((S)-3-amino-2,5- dioxopyrrolidin-1-yl)-5- guanidinopentanoic acid
Dim-Arg






 


rac-2-amino-4- morpholinobutanoic acid
AR-385- 017






 


(S)-2-amino-3-(2H- tetrazol-5-yl) propanoic acid
AR-314- 145






 


rac (1S,2S)-2- (octylcarbamoyl) cyclohexane carboxylic acid
AR-314- 171






 


rac (1S,2S)-2- (hexylcarbamoyl) cyclohexane carboxylic acid
AR-314- 170






 


rac (1R,2S)-2- octylcarbamoyl) cyclohexane carboxylic acid
AR-314- 169






 


(S)-2-(6-hexanamido-1- oxoisoindolin-2-yl)-3- phenylpropanoic acid
AR-385- 008






 


(S)-2-(4-octanamido-1,3- dioxoisoindolin-2-yl)-3- phenylpropanoic acid
AR-314- 172






 


(S)-2-(5-hexanamido-1,3- dioxoisoindolin-2-yl)-3- phenylpropanoic acid
AR-385- 042






 


(S,S)-2-(3-methyl-3- octanoylamino-2-oxo- pyrrolidin-1-yl)-3- phenyl-propionic acid
AR-314- 102






 


2-(7-Octanoyl-1-oxo-2,7- diaza-spiro[4.5]dec-2-yl)- 3-phenyl- propionic acid
AR-314- 087






 


1-(3-Methyl-butyl)- piperazine
AR-201- 124






 


Cycloheptyl-pyrrolidin-2- ylmethyl-amine
ES-283- 049






 


(S)-Amino-thiophen-2- yl-acetic acid
BB727






 


(R)-Amino-thiophen-2- yl-acetic acid
BB726






 


2-Octylsulfanyl- propionic acid
AR-201- 073






 


5-Pentylsulfanylmethyl- oxazole-2-carboxylic  acid
AR-201- 072






 


4-(4-Butyl-thiazol-2- ylamino)-benzoic acid
AR-201- 069






 


4-(5-Butyl-thiazol-2- ylamino)-benzoic acid
AR-201- 068






 


2-Hexylamino-oxazole-4- carboxylic acid
AR-201- 062






 


2-Hexanoylamino-oxazole- 4-carboxylic acid
AR-201- 059






 


3-Hexyloxy-isoxazole- 5-carboxylic acid
AR-201- 058






 


2-Hexanoylamino- isonicotinic acid
AR-201- 054






 


Octanoic acid 1-carboxy- ethyl ester
AR-201- 049






 


Dodecanoic acid 1-carboxy- 2-phenyl-ethyl ester
AR-201- 048






 


(R)-2-Amino-4- (piperidin-1-yl) butanoic acid
abu(pip)






 


8-amino-3,6- dioxaoctanoic acid
Adx






 


(2,3,4,5,6-Pentahydroxy- hexylidenaminooxy)- acetic acid
Gluc-Aoa






 


5-((4S)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol- 4-yl)pentanoic acid
 74






 


Adamantan-2-yl-amine
 504






 


Cyclohexylamine
 558






 


Cyclopentylamine
 559






 


2-((1S,2R,4R)-bicyclo [2.2.1]heptan-2-yl) acetic acid
 779






 


2-Phenethyl-benzoic acid
 785






 


Dodecanoic acid
 832






 


Aniline
 873






 


Octanesulfonyl chloride
 933






 


Hexyl chloroformate
1270






 


3-Phenyl-propionic acid
1281






 


4-Phenyl-butyric acid
1319






 


5-Phenyl-pentanoic acid
1320






 


4-Cyclohexyl-butyric acid
1339






 


3-Cyclohexyl-propionic acid
1340






 


(S)-3,3-dimethylbutan- 2-amine
1381






 


2-(hexylamino)acetic acid
1625-Ac






 


Piperidine-1,2-dicarboxylic acid 1-benzyl ester
1695






 


4-Methyl-cyclohexyl-amine
1859






 


(1R,2R)-2-methyl- cyclohexanamine
1860






 


2-(2-Methoxy-ethoxy)- ethoxy]-acetic acid
1888






 


(1R,2R,4R)-bicyclo[2.2.1] heptan-2-amine
1906






 


(2-Methoxy-ethoxy)-acetyl chloride
1913






 


(1R,2R)-2-(benzyloxy) cyclohexanamine
1934






 


(S)-1,2,3,4-tetrahydro- naphthalen-1-amine
2118






 


(S)-3-methylpiperidine
2137






 


4-(4-Methoxy-phenyl)- butyric acid
2553






 


(1R,2R,4R)-1,7,7- trimethylbicyclo[2.2.1] heptan-2-amine
2797






 


2-((2S,3R,4R,5R)-2,3,4,5,6- pentahydroxyhexylamino) acetic acid
2857-Ac






 


Cyclobutyl-amine
2906






 


(S)-2-cyclopentylhexanoic acid
3218






 


3-Amino-4-hydroxy- benzoic acid
3421






 


1-Ethyl-propyl-amine
3791






 


(R)-2-methylbutan-1- amine
3806






 


2-Ethyl-butyl-amine
3816






 


3-(4-Bromo-phenyl)- propionic acid
4703






 


(4-Butoxy-phenyl)-acetic acid
4734






 


(1S,2R)-2-aminocyclo- hexanecarboxamide
5116






 


(1R,2S)-ethyl 2-amino- cyclohexanecarboxylate
5118






 


(1R,2R)-ethyl 2-amino- cyclohexanecarboxylate
5119






 


1-Propyl-butyl-amine
5121






 


(S)-3-amino-1-ethyl- azepan-2-one
5164






 


Decanoic acid
5587






 


(2-Butoxy-ethoxy)-acetic acid
6013






 


(E)-dodec-2-enoic acid
6014






 


(Z)-dodec-5-enoic acid
6015






 


(2S)-2-octylcyclopropane- carboxylic acid
6056






 


3-Octylsulfanyl-propionic acid
6057






 


7-Butylsulfanyl-heptanoic acid
6058






 


3-(Octane-1-sulfinyl)- propionic acid
6059






 


3-(Octane-1-sulfonyl)- propionic acid
6059(O)






 


rac-6-Hydroxy-decanoic acid
(6071-OH)






 


rac-7-Hydroxy-dodecanoic acid
(6072-OH)






 


5-Butyl-2H-pyrrazole-3- carboxylic acid
6182






 


2-Pentyl-benzooxazole-5- carboxylic acid
6988






 


(R)-2-aminobutanoic acid
abu






 


3-Amino-1-carboxymethyl- pyridin-2-one
Acp






 


(S)-2-((S)-3-amino-2- oxopyrrollidin-1-yl)-3- phenylpropanoic acid
AFL






 


(S)-2-((R)-3-amino-2- oxopyrrollidin-1-yl)-3- phenylpropanoic acid
aFL






 


(R)-2-((R)-3-amino-2- oxopyrrolidin-1-yl)-3- phenylpropanoic acid
afL






 


2-Aminoisobutyric acid
Aib






 


2-Aminoindan-2-carboxylic acid
Aic






 


rac-α-Methyl-leucine
Aml






 


(R)-α-methyl-proline
Amp






 


1-Aminomethyl- cyclopropanecarboxylic  acid
Amcp






 


4-Amino-piperdine-4- carboxylic acid
Apc






 


4-Amino-1-(2-amino- ethyl)-piperidine-4- carboxylic acid
Apc(Ae)






 


4-Amino-1-ethyl- piperidine-4- carboxylic acid
Apc(Et)






 


4-Amino-1-methyl- piperidine-4- carboxylic acid
Apc(Me)






 


(2S,4S)-4-aminopyrrolidine- 2-carboxylic acid
Apr






 


Azetidine-3-carboxylic acid
Az3






 


(S)-azetidin-2-carboxylic acid
Aze






 


(R)-azetidin-2-carboxylic acid
aze






 


β-Alanine
Bal






 


(S)-β-Homolysine
Bhk






 


(2S,4R)-4-(benzyloxy) pyrrolidine-2- carboxylic acid
Bhp






 


(R)-β-homoleucine
Ble






 


rac-2-amino-3-phenyl- butyric acid
Bmf






 


(S)-2-((S)-3-(carboxy- methyl)-2- oxopiperazin-1-yl)-5- guanidinopentanoic acid
cDR






 


(S)-β-cyclohexylalanine
Cha






 


Cycloheptyl-amine
Che






 


(S)-Cyclohexylglycine
Chg






 


(2S,4S)-4-hydroxy- pyrrolidine-2- carboxylic acid
Chy






 


(S)-2-amino-2-cyclopropyl- acetic acid
Cpa






 


(S)-2-amino-2-cyclopentyl- acetic acid
Cpg






 


rac-(3R,4S)-cis-methano- proline
Cpp






 


(S)-2-amino-3-(tert-butylthio) propanoic acid
ctb






 


(S)-2-Amino-3-sulfo- propanoic acid
Cya






 


(R)-2,4-diaminobutanoic acid
dab






 


(R)-2-amino- 3-(neopentylamino) propanoic acid
dap(1464)






 


(R)-2-amino-3-(bis(2- aminoethyl) amino)propanoic acid
dap(6263)2






 


(R)-2-amino-3-(bis((1H- imidazol-2-yl)methyl) amino)propanoic acid
dap(3846)2






 


(R)-2-amino-3-(piperidin- 4-ylmethylamino) propanoic acid
dap(6238)






 


((R)-2-amino-4- (dimethylamino) butanoic acid
dab(Me2)






 


(R)-2,3-diaminopropanoic acid
dap






 


(S)-2-amino-3- (dimethylamino) propanoic acid
Dap(Me2)






 


(R)-2-amino-3- (dimethylamino) propanoic acid
dap(Me2)






 


2-Amino-2-ethyl-butyric acid
Deg






 


2-Aminoacrylic acid
Dha






 


(S)-2,5-dihydro-1H- pyrrole-2- carboxylic acid
Dhp






 


(R)-2,2-dimethyl- thiazolidine-4- carboxylic acid
Dtp






 


(S)-3,4-dichloro- phenylalanine
Eaa






 


(S)-2-(3-amino-2- oxoazepan-1-yl) acetic acid
Eah






 


rac-Imidazolidine-2- carboxylic acid
Eal






 


(S)-4-methyl-2-((S)-6- oxo-1,7-diazaspiro [4.4]nonan-7-yl) pentanoic acid
Eam






 


rac-1-amino-2,3- dihydro-1H-indene- 1-carboxylic acid
Eao






 


2,3-Dihydro-1H-indole- 2-carboxylic acid
Eat






 


(2S,4S)-4-phenyl- pyrrolidine-2- carboxylic acid
Eay






 


(R)-thiazolidine-4- carboxylic acid
Eaz






 


1-Aminocyclopropane- carboxylic acid
Ebc






 


(R)-2-amino-3- (methylsulfanyl) propanoic acid
Ebe






 


1-Amino-cyclopentane- carboxylic acid
Eca






 


2-Amino-3-piperidin-4-yl- propionic acid
Egg






 


1-aminocyclohexane- carboxylic acid
Egz






 


(1S,3R)-3-amino- cyclohexane carboxylic acid
Fio






 


trans-4-(aminomethyl) cyclohexane carboxylic acid
Fir






 


Amino-piperidin-3-yl- acetic acid
Fhy






 


(S)-2-amino-2-(piperidin-4- yl)acetic acid
Fhz






 


(2S,4S)-4-fluoropyrrolidine- 2-carboxylic acid
Fpr






 


4-aminobutyric acid
Gab






 


(R)-2-amino-3- guanidinopropanoic acid
gdp






 


(2S,4R)-4-guanidino- pyrrolidine-2- carboxylic acid
Gup






 


(2S,3S)-3-hydroxypyrrolidine-2- carboxylic acid
H3p






 


Hexanoic acid
Hex






 


(S)-homo-phenylalanine
Hfe






 


(S)-2-aminooctanoic acid
Hgl






 


(R)-2-aminooctanoic acid
hgl






 


(S)-2-amino-5- methylhexanoic acid
Hle






 


(S)-homo-serine
Hse






 


(R)-homo-serine
hse






 


(2S,4R)-4-hydroxypyrrolidine- 2-carboxylic acid
Hyp






 


Piperidine-4-carboxylic acid
Inp






 


Dodecane
Lau






 


(R)-2-amino-6-(dimethylamino) hexanoic acid
lys(Me2)






 


3-Aminomethyl-benzoic acid
Mam






 


(R)-2-amino-4-(methylsulfonyl) butanoic acid
metO2






 


(S)-meta-chloro-phenylalanine
Mcf






 


(S)-4-hydroxy-3-Iodo- phenylalanine
Miy






 


(S)-meta-methyl-phenylalanine
Mmf






 


(S)-3-(3-Pyridyl)-alanine
Mpa






 


(3-Amino-phenyl)-acetic acid
Mpe






 


(S)-meta-trifluoromethyl- phenylalanine
Mtf






 


(R)-2-amino-4- guanidinobutanoic acid
nar






 


rac-(2,3-Dihydroxy- propylamino)-acetic acid
Nbhp






 


4-Butyl-thiazole
Nbt






 


(3-Hydroxy-propylamino)acetic acid
Nhpr






 


Phenethylamino-acetic acid
NHfe






 


(S)-para-nitro-phenylalanine
Nif






 


rac-Nipecotic acid
Nip






 


(S)-Norleucine
Nle






 


(R)-Norleucine
nle






 


(S)-N-methyl-alanine
Nma






 


(S)-N-methyl-aspartic acid
Nmd






 


(S)-N-methyl-phenylalanine
Nmf






 


(S)-N-methyl-isoleucine
Nmi






 


(S)-N-methyl-lysine
Nmk






 


(S)-N-methyl-leucine
Nml






 


(S)-N-methyl-arginine
Nmr






 


(S)-2-amino-4,4-dimethyl- pentanoic acid
Npg






 


4,4-Dimethyl-2- methylamino-pentanoic acid
SH-112- 158






 


Benzylamino-acetic acid
NPhe






 


(S)-4-methyl-2-(propylamino) pentanoic acid
Npl






 


(S)-norvaline
Nva






 


(R)-norvaline
nva






 


Octanoic acid
Occ






 


octane
Oct






 


(2S,3aS,7aS)-octahydro-1H- indole-2-carboxylic acid
Oic






 


(S)-3-(2-pyridyl)-alanine
Opa






 


(S)-ornithine
Orn






 


(R)-ornithine
orn






 


(R)-2-amino-5-(dimethylamino) pentanoic acid
orn(Me2)






 


(S)-ortho-trifluoro-phenylalanine
Otf






 


Piperazin-1-yl-acetic acid
Paa






 


(S)-para-amino-phenylalanine
Paf






 


(4-Aminomethyl)-benzoic acid
Pam






 


(S)-para-bromo-phenylalanine
Pbf






 


(2S,3R)-3-aminopyrrolidine- 2-carboxylic acid
Pca






 


(S)-para-chloro-phenylalanine
Pcf






 


(S)-para-fluoro-phenylalanine
Pff






 


(S)-phenylglycine
Phg






 


(S)-pipecolinic acid
Pip






 


(R)-pipecolinic acid
pip






 


(S)-para-methyl-phenylalanine
Pmf






 


(S)-para-methoxy- phenylalanine
Pmy






 


(S)-3-(4-Pyridyl)-alanine
Ppa






 


(4-Amino-phenyl)-acetic acid
Ppe






 


(S)-2-amino-3-(phosphonooxy) propanoic acid
Pse






 


(2S,3R)-2-Amino-3- (phosphonooxy) butanoic acid
Pth






 


Sarcosine
Sar






 


5-Butyl-thiazole
Sbt






 


(S)-nipecotic acid
Sni






 


(2S,4R)-4-aminopyrrolidine-2- carboxylic acid
Tap






 


(2S,4R)-4-(dimethylamino) pyrrolidine-2-carboxylic acid
Tap(2Me)






 


(2S,4R)-4-acetamido- pyrrolidine-2-carboxylic acid
Tap(Ac)






 


(2S,4R)-4-(2-aminoethylamino) pyrrolidine-2-carboxylic acid
Tap(Ae)






 


(2S,4R)-4-(S)-3-amino-3- carboxypropaneamido) pyrrolidine-2-carboxylic  aicd
Tap(Asp (−))






 


4-(3-Amino-propylamino)- pyrrolidine-2-carboxylic acid
Tap(Ap)






 


(2S,4R)-4-(3-amino- propanamido)pyrrolidine- 2-carboxylic acid
Tap(Bal)






 


(2S,4R)-4- (diethylamino)pyrrolidine-2- carboxylic acid
Tap(Et2)






 


(2S,4R)-4-(ethylamino) pyrrolidine-2-carboxylic  acid
Tap(Et)






 


(2S,4R)-4-(2-aminoacetamido) pyrrolidine-2-carboxylic acid
Tap(G)






 


(S)-α-tert-butylglycine
Tbg






 


(R)-α-tert-butylglycine
tbg






 


(2S,4R)-4-fluoropyrrolidine-2- carboxylic acid
Tfp






 


(S)-2-thienyl-alanine
Thi






 


(S)-3-thienyl-alanine
Thk






 


(S)-thiazolidine-4-carboxylic acid
Thz






 


(S)-1,2,3,4-tetrahydroiso- quinoline-3-carboxylic acid
Tic






 


4-Amino-thiazol-2-carboxylic acid
Tnc






 


(S)-2,3-Diamino-propionic acid (side chain prolongation)
Udp













The novel NPR-B agonists of the invention comprise the general amino acid sequence of Formula I:

B-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Z  (I)

wherein
B is selected from the group consisting of H, Rb1—, Rb2—C(O)—, Rb2—S(O2)—, Rb3-Baa-;
Baa is a conventional α-amino acid, a non-conventional α-amino acid or a β-amino acid;
Rb1 is selected from C1-C12 alkyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; C1-C12 alkenyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; C1-C12 alkyl aryl optionally substituted by NR4bRb5, OH, or ORb6; C1-C12 alkynyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; aryl C1-C12 alkyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; C1-C12 alkyl C3-C8 cyclic alkyl optionally substituted by NRb4Rb5, OH, ORb6, aryl, heteroaryl, or heterocyclyl; C3-C6 cyclic alkyl C1-C12 alkyl optionally substituted by NRb4Rb5, OH, ORb6, aryl, heteroaryl, or heterocyclyl; C1-C9 alkylthio C2-C10 alkyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; C1-C9 alkylsulfonyl C1-C4 alkyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; C1-C9 alkylsulfoxyl C1-C10 alkyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; CH3—(CH2)qb—O—[—CH2—(CH2)nbO]mb—CH2—(CH2)pb—, 2-thiazolo optionally substituted by C1-8 alkyl;
qb=0-3
nb=1-3
mb=1-3
pb=1-3
Rb2 is selected from C1-C12 alkyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; C1-C12 alkenyl optionally substituted by NRb4Rb5, OH, ORb6 C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; aryl C1-C12 alkyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; C1-C12 alkynyl optionally substituted by NRb4Rb5, OH, ORb6 C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; C1-C12 alkyl aryl optionally substituted by NRb4Rb5, OH, or ORb6; C1-C12 alkyl C3-C8 cyclic alkyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; C3-C6 cyclic alkyl C1-C12 alkyl optionally substituted by NRb4Rb5, OH, ORb6 C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; C1-C9 alkylthio C1-C10 alkyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; C1-C9 alkylsulfonyl C1-C10 alkyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl; C1-C9 alkylsulfoxyl C1-C4 alkyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl, CH3—(CH2)qb—O—[—CH2—(CH2)nbO]mb—CH2—(CH2)pb—;
qb=0-3
nb=1-3
mb=1-3
pb=0-3
Rb3 is selected from H, Rb1—, Rb2—C(O)—, or Rb2—S(O2)—;
Rb4, Rb5 and Rb6 are, independently, selected from a group consisting of H, or C1-C4 alkyl, and
Xaa1 is selected from the group consisting of a direct bond, a conventional α-amino acid; a non-conventional α-amino acid; a β-amino acid; a γ-amino acid; or a residue of Formula IIa-y:










R1a is selected from H, C1-C6 alkyl;
R1b is selected from H, C1-C6 alkyl optionally substituted by OH, hydroxyC1-C6 alkyl optionally substituted by OH;
R1c is selected from H, C1-C6 alkyl;
R1d is selected from H, C1-C6 alkyl;
R1a and R1b together may form a heterocyclic ring;
n1 is 0 to 3;
Xaa2 is an amino acid residue of Formula IIIa-g:







wherein
R2a is selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, C1-C2 alkyl C3-C7 cycloalkyl and aryl C1-C2 alkyl;
R2b and R2c are, independently, selected from the group consisting of H, methyl, ethyl, propyl; and isopropyl, with the proviso that at least one of R2b and R2c is H;
R2d represents from 0 to 3 substituents, each such substituent being, independently, selected from the group consisting of H, Cl, F, Br, NO2, NH2, CN, CF3, OH, OR2e and C1-C4 alkyl;
R2a and R2b or R2a and R2c together may form a heterocyclic ring;
R2e is selected from the group consisting of methyl, ethyl, propyl, and isopropyl; or
Xaa1 and Xaa2 together may be selected from an amino acid residue of Formula IVa-b




Xaa3 is selected from the group consisting of Gly, Ala, a conventional D-α-amino acid, a non-conventional D-α-amino acid, and an amino acid residue of Formula Va:




wherein R3a is selected from the group consisting of H or C1-C4 alkyl;
R3b is selected from the group consisting of H, —(CH2)n3a—X3a;
n3a is 1 to 5;
X3a is selected from the group consisting of H, NR3cR3d;
R3c and R3d are independently selected from a group consisting of H, C1-C8 alkyl, —(C═N)—NH2 and —(CH2)n3bX3b;
n3b is 1 to 4;
X3b is selected from the group consisting of NR3eR3f, C5-C6 heteroaryl, C4-C7 heterocyclyl, —NHC(═N)NH2;
R3e and R3f are independently selected from a group consisting of H, C1-C8 alkyl,
wherein R3e and R3f can form a cyclic structure;
R3a and R3b can be linked to form a cyclic structure;
or R3a and R3b can be linked with a heteroatom selected from the group consisting of N, O, and S, to form a heterocyclic structure;
or
Xaa2 and Xaa3 together may be selected from an amino acid residue of Formula Vb:




wherein R3g represents from 0 to 3 substituents, each such substituent being, independently, selected from the group consisting of H, Cl, F, Br, NO2, NH2, CN; CF3, OH, OR3h and C1-C4 alkyl;

R3h is selected from the group consisting of C1-C4 alkyl
Xaa4 is an amino acid residue of Formula VIa-h:




wherein R4a is selected from the group consisting of H, C1-C8 alkyl which may be substituted with a moiety selected from the group consisting of OH, CO2R4c, C(═O)—NH2, a 5-6 membered heteroaryl, C1-C10 alkyl, C5-C8 cycloalkylC1-C10 alkyl, and C5-C8 cycloalkyl, —(CH2)n4a—X4a;
n4a is 1 or 2;
R4b is selected from the group consisting of H and methyl;
R4c is selected from the group consisting of H, and C1-C3alkyl; and
X4a is OH, CO2R4d, NR4eR4f, SR4g, 4-imidazoyl, 4-hydroxyphenyl;
R4d, R4e and R4f independently are selected from the group consisting of H, and C1-C3 alkyl;
R4g is selected from the group consisting of C1-C3 alkyl;
m4a, and m4b are independently selected from 0 or 1;
R4h is C2-C6 alkyl;
or
Xaa3 and Xaa4 together may be selected from an amino acid residue of Formula VIb-h;
Xaa5 is an amino acid residue of Formula VII:




wherein R5a is (CH2)n5a—X5a;
n5a is 1 to 6;
X5a is selected from the group consisting of H, NH2, and a C4-7 amine-containing aliphatic heterocyclic ring;
R5b is selected from the group consisting of H and methyl;
R5c is selected from the group consisting of H and methyl;
and wherein R5c and R5a can combine to form a four to six membered heterocyclic ring or can be linked with a heteroatom selected from the group consisting of N, O, and S to form a monocyclic or bicyclic heterocyclic structure; wherein said heterocyclic ring may have from 0 to 3 substituents, each such substituent being, independently, selected from the group consisting of OH, OR5d, F, C1-C4 alkyl, —NHC(═NH)NH2, aryl and NR5eR5f;
R5d is selected from C1-C4 alkyl, C1-C4 alkylaryl;
R5e is selected from the group consisting of H, C1-C4 alkyl, —C(═O)(CH2)n5b—X5b, —CH2(CH2)n5c—X5b;
R5f is selected from the group consisting of H, C1-C4 alkyl, —CH2(CH2)n5d—X5c;
n5b is selected from the group consisting of 1, 2, 3, and 4;
n5c and n5d are independently selected from the group consisting of 2, 3, and 4;
X5b and X5c are independently selected from the group consisting of H, NR5gR5h;
R5g and R5h are independently selected from a group consisting of H, C1-C4 alkyl;
Xaa6 is an amino acid residue of Formula VIIIa-d:




wherein R6a is selected from the group consisting of C1-C8 alkyl, aryl C1-C4 alkyl, C4-C7 cycloalkyl C1-C4 alkyl, C1-C4 alkyl S(C1-C4alkyl), and C4-C7 cycloalkyl, wherein said C1-C8 alkyl and C4-C7 cycloalkyl may be substituted with a moiety selected from the group consisting of OH, O(C1-C4 alkyl), S(C1-C4 alkyl), and NR6dR6e;
R6b is H;
R6c is selected from the group consisting of H, and C1-C4alkyl;
R6d, and R6e are, independently, selected from the group consisting of H, and C1-C4 alkyl;
wherein R6a and R6c can form a cyclic structure, which may be substituted with a moiety selected from the group consisting of OH, C1-C4 alkyl, NH2 and F;
or R6a and R6c can be linked with a heteroatom selected from the group consisting of N, O, and S, to form a heterocyclic structure;
or
Xaa5 and Xaa6 together may be an amino acid residue of Formula VIIIe:




Xaa7 is an amino acid residue of Formula IXa-b:




wherein R7a is selected from the group consisting of C1-C4 alkyl, C3-C7 cycloalkyl, 2-thienyl, (CH2)n7a—X7a, and C1-C4 alkyl substituted with OH;
R7b is H, and 2-thienyl;
R7c is selected from a group consisting of H, and methyl;
R7d is C1-C4 alkyl;
n7d is selected from the group consisting of 1 and 2;
X7a is selected from the group consisting of 2-thienyl, C(═O)OR7e, C(═O)NH2, S(═O)2OH, OS(═O)2OH, B(OH)2, P(═O)(OH)2, and OP(═O)(OH)2;
wherein R7e is selected from the group consisting of H, and C1-C4 alkyl;
Xaa8 is an amino acid residue of Formula Xa-g:




wherein R8a is selected from the group consisting of (CH2)m8a—X8a, and a C4-C7 nitrogen-containing aliphatic heterocyclic ring;
m8a=1-5;
X8a is selected from the group consisting of H, NH2, and —NHC(═NH)NH2;
R8b is selected from the group consisting of H and methyl;
R8c is selected from the group consisting of H, NH2, and OH;
Y8a is selected from the group consisting of CH(R8d), and S;
R8d is selected from the group consisting H, aryl, and OH;
Y8b is selected from the group consisting of CH(R8e), and NH;
R8e is selected from the group consisting H, NH2 and OH;
Y8c is selected from the group CH2, and NR8f;
R8f is selected from the group H, —C(═NH)NH2, and —C(═O)CH2NH2;
or
Xaa7 and Xaa8 together may be an amino acid residue of Formula Xh:




Xaa9 is selected from the group consisting of a direct bond, and an amino acid residue of Formula XIa-c,




wherein R9a is selected from the group consisting of C1-C5 alkyl, and C4-C7 cycloalkyl;
R9b is selected from the group consisting of H, C1-C5 alkyl;
and wherein R9a and R9b can form a 5-7 membered cycloalkyl ring;
R9c is selected from the group consisting of H, methyl;
or
Xaa8 and Xaa9 together may be a residue of Formula XId:




and
Z is selected from the group consisting of H, OR11a, NHR11b a conventional α-amino acid, a non-conventional α-amino acid, a α-amino acid; and a peptide consisting of from 2 to 30 amino acids selected from the group consisting of conventional α-amino acids, non-conventional α-amino acids, and α-amino acids;
wherein R11a and R11b are independently selected from the group consisting of H, C1-C8 alkyl, C4-C8 cycloalkyl, C7-C12 bicycloalkyl, C7-C12 cycloalkylaryl, C1-C4 alkyl C4-C8 cycloalkyl, or a residue of formula XIIa-c:




As used herein, the phrase “optionally substituted” shall be understood by the skilled artisan to mean that the moiety to which the phrase refers may be unsubstituted, or it may be substituted with certain specified additional moieties. For example, the phrase “C1-C12 alkyl optionally substituted by NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, or heterocyclyl” refers to a C1-C12 alkyl compound that is either non-substituted or is substituted by a moiety selected from the group consisting of NRb4Rb5, OH, ORb6, C3-C8 cyclic alkyl, aryl, heteroaryl, and heterocyclyl. The compound, hexane, would be considered a C6 alkyl compound that is not substituted, while the compound 3-hexanol is a C6 alkyl compound that is substituted on the third carbon atom with an OH moiety.
In certain preferred NPR-B agonists of the invention:
B is selected from the group consisting of Rb1—, Rb2—C(O)—;
Rb1 is selected from C1-C12 alkyl optionally substituted by NRb4Rb5;
Rb2 is selected from C1-C12 alkyl optionally substituted by NRb4Rb5;
Rb4, and Rb5 are, independently, selected from a group consisting of H, and C1-C4 alkyl, and
Xaa1 is selected from the group consisting of a direct bond, a conventional α-amino acid; a non-conventional α-amino acid; β-amino acid; or a residue selected from the group consisting of Formula IIa, IIs, IIt, IIu, and IIv:




R1a is selected from H, C1-C6 alkyl;
R1b is selected from H, C1-C6 alkyl optionally substituted by OH, hydroxyC1-C6 alkyl optionally substituted by OH;
R1c is selected from H, C1-C6 alkyl;
R1a and R1b together may form a heterocyclic ring;
n1 is 0 to 3; and
Xaa2 is an amino acid residue of Formula IIIa or Formula IIIb:




wherein

R2a is selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, C1-C2 alkyl C3-C7 cycloalkyl and aryl C1-C2 alkyl;
R2b and R2c are, independently, selected from the group consisting of H, methyl, ethyl, propyl; and isopropyl, with the proviso that at least one of R2b and R2c is H;
R2d represents from 0 to 3 substituents, each such substituent being, independently, selected from the group consisting of H, Cl, F, Br, NO2, NH2, CN, CF3, OH, OR2e and C1-C4 alkyl;
R2a and R2b or R2a and R2c together may form a heterocyclic ring;
R2e is selected from the group consisting of methyl, ethyl, propyl, and isopropyl; and
Xaa3 is an amino acid residue of Formula Va:




wherein R3a is selected from the group consisting of H or C1-C4 alkyl;
R3b is selected from the group consisting of H, —(CH2)n3a—X3a;
n3a is 1 to 5;
X3a is selected from the group consisting of H, NR3cR3d;
R3c and R3d are independently selected from a group consisting of H, C1-C8 alkyl, —(C═N)—NH2 and —(CH2)n3bX3b;
n3b is 1 to 4;
X3b is selected from the group consisting of NR3eR3f, C5-C6 heteroaryl, C4-C7 heterocyclyl, —NHC(═N)NH2;
R3e and R3f are independently selected from a group consisting of H, C1-C8 alkyl,
wherein R3e and R3f can form a cyclic structure;
R3a and R3b can be linked to form a cyclic structure;
or R3a and R3b can be linked with a heteroatom selected from the group consisting of N, O, and S, to form a heterocyclic structure;
and
Xaa4 is an amino acid residue of Formula VIa:




wherein R4a is selected from the group consisting of H, C1-C8 alkyl which may be substituted with a moiety selected from the group consisting of OH, CO2R4c, C(═O)—NH2, a 5-6 membered heteroaryl, C1-C10 alkyl, C5-C8 cycloalkyl C1-C10 alkyl, and C5-C8 cycloalkyl;

    
    
        n4a is 1 or 2;
    
    


R4b is selected from the group consisting of H and methyl;
R4c is selected from the group consisting of H, and C1-3alkyl; and
and
Xaa5 is an amino acid residue of Formula VII:




wherein R5a is (CH2)n5a—X5a;
n5a is 1 to 6;
X5a is selected from the group consisting of H, NH2, and a C4-7 amine-containing aliphatic heterocyclic ring;
R5b is selected from the group consisting of H and methyl;
R5c is selected from the group consisting of H and methyl;
and wherein R5c and R5a can combine to form a four to six membered heterocyclic ring wherein said heterocyclic ring may have from 0 to 2 substituents, each such substituent being, independently, selected from the group consisting of OH, OR5d, F, C1-C4 alkyl, —NHC(═NH)NH2, aryl and NR5eR5f;
R5d is selected from C1-C4 alkyl, C1-C4 alkylaryl;
R5e is selected from the group consisting of H, C1-C4 alkyl, —C(═O)(CH2)n5b—X5b, —CH2(CH2)n5c—X5b;
R5f is selected from the group consisting of H, C1-C4 alkyl, —CH2(CH2)n5d—X5c;
n5b is selected from the group consisting of 1, 2, 3, and 4;
n5c and n5d are independently selected from the group consisting of 2, 3, and 4;
X5b and X5c are independently selected from the group consisting of H, NR5gR5h;
R5g and R5h are independently selected from a group consisting of H, C1-C4 alkyl and
Xaa6 is an amino acid residue of Formula VIIIa:




wherein R6a is selected from the group consisting of C1-C8 alkyl, aryl C1-C4 alkyl, C4-C7 cycloalkyl C1-C4 alkyl, C1-C4 alkyl S(C1-C4alkyl), and C4-C7 cycloalkyl, wherein said C1-C8 alkyl and C4-C7 cycloalkyl may be substituted with a moiety selected from the group consisting of OH, O(C1-C4 alkyl), and S(C1-C4 alkyl);
R6b is H;
R6c is selected from the group consisting of H, and C1-C4alkyl; and
Xaa7 is an amino acid residue of Formula IXa:




wherein R7a is selected from the group consisting of C1-C4 alkyl, C3-C7 cycloalkyl, 2-thienyl, and C1-C4 alkyl substituted with OH;
R7b is H, and 2-thienyl;
R7c is selected from a group consisting of H, and methyl;
and
Xaa8 is an amino acid residue of Formula X(a)-(g):




wherein R8a is (CH2)m8a—X8a;
m8a=1-5;
X8a is selected from the group consisting of H, NH2, and —NHC(═NH)NH2;
R8b is selected from the group consisting of H and methyl; and
Xaa9 is selected from the group consisting of a direct bond, and an amino acid residue of Formula XIa-c,




wherein R9a is selected from the group consisting of C1-C5 alkyl, and C4-C7 cycloalkyl;
R9b is selected from the group consisting of H, and C1-C5 alkyl;
or R9a and R9b can form a 5-7 membered cycloalkyl ring;
R9c is selected from the group consisting of H, and methyl;
and
Z is NHR11b;
wherein R11b is selected from the group consisting of H, C1-C8 alkyl, C4-C8 cycloalkyl, C7-C12 bicycloalkyl, C7-C12 cycloalkylaryl, C1-C4 alkyl C4-C8 cycloalkyl, or a residue of formula XIIa-c




In more preferred embodiments of the present invention, B is selected from the group consisting of Rb1—, and Rb2—C(O)—;
Rb1 is selected from the group consisting of C6-C10 alkyl and C6-C10 alkyl substituted by NRb4Rb5;
Rb2 is selected from the group consisting of C6-C10 alkyl and C6-C10 alkyl substituted by NRb4Rb5;
Rb4, and Rb5 are, independently, selected from a group consisting of H, and C1-C4 alkyl, and
Xaa1 is selected from the group consisting of a direct bond, a conventional α-amino acid; a non-conventional α-amino acid; a β-amino acid; a residue of Formula IIa, a residue of Formula IIs, a residue of Formula IIt, a residue of Formula IIu, and a residue of Formula IIv




wherein R1a is selected from H, and C1-C4 alkyl;
R1b is selected from H, C1-C4 alkyl optionally substituted by OH, and hydroxy C1-C4 alkyl optionally substituted by OH;
R1c is selected from H, C1-C6 alkyl;
R1a and R1b together may form a heterocyclic ring;
n1 is 0, 1; and
Xaa2 is an amino acid residue of Formula III:




wherein
R2a is selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, C1-C2 alkyl C3-C7 cycloalkyl and aryl C1-C2 alkyl;
R2b and R2c are, independently, selected from the group consisting of H, methyl, ethyl, propyl; and isopropyl, with the proviso that at least one of R2b and R2c is H;
R2d represents from 0 to 3 substituents, each such substituent being, independently, selected from the group consisting of H, Cl, F, Br, CN, CF3, OH, OR2e and C1-C4 alkyl;
R2e is selected from the group consisting of methyl, ethyl, propyl, and isopropyl; and
Xaa3 is an amino acid residue of Formula Va:




wherein R3a is selected from the group consisting of H and C1-C4 alkyl;
R3b is selected from the group consisting of H, and —(CH2)n3a—X3a;
n3a is 1 to 5;
X3a is selected from the group consisting of H, and NR3cR3d;
R3c and R3d are independently selected from a group consisting of H, C1-C8 alkyl, and —(C═N)—NH2;
R3a and R3b can be linked to form a cyclic structure;
or R3a and R3b can be linked with a heteroatom selected from the group consisting of N, O, and S, to form a heterocyclic structure;
and
Xaa4 is an amino acid residue of Formula VIa:




wherein R4a is selected from the group consisting of H, C1-C8 alkyl which may be substituted with a moiety selected from the group consisting of OH, and CO2R4c;
R4b is selected from the group consisting of H and methyl;
R4c is selected from the group consisting of H, and C1-C3alkyl; and
and
Xaa5 is an amino acid residue of Formula VII:




wherein R5a is (CH2)n5a—X5a;
n5a is 1 to 6;
X5a is selected from the group consisting of H, NH2, and a C4-7 amine-containing aliphatic heterocyclic ring;
R5b is selected from the group consisting of H and methyl;
R5c is selected from the group consisting of H and methyl;
and wherein R5c and R5a can combine to form a four to six membered heterocyclic ring wherein said heterocyclic ring may have from 0 to 2 substituents, each such substituent being independently selected from the group consisting of OH, F, C1-C4 alkyl, —NHC(═NH)NH2, aryl and NR5eR5f;
R5e is selected from the group consisting of H, C1-C4 alkyl, —C(═O)(CH2)n5b—X5b, and —CH2(CH2)n5c—X5b;
R5f is selected from the group consisting of H, C1-C4 alkyl, and —CH2(CH2)n5d—X5c;
n5b is selected from the group consisting of 1, 2, 3, and 4;
n5c and n5d are independently selected from the group consisting of 2, 3, and 4;
X5b and X5c are independently selected from the group consisting of H, and NR5gR5h;
R5g and R5h are independently selected from a group consisting of H, and C1-C4 alkyl and
Xaa6 is an amino acid residue of Formula VIIIa:




wherein R6a is selected from the group consisting of C1-C8 alkyl, aryl C1-C4 alkyl, C4-C7 cycloalkyl C1-C4 alkyl, and C4-C7 cycloalkyl, wherein said C1-C8 alkyl and C4-C7 cycloalkyl may be substituted with a moiety selected from the group consisting of OH, and O(C1-C4 alkyl);
R6b is H;
R6c is selected from the group consisting of H, and C1-C4alkyl; and
Xaa7 is an amino acid residue of Formula IX:




wherein R7a is selected from the group consisting of C1-C4 alkyl, C3-C7 cycloalkyl, 2-thienyl, and C1-C4 alkyl substituted with OH;
R7b is H, and 2-thienyl;
R7c is selected from a group consisting of H, and methyl;
and
Xaa8 is an amino acid residue of Formula Xa:




wherein R8a is (CH2)m8a—X8a;
m8a=1-5;
X8a is selected from the group consisting of H, NH2, and —NHC(═NH)NH2;
R8b is selected from the group consisting of H and methyl; and
Xaa9 is selected from the group consisting of a direct bond, and an amino acid residue of Formula XIa,




wherein R9a is selected from the group consisting of C1-C5 alkyl, and C4-C7 cycloalkyl;
R9b is selected from the group consisting of H, and C1-C5alkyl;
and wherein R9a and R9b can form a 5-7 membered cycloalkyl ring;
R9c is selected from the group consisting of H, and methyl;
and
Z is NHR11b;
wherein R11b is selected from the group consisting of H, C1-C8 alkyl, C4-C8 cycloalkyl, C7-C12 bicycloalkyl, C7-C12 cycloalkylaryl, and C1-C4 alkyl C4-C8 cycloakyl.
The sequences of the preferred novel NPR-B agonists of the invention are provided herein in typical peptide sequence format, as would be understood by the ordinary skilled artisan. For example, the three-letter code of a conventional amino acid, or the abbreviation for a non-conventional amino acid, indicates the presence of a particular amino acid in a specified position in the sequence of the molecule, each amino acid being connected to the next and/or previous amino acid by a hyphen. The hyphen, which represents a chemical bond, typically an amide bond, removes OH from the 1-carboxyl group of the amino acid when it is placed right of the abbreviation, and removes H from the 2-amino group (or the only present amino group in case of amino acids lacking a 2-amino group, e.g., Bal) of the amino acid when it is placed on the left of the abbreviation. It is understood that both modifications can apply to one amino acid.
In the case of additional functional groups in the side chains of conventional or non-conventional amino acids, only the 2-amino and/or the 1-carboxy group is used for the formation of peptide bonds.
The C-termini of the novel NPR-B agonists described herein are shown in explicit form by adding either OH, NH2 or an abbreviation for a specific terminating amine separated by a hyphen on the right of the abbreviation of the C-terminal amino acid.
These specific terminating amines are provided in Table 2 as full formulas and similar conventions with regard to hyphens and its structure in a peptide context apply to them, e.g.,
3791=NH2—CH(CH2—CH3)—CH2—CH3 
−3791=—NH—CH(CH2—CH3)—CH2—CH3 
The N-termini of the novel peptides described herein are shown in explicit form by adding either H (for a free N-terminus), or an abbreviation for a specific terminating carboxylic acid, sulfonic acid or another terminating group in front of the symbol of the N-terminal amino acid.
These specific terminating carboxylic acids, sulfonic acids or other terminating groups like alkyl are provided in Table 2 as full formulas and similar conventions with regard to hyphens and its structure in a peptide context apply to them, e.g.,
Hex=Hexanoic acid
Hex-=Hexanoyl-.
For conventional amino acids and some non-conventional amino acids, a 3-letter code was used where the first letter indicates the stereochemistry of the C-alpha-atom. For example, a capital first letter indicates that the L-form of the amino acid is present in the peptide sequence, while a lower case first letter indicates that the D-form of the correspondent amino acid is present in the peptide sequence.
In preferred embodiments of the present invention, the novel NPR-B agonist is an 8-13 amino acid peptide having a sequence as set forth in Table 3. The agonistic activity of the preferred compounds is also provided in Table 3 and was categorized based upon the following conventions:











NPR-B activation



(assayed with GTM-3 Cells)









EC50
Emax (CNP = 100%)
Group





≦1 μM
>50%
A


≦5 μM
>20%
B


≦15 μM 
>10%
C









The agonistic activity data of each compound was checked first to determine whether it fulfills the criteria for the activity group A. If it did not fulfill the criteria for activity group A, it was checked for group B criteria. If it did not fulfill the criteria for activity group A or activity group B, it was finally checked for group C criteria. If it did not fulfill the criteria for activity group C, it was not included in Table 3.
All examples in Table 3 are linear peptides written in three letter code where applicable. For non-conventional amino acids and other chemical moieties the abbreviations which are listed in Table 2 were used. In vitro activities reported in Table 3 resulted from experiments performed according to the methods described in Example 4.
In certain embodiments of the NPR-B agonists of the invention, in the compound of Formula I:
B will be selected from a bond, Occ, Oct, Sbt, 1319, 1320, and 5587;
Xaa1 will be selected from Gly, AR-201-49, AR-201-68, ala, abu, his, aze, pro, pip, thz, thi, asn, ser, His, Ala, Ser, Bal, Sni, Az3, and Gab;
Xaa2 will be selected from Phe, Pcf, Nmf, Pbf, Pff, Pmf, Eaa. Mcf, Thk, and Mtf;
Xaa3 will be selected from Gly, Aib, Ebc, a conventional D-α-amino acid, and a non-conventional D-α-amino acid, and will preferably be selected from Gly, Fhy, Apc, Egz, Aib, Ebc, ala, lys, lys(Me2), arg, leu, nle, ctb, abu, AR-385-12, Egg, ser, orn, orn(Me2), and dap(Me2);
Xaa4 will be selected from Leu, Nva, Nie, Hle, Npg, Cha, and Ala;
Xaa5 will be selected from Lys, Om, Hly, Hpa, Dab, Arg, N(alkyl) derivatives of any of the preceding amino acids, Nmk, Hpr, Pro, Tfp, Apr, Eaz, Hyp, Tap, Tap(G), Tap(Bal), Tap(Et), Tap(Ae), Tap(Ap), Amp, Pip, and Chy;
Xaa6 will be selected from a bond, Leu, Ile, Nml, Tap, Npg, SH-158, Dap(Me2), Cpg, Val, Tbg, Chg, Hle, Nle, and N(alkyl) derivatives of any of the preceding amino acids;
Xaa7 will be selected from Asp, Val, BB725, BB727, Ser, Thr, and Cya;
Xaa8 will be selected from Arg, Nmr, Pro, Eaz, Pca, Om, Fhz, Har, Nar, Cyr, Mmr, Dmr, Bmr, Opy, and N(alkyl) derivatives of any of the preceding amino acids;
Xaa9 will be selected from Ile, Tbg, Deg, Egz, Aml, 1860, Che, Nmi, Leu, Val, Ecb, and Eca; and
Xaa10 will be selected from a bond, Ser and a N(alkyl) derivative thereof.







TABLE 3







Preferred compounds according to the present invention and their agonistic


activity in in vitro assays.













SEQ ID
(M + H)+ in
Activity


Structure
JAL
NO:
MS [amu]
(group)














Hex-Ebe-pro-Phe-Gly-Leu-Pro-Ile-Asp-Arg-Ile-
JAL-
19
1446
C


Ser-Ebe-NH2;
0533





 


Hex-Ebe-pro-Phe-Gly-Leu-Lys-Ile-Asp-Arg-Ile-
JAL-
20
1477
C


Ser-Ebe-NH2;
0534





 


Hex-Ser-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
21
1391
C


Ser-Ser-NH2;
0535





 


Hex-Ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
22
1359
B


Ser-Ala-NH2;
0536





 


Hex-Ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
23
1345
C


Ser-Gly-NH2;
0537





 


Hex-Gly-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
24
1345
B


Ser-Ala-NH2;
0538





 


Hex-Gly-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
25
1331
B


Ser-Gly-NH2;
0539





 


Hex-Ebe-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
26
1334
C


Ser-NH2;
0540





 


Hex-Ebe-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
27
1247
C


NH2;
0541





 


Hex-Gab-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Ser-
JAL-
28
1348
C


Ebe-NH2;
0542





 


Hex-Mam-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Ser-
JAL-
29
1396
C


Ebe-NH2;
0543





 


Hex-Gly-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
30
1188
C


NH2;
0631





 


Hex-Ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
31
1202
C


NH2;
0632





 


Hex-Ser-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
32
1218
C


NH2;
0633





 


Hex-Pro-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
33
1228
C


NH2;
0634





 


Hex-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
34
1201
C


NH2;
0635





 


Hex-Gly-pro-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
35
1213
C


NH2;
0636





 


Hex-Ser-pro-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
36
1241
C


NH2;
0638





 


Hex-Mam-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
37
1193
C


NH2;
0647





 


Hex-Pam-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
38
1193
C


NH2;
0648





 


Hex-Mpe-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
39
1193
C


NH2;
0649





 


Hex-Ppe-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
40
1193
C



0650





 


Hex-Inp-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
41
1171
C



0651





 


Hex-Acp-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
42
1210
C



0652





 


Hex-Fir-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
43
1199
C



0653





 


Hex-Nip-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
44
1171
C



0654





 


Hex-Eah-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
45
1228
C



0656





 


Hex-Fio-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
46
1185
C



0657





 


Hex-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Eca-
JAL-
47
1199
C


NH2;
0692





 


1339-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
48
1255
C


NH2;
0693





 


Occ-pro-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
49
1184
C


NH2;
0694





 


1339-pro-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
50
1210
C


NH2;
0695





 


1320-pro-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
51
1218
C


NH2;
0696





 


Occ-Nip-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
52
1198
B



0697





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
53
1229
B


NH2;
0701





 


1340-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
54
1241
C


NH2;
0703





 


Hex-Tnc-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
55
1186
C


NH2;
0713





 


Hex-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Chg-
JAL-
56
1227
C


NH2;
0718





 


Hex-ala-ala-Phe-Paa-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
57
1157
C



0731





 


Occ-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
58
1158
C



0738





 


Occ-thz-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
59
1202
C


NH2;
0739





 


Occ-aze-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
60
1170
C



0740





 


Occ-Az3-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
61
1170
C


NH2;
0742





 


Occ-Sni-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
62
1198
B



0743





 


Occ-Rni-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
63
1198
C



0744





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-2137;
JAL-
64
1199
C



0748





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-3816;
JAL-
65
1201
C



0749





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-3806;
JAL-
66
1187
C



0751





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-565;
JAL-
67
1200
B



0752





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-2797;
JAL-
68
1252
B



0754





 


Occ-val-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
69
1186
C



0756





 


Occ-tbg-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
70
1200
C



0758





 


Occ-Amcp-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-
JAL-
71
1184
C


NH2;
0760





 


Occ-Ebc-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
72
1170
C



0761





 


Occ-abu-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
73
1171
C



0762





 


Occ-ser-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
74
1174
C



0763





 


Occ-ala-ala-Phe-Gly-Leu-Lys-leu-Asp-Arg-Ile-
JAL-
75
1229
C


NH2;
0769





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Ile-Asp-Arg-Ile-
JAL-
76
1229
C


NH2;
0770





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Val-Asp-Arg-Ile-
JAL-
77
1215
C


NH2;
0771





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Chg-Asp-Arg-Ile-
JAL-
78
1255
C


NH2;
0772





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Nle-Asp-Arg-Ile-
JAL-
79
1229
C


NH2;
0775





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Nml-Asp-Arg-Ile-
JAL-
80
1243
C


NH2;
0776





 


Occ-ala-ala-Phe-Gly-Leu-Pro-Leu-Asp-Arg-Ile-
JAL-
81
1214
B


NH2;
0781_






01





 


Occ-ala-ala-Phe-Gly-Leu-Nmk-Leu-Asp-Arg-Ile-
JAL-
82
1243
C


NH2;
0782





 


933-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
83
1208
C



0786





 


1270-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
84
1160
C



0787





 


4956-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-NH2;
JAL-
85
1144
C



0788





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-1860;
JAL-
86
1213
B



0789





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-504;
JAL-
87
1251
C



0790





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-559;
JAL-
88
1185
C



0791





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-3791;
JAL-
89
1187
C



0792





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Che;
JAL-
90
1212
B



0797





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-1859;
JAL-
91
1211
C



0798





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-1934;
JAL-
92
1304
B



0799





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-1906;
JAL-
93
1209
B



0801





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-873;
JAL-
94
1192
C



0824





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-5116;
JAL-
95
1241
C



0825





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-5119;
JAL-
96
1270
B



0826





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-5118;
JAL-
97
1270
C



0831





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-5163;
JAL-
98
1227
C



0833





 


Occ-ala-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-5164;
JAL-
99
1255
C



0834





 


Occ-ala-Phe-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
100
1127
C



0835





 


Occ-pro-Phe-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
101
1153
C



0836





 


Occ-Sni-Phe-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
102
1167
C



0837





 


Occ-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-1860;
JAL-
103
1141
B



0839





 


Occ-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Che;
JAL-
104
1141
C



0840





 


Occ-ala-Phe-Gly-Leu-Lys-Leu-Asp-Arg-5121;
JAL-
105
1143
C



0841





 


Occ-ala-Phe-Gly-Leu-Pro-Ile-Asp-Arg-Ile-NH2;
JAL-
106
1127
C



0894





 


Occ-ala-Phe-Gly-Leu-Pro-Nml-Asp-Arg-Ile-NH2;
JAL-
107
1141
B



0895





 


Occ-ala-Phe-Gly-Leu-Pro-Npg-Asp-Arg-Ile-NH2;
JAL-
108
1141
C



0896





 


Occ-ala-Phe-Gly-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
109
1143
B



0898





 


Occ-ala-Phe-Gly-Npg-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
110
1141
C



0903





 


Occ-ala-Nmf-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
111
1141
C



0906





 


Occ-ala-Phe-Gly-Leu-Pro-Leu-Asp-Nmr-Ile-NH2;
JAL-
112
1141
C



0921





 


Occ-ala-Phe-Gly-Leu-Pro-Leu-Asn-Arg-Ile-NH2;
JAL-
113
1127
C



0924





 


Occ-ala-Phe-Gly-Leu-Pro-Leu-Nva-Arg-Ile-NH2;
JAL-
114
1111
C



0926





 


Occ-ala-Phe-Gly-Leu-Pro-Leu-Val-Arg-Ile-NH2;
JAL-
115
1111
C



0927





 


Occ-ala-Phe-Gly-Leu-Pro-Leu-Thr-Arg-Ile-NH2;
JAL-
116
1113
C



0929





 


Occ-ala-Phe-Gly-Cha-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
117
1167
C



0940





 


Occ-ala-Phe-Gly-Nle-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
118
1127
C



0942





 


Occ-ala-Phe-Aib-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
119
1155
C



0943





 


Occ-ala-Phe-ala-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
120
1141
C



0944





 


Occ-ala-Phe-Ebc-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
121
1153
C



0945





 


Occ-ala-Mcf-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
122
1161
C



0946





 


Occ-Sar-Phe-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
123
1127
C



0950





 


Occ-Gly-Phe-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
124
1113
C



0951





 


Occ-aze-Phe-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
125
1139
B



0953





 


Occ-ala-Nmf-Gly-Leu-Pro-Nml-Asp-Arg-Ile-
JAL-
126
1155
B


NH2;
0954





 


Occ-pro-Phe-Gly-Leu-Pro-Nml-Asp-Arg-Ile-NH2;
JAL-
127
1167
B



0955_






01





 


Occ-Sni-Phe-Gly-Leu-Pro-Nml-Asp-Arg-Ile-NH2;
JAL-
128
1181
B



0956





 


Occ-pro-Nmf-Gly-Leu-Pro-Nml-Asp-Arg-Ile-
JAL-
129
1181
C


NH2;
0957





 


Occ-Sni-Nmf-Gly-Leu-Pro-Nml-Asp-Arg-Ile-
JAL-
130
1195
B


NH2;
0958_






01





 


Occ-ala-Phe-Gly-Leu-Pro-Hle-Asp-Arg-Ile-NH2;
JAL-
131
1141
C



0959





 


Occ-ala-Phe-Gly-Leu-Amp-Leu-Asp-Arg-Ile-
JAL-
132
1141
C


NH2;
0962





 


Occ-ala-Phe-Gly-Leu-Chy-Leu-Asp-Arg-Ile-NH2;
JAL-
133
1143
C



0964





 


Occ-pro-Nmf-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
134
1167
C



0966





 


Occ-Sni-Nmf-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
135
1181
C



0967_






01





 


Occ-ala-Phe-Gly-Leu-Apr-Leu-Asp-Arg-Ile-NH2;
JAL-
136
1142
B



0974





 


Occ-ala-Phe-Gly-Leu-Eay-Leu-Asp-Arg-Ile-NH2;
JAL-
137
1204
C



0975





 


Occ-ala-Phe-Gly-Leu-Fpr-Leu-Asp-Arg-Ile-NH2;
JAL-
138
1145
C



0978





 


Occ-ala-Phe-Gly-Leu-Dtp-Leu-Asp-Arg-Ile-NH2;
JAL-
139
1174
C



0979





 


Occ-ala-Phe-Gly-Leu-Eaz-Leu-Asp-Arg-Ile-NH2;
JAL-
140
1146
C



0980





 


Occ-Az3-Phe-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
141
1139
C



0985





 


Occ-ala-Phe-Gly-Leu-Pro-Leu-Asp-Arg-Tbg-
JAL-
142
1127
C


NH2;
0989





 


Occ-ala-Phe-Gly-Leu-Pro-Leu-Ser-Arg-Ile-NH2;
JAL-
143
1099
C



0992





 


Occ-ala-Phe-Gly-Leu-Pro-Leu-Hse-Arg-Ile-NH2;
JAL-
144
1113
C



0993





 


Occ-ala-Phe-Gly-Ile-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
145
1127
C



0995





 


Occ-ala-Phe-Gly-Nva-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
146
1113
C



0996





 


Occ-ala-Phe-Gly-Hle-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
147
1141
C



0998





 


Occ-ala-Thi-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
148
1133
C



1000





 


Occ-ala-Pcf-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
149
1161
C



1002





 


Occ-ala-Thk-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
150
1133
C



1003





 


Occ-ala-Mtf-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
151
1195
C



1005





 


Occ-ala-Mmf-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
152
1141
C



1006





 


Occ-ala-Phe-ser-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
153
1157
B



1010





 


Occ-ala-Phe-thr-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
154
1171
B



1011





 


Occ-ala-Phe-val-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
155
1169
C



1012





 


Occ-ala-Phe-leu-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
156
1183
B



1013





 


Occ-ala-Phe-nle-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
157
1183
B



1014





 


Occ-Sni-Phe-Gly-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
158
1197
B


NH2;
1015





 


Occ-ala-Phe-Gly-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
159
1157
B


NH2;
1016





 


Occ-ala-Phe-asn-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
160
1184
B



1017





 


Occ-ala-Phe-met-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
161
1201
B



1018





 


Occ-ala-Phe-abu-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
162
1155
B



1019





 


Occ-ala-Phe-dap-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
163
1156
B



1020





 


Occ-Sni-Phe-nle-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
164
1223
B



1021





 


Occ-Sni-Nmf-nle-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
165
1237
B



1022





 


Occ-Sni-Phe-nle-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
166
1239
A



1024





 


Occ-ala-Phe-nle-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
167
1199
B



1025





 


Occ-ala-Phe-leu-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
168
1199
B



1026





 


Occ-ala-Phe-nva-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
169
1185
B



1027





 


Occ-ala-Phe-phe-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
170
1029
B



1028





 


Occ-ala-Phe-ctb-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
171
1244
B



1029





 


Occ-ala-Phe-lys-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
172
1198
B



1030





 


Occ-ala-Phe-arg-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
173
1226
B



1031





 


Occ-ala-Phe-his-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
174
1207
B



1032





 


Ac-Hgl-ala-ala-Phe-Gly-Leu-Pro-Leu-Asp-Arg-
JAL-
175
1255
B


Ile-NH2;
1033





 


Ac-hgl-ala-ala-Phe-Gly-Leu-Pro-Leu-Asp-Arg-
JAL-
176
1255
B


Ile-NH2;
1034





 


Occ-pip-Phe-Gly-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
177
1167
C



1035





 


Occ-ala-Phe-Gly-Leu-Pro-Leu-cDR-Ile-NH2;
JAL-
178
1153
C



1037





 


Occ-ala-Phe-Gly-Leu-Bhp-Leu-Asp-Arg-Ile-NH2;
JAL-
179
1234
C



1038





 


Occ-ala-Phe-leu-Leu-Pro-Nml-Asp-Arg-Ile-NH2;
JAL-
180
1196
A



1039





 


Occ-Sni-Phe-leu-Leu-Pro-Nml-Asp-Arg-Ile-NH2;
JAL-
181
1236
A



1040





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
182
1253
A



1041





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
183
1213
A



1042





 


Occ-ala-Pcf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
184
1247
A



1043





 


Occ-ala-Phe-nle-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
185
1213
A



1044





 


Occ-ala-Phe-Gly-Leu-Pro-Npl-Asp-Arg-Ile-NH2;
JAL-
186
1169
C



1045





 


Occ-ala-Phe-arg-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
187
1242
A



1047





 


Occ-ala-Phe-asp-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
188
1201
C



1048





 


Occ-ala-Phe-glu-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
189
1215
C



1049





 


Occ-ala-Pcf-leu-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
190
1233
A



1050





 


Occ-ala-Pmf-leu-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
191
1213
B



1051





 


Occ-ala-Nmf-leu-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
192
1213
A



1052





 


Occ-pro-Phe-leu-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
193
1225
A



1053





 


Occ-pip-Phe-leu-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
194
1239
A



1054





 


Occ-ala-Phe-lys-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
195
1228
A



1060





 


Occ-ala-Phe-orn-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
196
1214
A



1061





 


Occ-ala-Phe-lys-Leu-Pro-Nml-Asp-Arg-Ile-NH2;
JAL-
197
1212
B



1065





 


Occ-ala-Phe-lys-Leu-Pro-Nml-Ala-Arg-Ile-NH2;
JAL-
198
1168
C



1068





 


Occ-ala-Phe-arg-Leu-Pro-Nml-Asp-Arg-Ile-NH2;
JAL-
199
1240
B



1075





 


Occ-ala-Nmf-arg-Leu-Pro-Nml-Asp-Arg-Ile-NH2;
JAL-
200
1254
B



1076





 


Occ-pip-Nmf-arg-Leu-Pro-Nml-Asp-Arg-Ile-NH2;
JAL-
201
1294
A



1077





 


Occ-pip-Phe-arg-Leu-Pro-Nml-Asp-Arg-Ile-NH2;
JAL-
202
1280
A



1078





 


Occ-ala-Nmf-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
203
1270
A


NH2;
1085





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
204
1256
A



1086





 


Occ-pip-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
205
1296
A



1087





 


Occ-ala-Phe-arg-Leu-Tfp-Leu-Asp-Arg-Ile-NH2;
JAL-
206
1244
B



1114





 


Occ-ala-Phe-Gly-Leu-Tfp-Leu-Asp-Arg-Ile-NH2;
JAL-
207
1145
B



1115





 


Occ-ala-Pbf-arg-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
208
1321
A



1116





 


Occ-ala-Phe-dab-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
209
1169
B



1120





 


Occ-ala-Phe-nar-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
210
1212
B



1121





 


Occ-ala-Phe-gdp-Leu-Pro-Leu-Asp-Arg-Ile-NH2;
JAL-
211
1198
B



1122





 


Oct-ala-Phe-arg-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
212
1227
B



1156_






02





 


Oct-pip-Phe-arg-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
213
1267
C



1157_






02





 


Occ-ala-Phe-arg-(KM-116-167)-Nml-Asp-Arg-Ile-
JAL-
214
1226
C


NH2;
1159





 


832-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
215
1241
B



1214





 


Occ-ala-Phe-arg-Leu-Hyp-Ile-Asp-Arg-Ile-NH2;
JAL-
216
1242
B



1224





 


Occ-ala-Phe-arg-Leu-Hyp-Npg-Asp-Arg-Ile-NH2;
JAL-
217
1256
A



1225





 


Occ-ala-Phe-arg-Leu-Hyp-Tbg-Asp-Arg-Ile-NH2;
JAL-
218
1242
C



1226





 


Occ-ala-Phe-arg-Leu-Hyp-Ebe-Asp-Arg-Ile-NH2;
JAL-
219
1246
B



1227





 


Occ-ala-Phe-arg-Leu-Lys-Nml-Asp-Arg-Ile-NH2;
JAL-
220
1271
B



1228





 


Occ-ala-Phe-arg-Leu-Nmk-Nml-Asp-Arg-Ile-
JAL-
221
1285
B


NH2;
1229





 


Occ-ala-Phe-arg-Leu-Nma-Nml-Asp-Arg-Ile-
JAL-
222
1228
C


NH2;
1230





 


Occ-ala-Phe-arg-Leu-Sar-Nml-Asp-Arg-Ile-NH2;
JAL-
223
1214
B



1231





 


Occ-ala-Phe-arg-Nva-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
224
1242
B



1232





 


Occ-ala-Phe-arg-Ebe-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
225
1260
B



1233





 


6014-Phe-arg-Leu-Hyp-Nml-Asp-Arg-IleNH2;
JAL-
226
1239
B



1237





 


6015-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
227
1239
B



1238





 


6054-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
228
1241
B



1239





 


6056-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
229
1239
B



1240





 


6057-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
230
1259
B



1241





 


6058-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
231
1259
B



1242





 


6059-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
232
1274
B



1243





 


832-Nmf-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
233
1255
C



1244





 


832-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
234
1196
B



1245





 


832-Phe-arg-Leu-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
235
1225
C



1246





 


Oct-Sni-FrL-Hyp-Leu-Asp-Arg-Ile-NH2;
JAL-
236
1268
B



1248





 


Occ-ala-Phe-Gly-Leu-Tap-Leu-Asp-Arg-Ile-NH2;
JAL-
237
1142
A



1249





 


Occ-ala-Phe-arg-Leu-Tap-Leu-Asp-Arg-Ile-NH2;
JAL-
238
1241
A



1250





 


Occ-ala-Phe-leu-Leu-Tap-Asp-Arg-Ile-NH2;
JAL-
239
1198
A



1251





 


Occ-ala-Phe-ser-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
240
1187
A



1252





 


Occ-Sni-Phe-ser-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
241
1227
B



1253





 


Occ-Sni-Phe-lys-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
242
1268
A



1254





 


Occ-Sni-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
243
1296
A



1255





 


Occ-Sni-Mpa-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
244
1254
C


NH2;
1256





 


Occ-Sni-Ppa-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
245
1254
C



1257





 


(6071-OH)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
246
1230
C


NH2;
1259





 


(6072-OH)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
247
1258
B


NH2;
1260





 


5587-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
248
1214
C



1261





 


Occ-ala-Phe-Gly-Leu-Tap(2Me)-Leu-Asp-Arg-Ile-
JAL-
249
1170
B


NH2;
1262





 


Occ-ala-Phe-arg-Leu-Tap(2Me)-Leu-Asp-Arg-Ile-
JAL-
250
1269
B


NH2;
1263





 


Occ-ala-Phe-leu-Leu-Tap(2Me)-Leu-Asp-Arg-Ile-
JAL-
251
1226
B


NH2;
1264





 


Occ-Sni-Phe-orn-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
252
1254
A


NH2;
1265





 


Occ-Sni-Opa-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
253
1254
B


NH2;
1266





 


Occ-ala-Nmf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
254
1227
A


NH2;
1267





 


Occ-ala-Nmf-lys-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
255
1242
B


NH2;
1268





 


Occ-ala-Nmf-orn-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
256
1228
B


NH2;
1269





 


Occ-ala-Phe-Gly-Leu-Gup-Leu-Asp-Arg-Ile-NH2;
JAL-
257
1184
B



1270





 


Occ-ala-Phe-arg-Leu-Gup-Leu-Asp-Arg-Ile-NH2;
JAL-
258
1283
B



1271





 


Occ-ala-Phe-leu-Leu-Gup-Leu-Asp-Arg-Ile-NH2;
JAL-
259
1240
B



1272





 


Oct-Sar-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
260
1242
B



1273





 


Oct-aze-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
261
1254
B



1274





 


Oct-Az3-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
262
1254
B


NH2;
1275





 


Occ-ala-Phe-leu-Leu-Eal-Nml-Asp-Arg-Ile-NH2;
JAL-
263
1198
B



1281_






01





 


Occ-ala-Phe-Gly-Leu-Eal-Nml-Asp-Arg-Ile-NH2;
JAL-
264
1144
C



1282





 


Occ-ala-Phe-leu-Leu-Hyp-(SH-158)-Asp-Arg-Ile-
JAL-
265
1227
A


NH2;
1283





 


Occ-ala-Phe-arg-Leu-Hyp-(SH-158)-Asp-Arg-Ile-
JAL-
266
1271
A


NH2;
1284





 


Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
267
1254
A


Ile-NH2;
1287





 


Occ-ala-Phe-orn(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
268
1242
A


Ile-NH2;
1288





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
269
1282
A


Ile-NH2;
1289





 


(AR-201-48)-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
270
1242
C


NH2;
1291





 


(AR-201-49)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
271
1257
B


NH2;
1292





 


(AR-201-48)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
272
1199
C


NH2;
1293





 


(AR-201-49)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
273
1214
A


NH2;
1294





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Asp-Arg-Ile-NH2;
JAL-
274
1252
A



1295





 


Occ-ala-Phe-leu-Leu-Tap-Nml-Asp-Arg-Ile-NH2;
JAL-
275
1212
A



1296





 


Oct-Sni-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
276
1282
A



1297





 


6182-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
277
1280
B


NH2;
1298





 


Oct-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
278
1239
A



1302





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Tbg-
JAL-
279
1256
A


NH2;
1305





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Eca-
JAL-
280
1254
B


NH2;
1306





 


Occ-ala-Phe-arg-Leu-Hyp-Dap(Me2)-Asp-Arg-Ile-
JAL-
281
1242
B


NH2;
1314





 


Occ-ala-Phe-arg-Dap(Me2)-Hyp-Nml-Asp-Arg-
JAL-
282
1257
C


Ile-NH2;
1315





 


(AR-201-54)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
283
1277
B


NH2;
1316





 


Occ-Sni-Phe-arg-Leu-Tap-Nml-Asp-Arg-Ile-NH2;
JAL-
284
1295
A



1317





 


Occ-Sni-Phe-orn-Leu-Tap-Nml-Asp-Arg-Ile-NH2;
JAL-
285
1253
A



1318





 


Occ-Sni-Phe-nle-Leu-Tap-Nml-Asp-Arg-Ile-NH2;
JAL-
286
1252
B



1319





 


Occ-Sni-Phe-Gly-Leu-Tap-Nml-Asp-Arg-Ile-
JAL-
287
1196
A


NH2;
1320





 


Occ-Sni-Phe-leu-Leu-Tap(Ac)-Nml-Asp-Arg-Ile-
JAL-
288
1294
B


NH2;
1321





 


Occ-Sni-Phe-leu-Leu-Tap(G)-Nml-Asp-Arg-Ile-
JAL-
289
1309
A


NH2;
1322





 


Occ-Sni-Phe-leu-Leu-Tap(Bal)-Nml-Asp-Arg-Ile-
JAL-
290
1323
A


NH2;
1323





 


6059(O)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
291
1291
B


NH2;
1324





 


Occ-Sni-Phe-dap(Me2)-Leu-Tap-Nml-Asp-Arg-
JAL-
292
1253
A


Ile-NH2;
1325





 


Oct-Sni-Phe-leu-Leu-Tap-Nml-Asp-Arg-Ile-NH2;
JAL-
293
1238
A



1326





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Asp-Orn-Ile-NH2;
JAL-
294
1214
B



1327





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Orn-Ile-NH2;
JAL-
295
1171
B



1328





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Glu-Arg-Ile-NH2;
JAL-
296
1270
C



1329





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Glu-Arg-Ile-NH2;
JAL-
297
1227
B



1330





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Val-Arg-Ile-NH2;
JAL-
298
1240
B



1331





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Val-Arg-Ile-NH2;
JAL-
299
1197
A



1332





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Val-Arg-Ile-NH2;
JAL-
300
1197
B



1332_






02





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Thr-Arg-Ile-NH2;
JAL-
301
1242
B



1333





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Thr-Arg-Ile-NH2;
JAL-
302
1199
B



1334





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Eca-
JAL-
303
1211
B


NH2;
1335





 


Occ-ala-Phe-Fhy-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
304
1240
A


NH2;
1336





 


Occ-ala-Phe-Egg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
305
1254
B


NH2;
1337





 


Occ-ala-Phe-Apc-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
306
1226
A


NH2;
1338





 


(AR-201-58)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
307
1254
C


NH2;
1339





 


(AR-201-59)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
308
1267
C


NH2;
1340





 


(AR-201-62)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
309
1253
B


NH2;
1341





 


(AR-201-69)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
310
1317
B


NH2;
1342





 


Sbt-Sni-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
311
1309
A



1343





 


Nbt-Sni-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
312
1309
B



1344





 


Sbt-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
313
1269
C



1345





 


Nbt-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
314
1269
C



1346





 


Occ-ala-Phe-dap(Me2)-Leu-Tap-Nml-Asp-Arg-
JAL-
315
1213
B


Ile-NH2;
1347





 


Occ-Sni-Phe-leu-Leu-Tap(Et2)-Nml-Asp-Arg-Ile-
JAL-
316
1308
B


NH2;
1348





 


Occ-Sni-Phe-leu-Leu-Tap(Et)-Nml-Asp-Arg-Ile-
JAL-
317
1280
A


NH2;
1349





 


Occ-ala-Phe-Apc-Leu-Tap-Nml-Asp-Arg-Ile-
JAL-
318
1265
A


NH2;
1350





 


Occ-Sni-Phe-Apc-Leu-Tap-Nml-Asp-Arg-Ile-
JAL-
319
1265
A


NH2;
1351





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Tbg-
JAL-
320
1213
A


NH2;
1352





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Egz-
JAL-
321
1225
A


NH2;
1358





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Egz-
JAL-
322
1268
B


NH2;
1359





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Nle-Ile-NH2;
JAL-
323
1170
C



1360





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Asp-Nle-Ile-NH2;
JAL-
324
1213
C



1361





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Ile-Arg-Ile-NH2;
JAL-
325
1254
C



1362





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Ile-Arg-Ile-NH2;
JAL-
326
1211
B



1363





 


Occ-ala-Phe-arg-Leu-Hyp-Oic-Asp-Arg-Ile-NH2;
JAL-
327
1280
B



1364





 


Occ-ala-Phe-arg-Leu-Hyp-Pip-Asp-Arg-Ile-NH2;
JAL-
328
1240
C



1365





 


Occ-ala-Phe-leu-Leu-Hyp-Pip-Asp-Arg-Ile-NH2;
JAL-
329
1197
B



1366





 


Occ-ala-Phe-leu-Leu-Hyp-Dap(Me2)-Asp-Arg-Ile-
JAL-
330
1200
A


NH2;
1367





 


Occ-ala-Phe-leu-Dap(Me2)-Hyp-Nml-Asp-Arg-
JAL-
331
1214
B


Ile-NH2;
1368





 


Oct-Sni-Phe-dap(Me2)-Leu-Tap-Nml-Asp-Arg-
JAL-
332
1239
A


Ile-NH2;
1369





 


Occ-Sni-Phe-dap(6263)2-Leu-Tap-Nml-Asp-Arg-
JAL-
333
1311
B


Ile-NH2;
1370





 


Occ-Sni-Phe-leu-Leu-Tap(Ae)-Nml-Asp-Arg-Ile-
JAL-
334
1295
A


NH2;
1371





 


Occ-Sni-Phe-leu-Leu-Tap(Ap)-Nml-Asp-Arg-Ile-
JAL-
335
1309
A


NH2;
1372





 


(AR-201-58)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
336
1211
B


NH2;
1373





 


(AR-201-62)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
337
1210
B


NH2;
1374





 


(AR-201-69)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
338
1274
B


NH2;
1375





 


(AR-201-72)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
339
1227
C


NH2;
1376





 


(AR-201-72)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
340
1270
C


NH2;
1377





 


(AR-201-73)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
341
1216
B


NH2;
1378





 


(AR-201-73)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
342
1259
B


NH2;
1379





 


(AR-201-68)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
343
1274
A


NH2;
1380





 


(AR-201-68)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
344
1317
B


NH2;
1381





 


Sbt-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
345
1266
A



1382





 


Nbt-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
346
1266
B



1383





 


Occ-ala-Phe-leu-Leu-Hyp-Oic-Asp-Arg-Ile-NH2;
JAL-
347
1237
B



1386





 


Occ-ala-Phe-arg-Leu-Hyp-Pro-Asp-Arg-Ile-NH2;
JAL-
348
1226
C



1387





 


Occ-ala-Phe-arg-Leu-Hyp-Aze-Asp-Arg-Ile-NH2;
JAL-
349
1212
C



1393





 


Occ-ala-Phe-arg-Leu-Hyp-Eat-Asp-Arg-Ile-NH2;
JAL-
350
1244
C



1394





 


Occ-ala-Phe-arg-Leu-Hyp-Eaz-Asp-Arg-Ile-NH2;
JAL-
351
1244
C



1395





 


Occ-ala-Phe-arg-Leu-Hyp-Tic-Asp-Arg-Ile-NH2;
JAL-
352
1288
B



1396





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Val-Arg-Ile-NH2;
JAL-
353
1237
A



1398





 


Oct-Sni-Phe-leu-Leu-Hyp-Nml-Val-Arg-Ile-NH2;
JAL-
354
1223
B



1399





 


Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Val-Arg-
JAL-
355
1238
C


Ile-NH2;
1400





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Val-Arg-Ile-NH2;
JAL-
356
1236
A



1401





 


Oct-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Val-Arg-
JAL-
357
1224
B


Ile-NH2;
1402





 


Occ-Sni-Phe-dap(Me2)-Leu-Tap-Nml-Val-Arg-
JAL-
358
1237
A


Ile-NH2;
1403





 


Oct-Sni-Phe-leu-Leu-Tap-Nml-Val-Arg-Ile-NH2;
JAL-
359
1222
B



1404





 


Oct-Sni-Phe-dap(Me2)-Leu-Tap-Nml-Val-Arg-Ile-
JAL-
360
1223
A


NH2;
1405





 


Occ-ala-Phe-Apc(Me)-Met-glu--Leu-Hyp-Nml-
JAL-
361
1240
A


Asp-Arg-Ile-NH2;
1406





 


Occ-ala-Phe-Apc(Et)-Glu-thr--Leu-Hyp-Nml-Asp-
JAL-
362
1254
A


Arg-Ile-NH2;
1407





 


Occ-ala-Phe-Apc(Ae)-Ala-glu--Leu-Hyp-Nml-
JAL-
363
1269
B


Asp-Arg-Ile-NH2;
1408





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Aib-
JAL-
364
1185
B


NH2;
1413





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Aml-
JAL-
365
1227
A


NH2;
1414





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Deg-
JAL-
366
1213
A


NH2;
1416





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Nmr-Ile-
JAL-
367
1227
A


NH2;
1417





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Pro-Ile-NH2;
JAL-
368
1254
B



1418





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Tbg-Arg-Ile-NH2;
JAL-
369
1211
C



1420





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Chg-Arg-Ile-NH2;
JAL-
370
1237
C



1421





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Cpa-Arg-Ile-NH2;
JAL-
371
1195
C



1424





 


Oct-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
372
1240
A


Ile-NH2;
1429





 


Miy-Hgl-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
373
1561
A


NH2;
1430





 


Miy-Gab-Hgl-ala-Phe-arg-Leu-Hyp-Nml-Asp-
JAL-
374
1647
C


Arg-Ile-NH2;
1431





 


Ac-Miy-Gab-Hgl-ala-Phe-arg-Leu-Hyp-Nml-Asp-
JAL-
375
1730
C


Arg-Ile-NH2;
1432





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Asp-Pro-Ile-NH2;
JAL-
376
1198
B



1434





 


Occ-ala-Phe-Apc-Leu-Hyp-Nml-Asp-Pro-Ile-
JAL-
377
1167
B


NH2;
1435





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-Ile-NH2;
JAL-
378
1194
B



1436





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Aze-Ile-NH2;
JAL-
379
1140
B



1437





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Pip-Ile-NH2;
JAL-
380
1168
B



1438





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Hyp-Ile-NH2;
JAL-
381
1170
C



1441





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Eaz-Ile-NH2;
JAL-
382
1173
B



1442





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Cpp-Ile-NH2;
JAL-
383
1167
B



1443





 


Occ-ala-Phe-leu-Leu-Tap-Nml-Asp-Pro-Ile-NH2;
JAL-
384
1153
B



1450





 


Occ-ala-Phe-Apc-Leu-Hyp-Nml-Asp-Pro-Ile-
JAL-
385
1207
B


NH2;
1451





 


Occ-ala-Phe-Apc-Leu-Tap-Nml-Asp-Pro-Ile-NH2;
JAL-
386
1166
A



1452





 


Occ-ala-Phe-dap(Me2)-Leu-Tap-Nml-Asp-Pro-Ile-
JAL-
387
1154
A


NH2;
1453





 


Occ-ala-Phe-Egz-Leu-Tap-Nml-Asp-Arg-Ile-NH2;
JAL-
388
1224
A



1454





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Eay-Ile-NH2;
JAL-
389
1230
C



1456





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Egz-Ile-NH2;
JAL-
390
1182
C



1457





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Apc-Ile-NH2;
JAL-
391
1183
B



1458





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Tap-Ile-NH2;
JAL-
392
1169
C



1459





 


Occ-ala-Phe-dap(6238)2-Leu-Tap-Nml-Asp-Arg-
JAL-
393
1380
B


Ile-NH2;
1460





 


Occ-ala-Phe-dap(6238)-Leu-Tap-Nml-Asp-Arg-
JAL-
394
1282
B


Ile-NH2;
1461





 


Occ-ala-Phe-dap(3846)2-Leu-Tap-Nml-Asp-Arg-
JAL-
395
1345
B


Ile-NH2;
1462





 


Occ-ala-Phe-dap(1464)-Leu-Tap-Nml-Asp-Arg-
JAL-
396
1255
A


Ile-NH2;
1463





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-558;
JAL-
397
1162
B



1464





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-Ile-OH;
JAL-
398
1194
C



1474





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-Ile-(NH-
JAL-
399
1207
B


CH3;
1475





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Chy-Ile-NH2;
JAL-
400
1170
B



1476





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-H3p-Ile-NH2;
JAL-
401
1170
B



1477





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Dhp-Ile-NH2;
JAL-
402
1152
B



1479





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Udp-Ile-NH2;
JAL-
403
1143
B



1482





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Bhk-Ile-NH2;
JAL-
404
1199
B



1483





 


Occ-Sni-Nif-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
405
1298
B



1486





 


Occ-Sni-Pff-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
406
1271
A



1487





 


Occ-Sni-Pmy-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
407
1283
B


NH2;
1488





 


Occ-Sni-Tyr-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
408
1269
C



1489





 


Occ-Sni-Bmf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
409
1267
C


NH2;
1490





 


Occ-Sni-Eay-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
410
1279
B



1491





 


Occ-Sni-Paf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
411
1268
B



1492





 


Occ-Sni-Pcf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
412
1287
A



1493





 


Occ-Sni-Pmf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
413
1267
A


NH2;
1494





 


Occ-Sni-Eaa-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
414
1322
A



1496





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-2118;
JAL-
415
1210
B



1506





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-2906;
JAL-
416
1134
C



1508





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-1381;
JAL-
417
1164
B



1509





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-1381;
JAL-
418
1164
B



1509_






02





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-1860;
JAL-
419
1176
A



1510





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-1906 
JAL-
420
1174
B



1511





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-Che;
JAL-
421
1176
A



1512_






02





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-5121 
JAL-
422
1178
C



1513





 


Occ-Sni-Phe-Ala-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
423
1211
C


NH2;
1553





 


Occ-Sni-Phe-Leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
424
1253
B


NH2;
1554





 


Occ-Sni-Phe-Apc-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
425
1266
A


NH2;
1555





 


Occ-ala-Phe-leu-Leu-Hyp-Nm1-(BB725)-Arg-Ile-
JAL-
426
1224
A


NH2;
1556





 


Occ-ala-Phe-leu-Leu-Hyp-Nm1-(BB726)-Arg-Ile-
JAL-
427
1238
C


NH2;
1557





 


Occ-ala-Phe-leu-Leu-Hyp-Nm1-(BB727)-Arg-Ile-
JAL-
428
1238
A


NH2;
1558





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Asp-Pro-Ile-NH2;
JAL-
429
1194
A



1559





 


Occ-Sni-Phe-Gly-Leu-Tap-Nml-Asp-Pro-Ile-NH2;
JAL-
430
1138
B



1560





 


Occ-Sni-Phe-Apc-Leu-Tap-Nml-Asp-Pro-Ile-
JAL-
431
1207
A


NH2;
1561





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Asp-Pro-Che;
JAL-
432
1176
A



1568





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Nmi-
JAL-
433
1267
A


NH2;
1569





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Nmr-Ile-
JAL-
434
1267
B


NH2;
1570





 


Occ-Sni-Phe-leu-Nml-Hyp-Nml-Asp-Arg-Ile-
JAL-
435
1267
C


NH2;
1572





 


Occ-Sni-Nmf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
436
1267
A


NH2;
1573





 


Occ-Sni-Phe-nml-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
437
1267
C


NH2;
1574





 


Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
438
1268
C


Nmi-NH2;
1575





 


Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-Nmr-
JAL-
439
1268
A


Ile-NH2;
1576





 


Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Nmd-Arg-
JAL-
440
1268
C


Ile-NH2;
1577





 


Occ-Sni-Phe-dap(Me2)-Nml-Hyp-Nml-Asp-Arg-
JAL-
441
1268
B


Ile-NH2;
1578





 


Occ-Sni-Nmf-dap(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
442
1268
A


Ile-NH2;
1579





 


Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-Pro-
JAL-
443
1195
A


Ile-NH2;
1580





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Val-Pro-Che;
JAL-
444
1160
B



1594





 


Occ-Sni-Phe-leu-Leu-Hyp-Npg-Asp-Pro-Che;
JAL-
445
1177
A



1595





 


Occ-Sni-Phe-leu-Leu-Hyp-Ile-Asp-Pro-Che;
JAL-
446
1162
B



1596





 


Occ-Sni-Nmf-leu-Leu-Hyp-Nml-Asp-Pro-Che;
JAL-
447
1191
A



1597





 


Occ-Sni-Phe-leu-Nml-Hyp-Nml-Asp-Pro-Che;
JAL-
448
1190
C



1598





 


Occ-Sni-Eaa-leu-Leu-Hyp-Nml-Asp-Pro-Che;
JAL-
449
1245
A



1599





 


Occ-Sni-Phe-Gly-Leu-Hyp-Nml-Asp-Pro-Che;
JAL-
450
1120
B



1600





 


Occ-Sni-Phe-Apc-Leu-Hyp-Nml-Asp-Pro-Che 
JAL-
451
1190
B



1601





 


Occ-Sni-Phe-Apc-Leu-Tap-Nml-Asp-Pro-Che;
JAL-
452
1190
A



1602





 


Occ-Sni-Phe-dap(Me2)-Leu-Tap-Nml-Asp-Pro-
JAL-
453
1177
A


Che;
1603





 


Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-Pro-
JAL-
454
1177
A


Che;
1604





 


1319-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
455
1272
A


NH2;
1605





 


1320-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
456
1286
A


NH2;
1606





 


2553-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
457
1302
C


NH2;
1607





 


4734-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
458
1316
B


NH2;
1609





 


4703-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
459
1339
B


NH2;
1612





 


6988-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
460
1342
C


NH2;
1615





 


Hex-(3421)-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
461
1360
B


Ile-NH2;
1616





 


1695-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
462
1372
C


NH2;
1617





 


Occ-Sni-Mcf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
463
1287
A


NH2;
1618





 


Occ-Sni-Pbf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
464
1332
A



1619





 


Occ-Sni-Thk-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
465
1259
A


NH2;
1620





 


Occ-Sni-Mtf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
466
1321
A



1621





 


Occ-Sni-Otf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
467
1321
C



1622





 


Occ-Sni-Phe-ctb-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
468
1299
A



1623





 


Occ-Sni-Phe-leu-Nle-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
469
1253
A



1624





 


Occ-Sni-Phe-leu-Leu-Hyp-Ile-Asp-Arg-Ile-NH2;
JAL-
470
1239
A



1625





 


Occ-Sni-Phe-leu-Leu-Hyp-Cpg-Asp-Arg-Ile-NH2;
JAL-
471
1251
A



1626





 


Occ-Sni-Phe-leu-Leu-Hyp-Chg-Asp-Arg-Ile-NH2;
JAL-
472
1265
B



1627





 


Occ-Sni-NPhe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
473
1253
C


NH2;
1634





 


Occ-Sni-NHfe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
474
1267
C


NH2;
1635





 


Occ-(aFL)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
475
1225
B



1636





 


Occ-(afL)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
476
1225
B



1637





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Eaz-Che;
JAL-
477
1195
A



1638





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Eal-Che;
JAL-
478
1177
B



1639





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-(ES-283-049);
JAL-
479
1163
B



1646





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Glu-Pro-Che;
JAL-
480
1191
B



1652





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Val-Pro-Che;
JAL-
481
1160
A



1654





 


Occ-Sni-Phe-leu-Nle-Hyp-Nml-Asp-Pro-Che;
JAL-
482
1177
A



1657





 


779-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
483
1263
B



1659





 


785-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
484
1335
C



1660





 


1281-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
485
1259
B


NH2;
1661





 


3218-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
486
1293
C


NH2;
1664





 


6013-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
487
1285
B


NH2;
1665





 


5587-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
488
1281
A


NH2;
1666





 


1281-G-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
489
1316
C


NH2;
1668





 


1281-Bal-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
490
1330
C


NH2;
1669





 


Occ-(AFL)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
491
1225
A



1671





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Apc-Che;
JAL-
492
1204
C



1672





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-NP-Che;
JAL-
493
1176
C



1673





 


Occ-Sni-Phe-leu-Leu-Tap-Nm1-(BB726)-Pro-Che;
JAL-
494
1200
B



1676





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pca-Che;
JAL-
495
1192
A



1679





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Che;
JAL-
496
1236
A



1680





 


Occ-Sni-Phe-leu-Leu-Tap(Ae)-Nml-Asp-Arg-
JAL-
497
1278
A


Che;
1681





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Asp-Arg-Che;
JAL-
498
1235
A



1682





 


Occ-Sni-Phe-leu-Leu-Tap(Ae)-Nml-Val-Arg-Che;
JAL-
499
1262
C



1683





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Apc(Gua)-
JAL-
500
1248
C


Che;
1685





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Apc(Gly)-
JAL-
501
1263
B


Che;
1687





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-(BB394)-
JAL-
502
1166
C


Che;
1694





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-(BB785)-
JAL-
503
1192
B


Che;
1697





 


Occ-Sni-Hfe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
504
1267
C


NH2;
1701





 


Occ-ala-Nmf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
505
1227
C


NH2;
1702





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Val-Arg-Che;
JAL-
506
1218
A



1729





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Val-Arg-Che;
JAL-
507
1219
A



1730





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Ala-Arg-Che;
JAL-
508
1193
C



1750





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asn-Arg-Che;
JAL-
509
1236
C



1751





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Ser-Arg-Che;
JAL-
510
1209
A



1752





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Thr-Arg-Che;
JAL-
511
1223
A



1753





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Nle-Arg-Che;
JAL-
512
1235
C



1755





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Ble-Arg-Che;
JAL-
513
1235
B



1756





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Thi-Arg-Che;
JAL-
514
1275
C



1758





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Chg-Arg-Che;
JAL-
515
1261
C



1763





 


(AR-314-87)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
516
1279
A


NH2;
1765-






2





 


(AR-314-102)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
517
1239
A


NH2;
1774





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
518
1265
A


Che;
1776





 


Occ-Sni-Phe-lys(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
519
1279
A


Che;
1777





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Val-Arg-
JAL-
520
1249
B


Che;
1778





 


Occ-Sni-Phe-lys(Me2)-Leu-Hyp-Nml-Val-Arg-
JAL-
521
1263
A


Che;
1779





 


Occ-Sni-Phe-lys(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
522
1296
B


Ile-NH2;
1781





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Val-Arg-
JAL-
523
1266
A


Ile-NH2;
1782





 


Occ-Sni-Phe-lys(Me2)-Leu-Hyp-Nml-Val-Arg-Ile-
JAL-
524
1280
C


NH2;
1783





 


Occ-Sni-Phe-dab(Me2)-Leu-Hyp-Nml-Val-Arg-
JAL-
525
1235
A


Che;
1784





 


Occ-Sni-Phe-dab(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
526
1268
A


Ile-NH2;
1785





 


Occ-Sni-Phe-dab(Me2)-Leu-Hyp-Nml-Val-Arg-
JAL-
527
1252
B


Ile-NH2;
1786





 


Occ-Nhpr-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
528
1257
B


NH2;
1798





 


Occ-Nbhp-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
529
1273
B


NH2;
1799





 


Occ-ser-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
530
1229
B



1800





 


Occ-hse-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
531
1243
B



1801





 


Gluc-Aoa-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-
JAL-
532
1503
B


Arg-Ile-NH2;
1802





 


Gluc-Aoa-hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-
JAL-
533
1503
A


Arg-Ile-NH2;
1803





 


(1913)-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
534
1384
B


Ile-NH2;
1804





 


(1270)-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
535
1396
C


Ile-NH2;
1805





 


(1888)-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
536
1428
B


Ile-NH2;
1806





 


Occ-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
537
1394
C


NH2;
1807





 


H-Adx-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
538
1413
A


Ile-NH2;
1808





 


1888-hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
539
1428
B


NH2;
1837





 


H-Adx-hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
540
1413
B


Ile-NH2;
1838





 


Oct-Sni-Phe-leu-Leu-Tap-Nml-Asp-Arg-Che;
JAL-
541
1221
A



1843





 


Oct-Sni-Phe-leu-Leu-Tap-Nml-Val-Pro-Che;
JAL-
542
1146
B



1844





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Val-Pro-
JAL-
543
1190
B


Che;
1845





 


Occ-Sni-Phe-orn(Me2)-Leu-Tap-Nml-Val-Pro-
JAL-
544
1189
B


Che;
1846





 


Oct-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Val-Pro-
JAL-
545
1176
C


Che;
1847





 


Oct-Sni-Phe-leu-Leu-Hyp-Nml-Val-Arg-Che;
JAL-
546
1206
B



1848





 


Oct-Sni-Phe-leu-Leu-Tap-Nml-Val-Arg-Che;
JAL-
547
1205
B



1849





 


Occ-Sni-Phe-orn(Me2)-Leu-Tap-Nml-Val-Arg-
JAL-
548
1248
A


Che;
1850





 


Oct-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Val-Arg-
JAL-
549
1235
B


Che;
1851





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Bmf-Arg-Ile-
JAL-
550
1299
C


NH2;
1857





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Phg-Arg-Ile-NH2;
JAL-
551
1859
C



1858





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Cpg-Arg-Ile-NH2;
JAL-
552
1263
B



1859





 


Occ-Sni-Phe-leu-Leu-Hyp-Nm1-(AR-314-145)-
JAL-
553
1277
C


Arg-Ile-NH2;
1864





 


(AR-314-169)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
554
1281
B


Ile-NH2;
1868-






2





 


(AR-314-170)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
555
1253
C


Ile-NH2;
1869-






2





 


(AR-314-171)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
556
1281
C


Ile-NH2;
1870-






2





 


(AR-385-008)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
557
1273
C


NH2;
1873





 


(AR-314-172)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
558
1287
B


NH2;
1874





 


Occ-Sni-Phe-(AR-385-12)-Leu-Hyp-Nml-Asp-
JAL-
559
1294
A


Arg-Ile-NH2;
1877





 


Occ-Sni-Phe-hse-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
560
1241
B


NH2;
1878





 


Occ-Sni-Phe-abu(pip)-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
561
1308
B


NH2;
1879





 


(AR-385-042)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
562
1287
B


NH2;
1880





 


Occ-Sni-Phe-Fhz-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
563
1280
B


NH2;
1881





 


Occ-Sni-Phe-Fhy-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
564
1280
B


NH2;
1882





 


Occ-Sni-Phe-thr-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
565
1241
C



1883





 


Occ-Sni-Phe-his-Leu-Hyp-Nml-Asp-Arg-Ile-NH2;
JAL-
566
1277
B



1884





 


Occ-Sni-Phe-metO2-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
567
1303
B


NH2;
1885





 


Occ-Sni-Phe-(AR-385-017)-Leu-Hyp-Nml-Asp-
JAL-
568
1310
B


Arg-Ile-NH2;
1886





 


Occ-Sni-Phe-opa-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
569
1288
B


NH2;
1887





 


Occ-Sni-Phe-mpa-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
570
1288
B


NH2;
1888





 


Occ-Sni-Phe-ppa-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
571
1288
B


NH2;
1889





 


Occ-Sni-Phe-Egg-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
572
1294
A


NH2;
1890





 


Occ-Sni-Phe-Eao-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
573
1299
B


NH2;
1892





 


Occ-Sni-Phe-Aic-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
574
1299
B


NH2;
1893





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Ser-Arg-
JAL-
575
1237
B


Che;
1894





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Thr-Arg-
JAL-
576
1251
A


Che;
1895





 


H-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
577
1268
B


NH2;
1896





 


H-hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
578
1268
B


NH2;
1897





 


H-Lys-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
579
1396
B


Ile-NH2;
1898





 


H-Lys-hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
580
1396
B


Ile-NH2;
1899





 


H-Lys-Pro-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-
JAL-
581
1493
A


Arg-Ile-NH2;
1900





 


(2857-Ac)-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-
JAL-
582
1489
B


Arg-Ile-NH2;
1901





 


(1625-Ac)-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
583
1268
B


Ile-NH2;
1907





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Dim-Arg-
JAL-
584
1264
B


Ile-NH2;
1910





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Pse-Arg-Ile-NH2;
JAL-
585
1305
C



1912





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Pth-Arg-Ile-
JAL-
586
1348
C


NH2;
1913





 


Occ-Sni-Phe-Dha-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
587
1209
B


NH2;
1915-






2





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Pse-Arg-
JAL-
588
1316
C


Che;
1916





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Pse-Arg-Che;
JAL-
589
1288
C



1917





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Pth-Arg-
JAL-
590
1330
B


Che;
1918





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Pth-Arg-Che;
JAL-
591
1302
B



1919





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Ser-Arg-Ile-NH2;
JAL-
592
1225
B



1920





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Ser-Arg-Ile-
JAL-
593
1254
C


NH2;
1921





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Cya-Arg-Ile-NH2;
JAL-
594
1289
B



1922





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Cya-Arg-
JAL-
595
1318
B


Ile-NH2;
1923





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Thr-Arg-
JAL-
596
1268
B


Ile-NH2;
1924





 


Occ-Sni-Phe-leu-Leu-Hyp(Asp(-))-Nml-Asp-Arg-
JAL-
597
1368
B


Ile-NH2;
1928





 


Occ-Sni-Phe-leu-Leu-Hyp(2581)-Nml-Asp-Arg-
JAL-
598
1338
B


Ile-NH2;
1929





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-OH;
JAL-
599
1254
B



1930





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
600
1283
B


Ile-OH;
1931





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Thr-Arg-Ile-NH2;
JAL-
601
1239
A



1932





 


Occ-Sni-Phe-leu-Leu-Tap(Asp(-))-Nml-Asp-Arg-
JAL-
602
1367
B


Ile-NH2;
1935





 


Occ-Sni-Phe-orn(Me2)-Leu-Tap-Nml-Asp-Arg-
JAL-
603
1281
A


Ile-NH2;
1936









Preferred NPR-B agonists of the present invention are those peptides within activity group B, as presented in Table 3, above. Most preferred NPR-B agonists of the present invention are those peptides within activity group A, as presented in Table 4, below.







TABLE 4







Most preferred compounds according to the present invention andtheir


agonistic activity in in vitro assays.













SEQ
(M + H)+ in
Activity


Structure
JAL
ID NO:
MS [amu]
(group)





Occ-Sni-Phe-nle-Leu-Hyp-Leu-Asp-Arg-Ile-NH2
JAL-
166
1239
A



1024





 


Occ-ala-Phe-leu-Leu-Pro-Nml-Asp-Arg-Ile-NH2
JAL-
180
1196
A



1039





 


Occ-Sni-Phe-leu-Leu-Pro-Nml-Asp-Arg-Ile-NH2
JAL-
181
1236
A



1040





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
182
1253
A



1041





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
183
1213
A



1042





 


Occ-ala-Pcf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
184
1247
A



1043





 


Occ-ala-Phe-nle-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
185
1213
A



1044





 


Occ-ala-Phe-arg-Leu-Hyp-Leu-Asp-Arg-Ile-NH2
JAL-
187
1242
A



1047





 


Occ-ala-Pcf-leu-Leu-Hyp-Leu-Asp-Arg-Ile-NH2
JAL-
190
1233
A



1050





 


Occ-ala-Nmf-leu-Leu-Hyp-Leu-Asp-Arg-Ile-NH2
JAL-
192
1213
A



1052





 


Occ-pro-Phe-leu-Leu-Hyp-Leu-Asp-Arg-Ile-NH2
JAL-
193
1225
A



1053





 


Occ-pip-Phe-leu-Leu-Hyp-Leu-Asp-Arg-Ile-NH2
JAL-
194
1239
A



1054





 


Occ-ala-Phe-lys-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
195
1228
A



1060





 


Occ-ala-Phe-orn-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
196
1214
A



1061





 


Occ-pip-Nmf-arg-Leu-Pro-Nml-Asp-Arg-Ile-NH2
JAL-
201
1294
A



1077





 


Occ-pip-Phe-arg-Leu-Pro-Nml-Asp-Arg-Ile-NH2
JAL-
202
1280
A



1078





 


Occ-ala-Nmf-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
203
1270
A



1085





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
204
1256
A



1086





 


Occ-pip-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
205
1296
A



1087





 


Occ-ala-Pbf-arg-Leu-Hyp-Leu-Asp-Arg-Ile-NH2
JAL-
208
1321
A



1116





 


Occ-ala-Phe-arg-Leu-Hyp-Npg-Asp-Arg-Ile-NH2
JAL-
217
1256
A



1225





 


Occ-ala-Phe-Gly-Leu-Tap-Leu-Asp-Arg-Ile-NH2
JAL-
237
1142
A



1249





 


Occ-ala-Phe-arg-Leu-Tap-Leu-Asp-Arg-Ile-NH2
JAL-
238
1241
A



1250





 


Occ-ala-Phe-leu-Leu-Tap-Asp-Arg-Ile-NH2
JAL-
239
1198
A



1251





 


Occ-ala-Phe-ser-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
240
1187
A



1252





 


Occ-Sni-Phe-lys-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
242
1268
A



1254





 


Occ-Sni-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
243
1296
A



1255





 


Occ-Sni-Phe-orn-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
252
1254
A



1265





 


Occ-ala-Nmf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
254
1227
A



1267





 


Occ-ala-Phe-leu-Leu-Hyp-(SH-158)-Asp-Arg-
JAL-
265
1227
A


Ile-NH2
1283





 


Occ-ala-Phe-arg-Leu-Hyp-(SH-158)-Asp-Arg-
JAL-
266
1271
A


Ile-NH2
1284





 


Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
267
1254
A


Ile-NH2
1287





 


Occ-ala-Phe-orn(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
268
1242
A


Ile-NH2
1288





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
269
1282
A


Ile-NH2
1289





 


(AR-201-49)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
273
1214
A


NH2
1294





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Asp-Arg-Ile-NH2
JAL-
274
1252
A



1295





 


Occ-ala-Phe-leu-Leu-Tap-Nml-Asp-Arg-Ile-NH2
JAL-
275
1212
A



1296





 


Oct-Sni-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
276
1282
A



1297





 


Oct-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
278
1239
A



1302





 


Occ-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Tbg-NH2
JAL-
279
1256
A



1305





 


Occ-Sni-Phe-arg-Leu-Tap-Nml-Asp-Arg-Ile-NH2
JAL-
284
1295
A



1317





 


Occ-Sni-Phe-orn-Leu-Tap-Nml-Asp-Arg-Ile-NH2
JAL-
285
1253
A



1318





 


Occ-Sni-Phe-Gly -Leu-Tap-Nml-Asp-Arg-Ile-NH2
JAL-
287
1196
A



1320





 


Occ-Sni-Phe-leu-Leu-Tap(G)-Nml-Asp-Arg-Ile-
JAL-
289
1309
A


NH2
1322





 


Occ-Sni-Phe-leu-Leu-Tap(Bal)-Nml-Asp-Arg-
JAL-
290
1323
A


Ile-NH2
1323





 


Occ-Sni-Phe-dap(Me2)-Leu-Tap-Nml-Asp-Arg-
JAL-
292
1253
A


Ile-NH2
1325





 


Oct-Sni-Phe-leu-Leu-Tap-Nml-Asp-Arg-Ile-NH2
JAL-
293
1238
A



1326





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Val-Arg-Ile-NH2
JAL-
299
1197
A



1332





 


Occ-ala-Phe-Fhy -Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
304
1240
A



1336





 


Occ-ala-Phe-Apc-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
306
1226
A



1338





 


Occ-Sni-Phe-leu-Leu-Tap (Et) -Nml-Asp-Arg-
JAL-
317
1280
A


Ile-NH2
1349





 


Occ-ala-Phe-Apc-Leu-Tap-Nml-Asp-Arg-Ile-NH2
JAL-
318
1265
A



1350





 


Occ-Sni-Phe-Apc-Leu-Tap-Nml-Asp-Arg-Ile-NH2
JAL-
319
1265
A



1351





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Tbg-NH2
JAL-
320
1213
A



1352





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Egz-NH2
JAL-
321
1225
A



1358





 


Occ-ala-Phe-leu-Leu-Hyp-Dap(Me2)-Asp-Arg-
JAL-
330
1200
A


Ile-NH2
1367





 


Oct-Sni-Phe-dap(Me2)-Leu-Tap-Nml-Asp-Arg-
JAL-
332
1239
A


Ile-NH2
1369





 


Occ-Sni-Phe-leu-Leu-Tap(Ae)-Nml-Asp-Arg-
JAL-
334
1295
A


Ile-NH2
1371





 


Occ-Sni-Phe-leu-Leu-Tap(Ap)-Nml-Asp-Arg-
JAL-
335
1309
A


Ile-NH2
1372





 


(AR-201-68)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-
JAL-
343
1274
A


Ile-NH2
1380





 


Sbt-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
345
1266
A



1382





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Val-Arg-Ile-NH2
JAL-
353
1237
A



1398





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Val-Arg-Ile-NH2
JAL-
356
1236
A



1401





 


Occ-Sni-Phe-dap(Me2)-Leu-Tap-Nml-Val-Arg-
JAL-
358
1237
A


Ile-NH2
1403





 


Oct-Sni-Phe-dap(Me2)-Leu-Tap-Nml-Val-Arg-
JAL-
360
1223
A


Ile-NH2
1405





 


Occ-ala-Phe-Apc(Me)-Met-glu--Leu-Hyp-Nml-
JAL-
361
1240
A


Asp-Arg-Ile-NH2
1406





 


Occ-ala-Phe-Apc(E0-Glu-thr--Leu-Hyp-Nml-
JAL-
362
1254
A


Asp-Arg-Ile-NH2
1407





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ami-NH2
JAL-
365
1227
A



1414





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Deg-NH2
JAL-
366
1213
A



1416





 


Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Nmr-Ile-NH2
JAL-
367
1227
A



1417





 


Oct-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
372
1240
A


Ile-NH2
1429





 


Miy-Hgl-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-
JAL-
373
1561
A


Ile-NH2
1430





 


Occ-ala-Phe-Apc-Leu-Tap-Nml-Asp-Pro-Ile-NH2
JAL-
386
1166
A



1452





 


Occ-ala-Phe-dap(Me2)-Leu-Tap-Nml-Asp-Pro-
JAL-
387
1154
A


Ile-NH2
1453





 


Occ-ala-Phe-Egz-Leu-Tap-Nml-Asp-Arg-Ile-NH2
JAL-
388
1224
A



1454





 


Occ-ala-Phe-dap(1464)-Leu-Tap-Nml-Asp-Arg-
JAL-
396
1255
A


Ile-NH2
1463





 


Occ-Sni-Pff-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
406
1271
A



1487





 


Occ-Sni-Pcf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
412
1287
A



1493





 


Occ-Sni-Pmf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
413
1267
A



1494





 


Occ-Sni-Eaa-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
414
1322
A



1496





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-1860
JAL-
419
1176
A



1510





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-Che
JAL-
421
1176
A



1512_






02





 


Occ-Sni-Phe-Apc-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
425
1266
A



1555





 


Occ-ala-Phe-leu-Leu-Hyp-Nm1-(BB725)-Arg-Ile-
JAL-
426
1224
A


NH2
1556





 


Occ-ala-Phe-leu-Leu-Hyp-Nm1-(BB727)-Arg-Ile-
JAL-
428
1238
A


NH2
1558





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Asp-Pro-Ile-NH2
JAL-
429
1194
A



1559





 


Occ-Sni-Phe-Apc-Leu-Tap-Nml-Asp-Pro-Ile-NH2
JAL-
431
1207
A



1561





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Asp-Pro-Che
JAL-
432
1176
A



1568





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Nmi-
JAL-
433
1267
A


NH2
1569





 


Occ-Sni-Nmf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
436
1267
A



1573





 


Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-Nmr-
JAL-
439
1268
A


Ile-NH2
1576





 


Occ-Sni-Nmf-dap(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
442
1268
A


Ile-NH2
1579





 


Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-Pro-
JAL-
443
1195
A


Ile-NH2
1580





 


Occ-Sni-Phe-leu-Leu-Hyp-Npg-Asp-Pro-Che
JAL-
445
1177
A



1595





 


Occ-Sni-Nmf-leu-Leu-Hyp-Nml-Asp-Pro-Che
JAL-
447
1191
A



1597





 


Occ-Sni-Eaa-leu-Leu-Hyp-Nml-Asp-Pro-Che
JAL-
449
1245
A



1599





 


Occ-Sni-Phe-Apc-Leu-Tap-Nml-Asp-Pro-Che
JAL-
452
1190
A



1602





 


Occ-Sni-Phe-dap(Me2)-Leu-Tap-Nml-Asp-Pro-
JAL-
453
1177
A


Che
1603





 


Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-Pro-
JAL-
454
1177
A


Che
1604





 


1319-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
455
1272
A



1605





 


1320-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
456
1286
A



1606





 


Occ-Sni-Mcf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
463
1287
A



1618





 


Occ-Sni-Pbf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
464
1332
A



1619





 


Occ-Sni-Thk-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
465
1259
A



1620





 


Occ-Sni-Mtf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
466
1321
A



1621





 


Occ-Sni-Phe-ctb-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
468
1299
A



1623





 


Occ-Sni-Phe-leu-Nle-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
469
1253
A



1624





 


Occ-Sni-Phe-leu-Leu-Hyp-Ile-Asp-Arg-Ile-NH2
JAL-
470
1239
A



1625





 


Occ-Sni-Phe-leu-Leu-Hyp-Cpg-Asp-Arg-Ile-NH2
JAL-
471
1251
A



1626





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Eaz-Che
JAL-
477
1195
A



1638





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Val-Pro-Che
JAL-
481
1160
A



1654





 


Occ-Sni-Phe-leu-Nle-Hyp-Nml-Asp-Pro-Che
JAL-
482
1177
A



1657





 


5587-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
488
1281
A


NH2
1666





 


Occ-(AFL)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
491
1225
A



1671





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pca-Che
JAL-
495
1192
A



1679





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Che
JAL-
496
1236
A



1680





 


Occ-Sni-Phe-leu-Leu-Tap(Ae)-Nml-Asp-Arg-Che
JAL-
497
1278
A



1681





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Asp-Arg-Che
JAL-
498
1235
A



1682





 


Occ-Sni-Phe-leu-Leu-Tap-Nml-Val-Arg-Che
JAL-
506
1218
A



1729





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Val-Arg-Che
JAL-
507
1219
A



1730





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Ser-Arg-Che
JAL-
510
1209
A



1752





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Thr-Arg-Che
JAL-
511
1223
A



1753





 


(AR-314-87)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
516
1279
A



1765-






2





 


(AR-314-102)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
JAL-
517
1239
A


NH2
1774





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
518
1265
A


Che
1776





 


Occ-Sni-Phe-lys(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
519
1279
A


Che
1777





 


Occ-Sni-Phe-lys(Me2)-Leu-Hyp-Nml-Val-Arg-
JAL-
521
1263
A


Che
1779





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Val-Arg-
JAL-
523
1266
A


Ile-NH2
1782





 


Occ-Sni-Phe-dab(Me2)-Leu-Hyp-Nml-Val-Arg-
JAL-
525
1235
A


Che
1784





 


Occ-Sni-Phe-dab(Me2)-Leu-Hyp-Nml-Asp-Arg-
JAL-
526
1268
A


Ile-NH2
1785





 


H-Adx-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg- 
JAL-
538
1413
A


Ile-NH2
1808





 


Oct-Sni-Phe-leu-Leu-Tap-Nml-Asp-Arg-Che
JAL-
541
1221
A



1843





 


Occ-Sni-Phe-orn(Me2)-Leu-Tap-Nml-Val-Arg-
JAL-
548
1248
A


Che
1850





 


Occ-Sni-Phe-(AR-385-12)-Leu-Hyp-Nml-Asp-
JAL-
559
1294
A


Arg-Ile-NH2
1877





 


Occ-Sni-Phe-Egg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2
JAL-
572
1294
A



1890





 


Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Thr-Arg-
JAL-
576
1251
A


Che
1895





 


H-Lys-Pro-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp- 
JAL-
581
1493
A


Arg-Ile-NH2
1900





 


Occ-Sni-Phe-leu-Leu-Hyp-Nml-Thr-Arg-Ile-NH2
JAL-
601
1239
A



1932





 


Occ-Sni-Phe-orn(Me2)-Leu-Tap-Nml-Asp-Arg-
JAL-
603
1281
A


Ile-NH2
1936









B. Diseases to be Treated and/or Prevented
The present invention is also directed to methods of treating or preventing diseases in a subject that involve administering to the subject a therapeutically effective amount of a composition that includes one or more NPR-B agonists as described herein, wherein the disease is one of the following. The subject may be a mammal, such as a human, a primate, a cow, a horse, a dog, a cat, a mouse, or a rat. In particular embodiments, the subject is a human.
1. Definitions
“Treatment” and “treating” refer to administration or application of a drug to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. The term “therapeutic benefit” used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of his condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. Therapeutic benefit also includes reducing the signs or symptoms associated with glaucoma in a subject with glaucoma. For example, a therapeutic benefit in a patient with glaucoma is obtained where there is no further progression of visual field loss in the affected eye, or a slowing of the rate of progression of visual field loss in the affected eye, or an improvement in vision.
A “disease” or “health-related condition” can be any pathological condition of a body part, an organ, or a system resulting from any cause, such as infection, trauma, genetic defect, age-related deterioration of bodily functions, and/or environmental stress. The cause may or may not be known. Examples of diseases include glaucoma, retinopathies, ocular trauma, and optic neuropathies. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease.
The terms “prevention” and “preventing” are used herein according to their ordinary and plain meaning to mean “acting before” or such an act. In the context of a particular disease or health-related condition, those terms refer to administration or application of an agent, drug, or remedy to a subject or performance of a procedure or modality on a subject for the purpose of blocking or minimizing the onset of a disease or health-related condition. For example, an individual with an eye that is at risk of developing glaucoma (such as an individual with ocular hypertension) can be treated with a NPR-B agonist as set forth herein for the purpose of blocking or minimizing the onset of the signs or symptoms of glaucoma (i.e., prevention of glaucoma). In a specific embodiment, prevention pertains to lowering elevated intraocular pressure, blocking detectable optic nerve damage as a result of glaucoma in a subject, reducing the rate of vision loss in a subject, or halting loss of vision in a subject. The subject can be a subject who is known or suspected of being free of a particular disease or health-related condition at the time the relevant preventive agent is administered. The subject, for example, can be a subject with no known disease or health-related condition (i.e., a healthy subject). In some embodiments, the subject had a previous disease that has been treated in the past and is now known or suspected to be disease-free.
For those skilled in the art it is easy to understand, that different diseases are summarized under certain terms or generic terms. These summaries are no limitation and each disease can be viewed on its own and can be treated or prevented with the compounds according to the present invention.
2. Glaucoma and Ocular Hypertension
Glaucoma is the second leading cause of blindness world-wide (Thylefors and Negrel 1994, Bull World Health Organ. 72:323-326). Open-angle glaucoma (OAG) and angle closure glaucoma combined represent the second leading cause of blindness worldwide (Quigley and Broman, 2006 Br J Ophthalmol. 90:262-267). Angle-closure glaucoma is more common in the Asian population (Foster et al. 2000, Arch Ophthalmol. 118:1105-11), while open-angle glaucoma is more commonly found in black patients (Leske et al. 2007, Ophthalmic Epidemiol. 14:166-172). Glaucoma is a progressive disease in which the risk of vision loss increases with disease duration. In light of an aging population world-wide, the impact of this blinding disorder can be expected to increase in the future.
The disease state referred to as glaucoma is a family of diseases characterized by a permanent loss of visual function due to irreversible damage to the optic nerve. More specifically, glaucoma results in optic neuropathy leading to the loss of retinal ganglion cell (RGC) function followed by apoptotic cell death and a progressive increase in vision loss. Morphologically or functionally distinct types of glaucoma are typically characterized by elevated intraocular pressure (TOP), which is considered to be an important risk factor of the pathological course of the disease. Disruption of normal aqueous outflow leading to elevated IOP is integral to glaucoma pathophysiology. Ocular hypertension is a condition wherein IOP is elevated but no apparent loss of visual function has occurred; such patients are considered to be at high risk for the eventual development of the visual loss associated with glaucoma. Some patients with glaucomatous field loss have relatively low IOPs. These so called normotension or low tension glaucoma patients can also benefit from agents that lower and control IOP.
Glaucoma is typically identified by changes in IOP, visual field deficits and/or fundus changes at the optic disk. Elevated IOP, found in most glaucoma patients, is a result of morphological and biochemical changes in the trabecular meshwork (TM), an aqueous humor filtering tissue located at the iris-cornea angle of the eye. As glaucoma progresses, there is a loss of TM cells and a buildup of extracellular products which inhibit the normal aqueous humor outflow resulting in IOP elevation. In addition to elevated IOP, other factors, such as genetic defects, may lead to mechanical distortion of the optic nerve head (ONH) ultimately resulting in ONH cupping and loss of RGC and their axons. The exact mechanism of this pathological process is currently unknown. It has been suggested that lowering the IOP of patients diagnosed with glaucoma by at least 20-30% will decrease the progressive worsening of the disease by 50-60% (Quigley 2005 Ophthalmology 112:1642-1643). Without proper diagnosis and treatment, glaucoma can progress to total irreversible blindness.
Initially, most open-angle glaucoma patients are managed with one or more of a wide variety of topical ocular or oral hypotensive medications that act to increase aqueous fluid outflow and/or decrease aqueous fluid production, or with surgical procedures such as laser trabeculoplasty and filtration surgery. Treatment regimens currently available for patients exhibiting elevated IOP, regardless of cause, typically include the topical application, from once daily to multiple times per day, of one or multiple eyedrops or pills containing a small molecule IOP-lowering compound. Also, pills that decrease the amount of aqueous humor created can be given between two and four times daily. Glaucoma medications typically prescribed include cholinergic agonists, adrenergic agonists, beta adrenergic blockers, carbonic anhydrase inhibitors and prostaglandin analogs. Although these classes of medications are effective in controlling IOP, each of them has certain limitations in efficacy and untoward effects. For example, beta adrenergic blockers do not lower IOP at night; many glaucoma patients do not respond to a particular drug class; and a majority of glaucoma patients require the use of a combination of drugs. In addition, many of the drugs cause local irritation of the eye, such as burning, stinging, itching, tearing, conjunctival hyperemia, foreign body sensation, blurred vision, and eye pain. Some occasionally induce systemic side effects. Hence, there is a genuine and continuous need for novel and improved glaucoma medications.
“Glaucoma” and “glaucomatous optic neuropathy” and “glaucomatous retinopathy,” as used herein, are interchangeable. Glaucoma refers to a disease characterized by the permanent loss of visual function due to irreversible damage to the retinal ganglion cells in the retina and optic nerve. The major risk factor for glaucoma and the related loss of visual function is elevated intraocular pressure. There are different types of glaucoma, including primary open angle glaucoma (POAG), angle closure glaucoma, and congenital/developmental glaucoma.
As used herein, the term “intraocular pressure” or “IOP” refers to the pressure of the content inside the eye. In a normal human eye, IOP is typically in the range of 10 to 21 mm Hg. IOP varies among individuals, for example, it may become elevated due to anatomical problems, inflammation of the eye, as a side-effect from medication or due to genetic factors. “Elevated” intraocular pressure is currently considered to be ≧21 mm Hg, which is also considered to be a major risk factor for the development of glaucoma.
However, some individuals with an elevated IOP may not develop glaucoma and are considered to have ocular hypertension. “Ocular hypertension” as used herein refers to a condition in which the intraocular pressure in the eye of a subject is higher than normal but the optic nerve and visual fields are within normal limits. These individuals may be susceptible to developing the loss of visual function that is typically associated with glaucoma. As used herein, the terms “susceptible,” or “susceptibility” refers to an individual or subject that is or at risk of developing optic nerve damage or retinal damage that is associated with elevated intraocular pressure.
Thus, the present invention is directed to methods of treating or preventing an ophthalmic disease in a subject that involve administering to the subject a therapeutically effective amount of a composition that includes one or more NPR-B agonists as described herein, wherein the ophthalmic disease is glaucoma, elevated intraocular pressure or ocular hypertension. The subject may be a mammal, such as a human, a primate, a cow, a horse, a dog, a cat, a mouse, or a rat. In particular embodiments, the subject is a human.
In preferred aspects, the NPR-B agonists of the invention will lower intraocular pressure associated with glaucoma. The glaucoma may be any type of glaucoma, such as primary open angle glaucoma, angle closure glaucoma, normal tension glaucoma, congenital glaucoma, neovascular glaucoma, steroid-induced glaucoma, or glaucoma related to ocular trauma (e.g., ghost cell glaucoma or glaucoma related to choroidal detachment).
The present invention is also directed to methods of lowering intraocular pressure in a subject, comprising administering to the subject a pharmaceutically effective amount of a composition comprising a NPR-B agonist described herein, wherein intraocular pressed is lowered. In particular embodiments, the subject is a human. For example, in specific embodiments, the human is a patient with ocular hypertension or elevated IOP.
3. CNP Deficiencies as in Diabetes
Diabetic nephropathy is a progressive kidney disease, resulting from longstanding diabetes mellitus. Experimental evidence shows that natriuretic peptides play a pathophysiological role in the glomerular abnormalities seen in diabetes mellitus. BNP overexpression prevented diabetic nephropathy in a streptozotocin-induced mouse model of diabetes (Makino et al. 2006, Diabetologia. 49:2514-2524). In another study with streptozotocin-induced diabetic rats, cardiac CNP mRNA concentrations were decreased 2.6-fold (Walther et al. 2000, J Mol Endocrinol. 24:391-395). In a genetic model of diabetes, the non-obese diabetic mouse, mesangial cells derived from diabetic mice showed constitutive overexpression of NPR-C; this was associated with a reduced response of cGMP production to ANP or CNP treatment (Ardaillou et al. 1999, Kidney Int 55:1293-1302).
4. Conditions with Hyperproliferation of Vascular Smooth Muscle Cells
The abnormal growth of vascular smooth muscle cells (VSMC) is a common cause of many vascular diseases. A disturbance of the balance between growth inhibitors and growth promoters results in the hyperproliferation of those cells, and vasoactive substances, including natriuretic peptides, seem to play a major role in this process. Early experimental findings indicate that the guanylyl-cyclase-linked natriuretic peptide receptors mediate anti-proliferative activity of the natriuretic peptides on vascular smooth muscle cell growth (Hutchinson et al. 1997, Cardiovasc Res. 35:158-167). Ex vivo experiments showed a direct inhibition of growth in rat VSMCs by CNP (Furuya et al. 1991, Biochem Biophys Res Commun. 177:927-931). Furthermore, migration of rat VSMCs could be inhibited by CNP (Ikeda et al. 1997, Arterioscler Thromb Vasc Biol. 17:731-736). CNP gene transfer resulted in a reduction of the VSMC proliferation in pig femoral arteries in vivo, and the effect was even superior over CNP peptide application (Pelisek et al. 2006, J Gene Med. 8:835-844). In another report, CNP gene transfer resulted in the suppression of vascular remodelling in porcine coronary arteries in vivo (Morishige et al. 2000, J Am Coll Cardiol. 35:1040-1047), thus further strengthening the rationale of using CNP to offset the hyperproliferation of VSMCs.
5. Cardiac Pathologies, Especially Heart Failure and Hypertrophy
Considerable evidence supports a central pathophysiological role for natriuretic peptides in cardiovascular diseases, and in particular heart failure. The advantage of focusing on CNP in this indication is the unchanged reactivity of NPR-B, while NPR-A activity was shown to be reduced in this condition (Dickey et al. 2007, Endocrinology. 148:3518-3522, Nakamura et al. 1994, Circulation. 90:1210-1214). The fact that plasma CNP is elevated in heart failure patients (Del Ry et al. 2005, Eur J Heart Fail. 7:1145-1148, Del Ry et al. 2007, Peptides. 28:1068-1073) is interpreted as part of a compensatory vasodilating response in the peripheral vasculature (Del Ry et al. 2005, Eur J Heart Fail. 7:1145-1148, Wright et al. 2004, Hypertension. 43:94-100). Traditional treatment of heart failure aims at the support of cardiac function by preventing cardiomyocyte loss and hypertrophy. CNP is able to support cardiac function via a positive effect on the vitality of cardiomyocytes (Rosenkranz et al. 2003, Cardiovasc Res. 57:515-522, Tokudome et al. 2004, Endocrinology. 145:2131-2140). Also, CNP reduced cardiac fibrosis (Horio et al. 2003, Endocrinology. 144:2279-2284), the effect being stronger than that by ANP or BNP. Results from studies on dogs showed a potential inotropic effect of CNP (Beaulieu et al. 1997, Am J Physiol. 273:H1933-1940), supporting the potential of CNP to treat heart failure.
Hypertrophy of the heart is an enlargement of the organ, due to an increase in the volume of its muscular fibres. Experimental evidence suggests that CNP exhibits important autocrine and paracrine functions within the heart and the coronary circulation (D'Souza et al. 2004, Pharmacol Ther. 101:113-129). In vivo administration of CNP has been shown to improve cardiac function and attenuate cardiac remodelling after myocardial infarction in rats (Soeki et al. 2005, J Am Coll Cardiol 45:608-616). Another recent study shows that CNP is able to reduce reactive hypertrophy of cardiomyocytes after an experimental myocardial infarction in transgenic mice over-expressing CNP in cardiomyocytes (Wang et al. 2007, Eur J Heart Fail. 9:548-557).
6. Cardiovascular Pathologies, Especially Atherosclerosis, Hypertension, Endothelial Dysfunction and Thrombotic Events
Atherosclerosis is a chronic inflammatory response in the walls of arterial blood vessels. In vitro evidence suggests that CNP has an inhibitory role in vascular smooth muscle cell proliferation and migration (Furuya et al. 1991, Biochem Biophys Res Commun. 177:927-931, Shinomiya et al. 1994, Biochem Biophys Res Commun. 205:1051-1056). Type-C natriuretic peptide inhibited neointimal thickening in injured arteries of rabbits and rats in vivo (Furuya et al. 1995, Ann N Y Acad Sci. 748:517-523, Ueno et al. 1997, Circulation. 96:2272-2279). In an experimental model of atherosclerosis in rabbits, local infusion of CNP resulted in the preservation of endothelial function and the prevention of neointimal thickening, which normally results from endothelial injury (Gaspari et al. 2000, Clin Exp Pharmacol Physiol. 27:653-655).
Pulmonary hypertension is a progressive disease, characterized by an elevated pressure in the pulmonary arterial system. Common treatment is the use of vasodilatory substances. The ability of CNP to relax arteries, possibly via direct interaction with the VSMCs, has been show before in isolated pig coronary arteries (Marton et al. 2005, Vascul Pharmacol. 43:207-212). More specifically, CNP was able to ameliorate monocrotaline-induced pulmonary hypertension in rats and improved survival (Itoh et al. 2004, Am J Respir Crit Care Med. 170:1204-1211), even if treatment with CNP started 3 weeks after the onset of symptoms.
Endothelial dysfunction plays a fundamental role in the development of atherosclerosis and restenosis. In a rabbit model with features similar to those of the early stage of atherosclerosis or restenosis, chronic pen-arterial administration of ANP or CNP prevented endothelial dysfunction and development of neointima (Gaspari et al. 2000, Clin Exp Pharmacol Physiol. 27:653-655, Barber et al. 2005, J Vasc Res. 42:101-110).
Prevention of thrombotic events is critical to the management of cardiovascular diseases. The anti-thrombotic effect of CNP is well known (Ahluwalia et al. 2004, Basic Res Cardiol. 99:83-89). Thrombus formation was significantly suppressed in the presence of CNP in antilogous rabbit jugular vein grafts (Ohno et al. 2002, Circulation. 105:1623-1626). In a model of balloon-injured rabbit carotid arteries CNP was shown to exert anti-thrombotic activity, probably via an increase in the NO production by enhancing the expression of inducible NO synthase (Qian et al. 2002, Circ Res 91:1063-1069).
7. Stimulation of Arteriogenesis
Arteriogenesis refers to the growth of collateral arterioles into functional collateral arteries, and is linked to elevated blood pressure, and elevated flow, causing shear stress against the wall of the arterioles. The stimulation of this event presents a strategy to treat arterial occlusive diseases (van Royen et al. 2001, Cardiovasc Res. 49:543-553). A beneficial effect of ANP on coronary collateral blood flow has been shown earlier (Kyriakides et al. 1998, Clin Cardiol. 21:737-742).
8. Inflammation, Especially Reduction of Inflammatory Mediators, e.g. TNF-Alpha, Other Cytokines or any Kind of Inflammatory Mediator
Several publications suggest a role of CNP in the modulation of inflammatory responses: in a model of balloon-injured rabbit carotid arteries, in vivo expression of CNP lowered the expression of the inflammatory marker ICAM-1, and reduced the infiltration of macrophages, supposedly via enhancement of NO generation (Qian et al. 2002, Circ Res 91:1063-1069). In another study, in rat aortic smooth muscle cells in vitro, CNP augmented the transcriptional activation of iNOS induced by inflammatory cytokines (interleukin-1 and tumour necrosis factor-α) and hence the production of NO (Marumo et al. 1995, Endocrinology. 136:2135-2142). CNP infusion in rats with an acute experimental myocarditis led to a reduction of CD68-positive inflammatory cell infiltration, and lowered myocardial and serum levels of monocyte chemoattractant protein-1 (Obata et al. 2007, Biochem Biophys Res Commun. 356:60-66). By selectively attenuating the expression of P-selectin, CNP suppressed leukocyte rolling induced by IL-113 or histamine in a rapid, reversible, and concentration-dependent manner in mice (Scotland et al. 2005, Proc Natl Acad Sci USA. 102:14452-14457). In a model of bleomycin-induced pulmonary fibrosis in mice, infusion of CNP markedly reduced bronchoalveolar lavage fluid IL-1β levels (Murakami et al. 2004, Am J Physiol Lung Cell Mol Physiol. 287:L1172-1177).
9. Pathological Leukocyte Adhesion to Endothelium and Diapedesis into Tissue
In mouse mesenteric postcapillary venules in vivo in animals with high basal leukocyte activation (endothelial nitric oxide synthase knockout mice) or under acute inflammatory conditions (induced by IL-1β or histamine), CNP suppressed basal leukocyte rolling in a rapid, reversible, and concentration-dependent manner. CNP was also able to inhibit platelet-leukocyte interactions (Scotland et al. 2005, Proc Natl Acad Sci USA. 102:14452-14457). In a model of bleomycin-induced pulmonary fibrosis in mice, infusion of CNP for 14 days significantly inhibited infiltration of macrophages into the alveolar and interstitial regions (Murakami et al. 2004, Am J Physiol Lung Cell Mol Physiol. 287:L1172-1177). CNP is also known to lower the expression of cell adhesion molecules such as ICAM-1 (Qian et al. 2002, Circ Res 91:1063-1069), and P-Selectin (Scotland et al. 2005, Proc Natl Acad Sci USA. 102:14452-14457), further strengthening its role in adhesion molecule modulation.
10. Kidney Disease, Especially Renal Insufficiency, Renal Failure Due to Reduced Renal Perfusion, Glomerulonephritis and Kidney Fibrosis
Local CNP production and CNP receptor expression have previously been demonstrated in glomeruli (Terada et al. 1994, Am J Physiol. 267:F215-222, Lohe et al. 1995, J Am Soc Nephrol. 6:1552-1558, Mattingly et al. 1994, Kidney Int. 46:744-747, Dean et al. 1994, Am J Physiol. 266:F491-496), in kidney cells (Zhao et al. 1994, Kidney Int. 46:717-725) and in mesangial cells (Suga et al. 1992, Hypertension. 19:762-765), suggesting a role in kidney physiology. In several conditions CNP levels in plasma or urine are altered. CNP in plasma and urine was increased in nephrotic syndrome (Cataliotti et al. 2002, Am J Physiol Renal Physiol 283:F464-472), CNP was increased in urine in cirrhosis with renal impairment (Gulberg et al. 2000, Gut. 47:852-857), renal and urine levels of CNP were increased in experimental diabetes (Shin et al. 1998, J Endocrinol. 158:35-42), and NP levels were elevated in chronic kidney disease, but decreased after hemodialysis or transplantation (Horl 2005, J Investig Med 53:366-370).
The benefit from using CNP in indications such as renal insufficiency, and renal failure, comes from its ability to relax smooth muscles in conduit arteries (Drewett et al. 1995, J Biol Chem. 270:4668-4674, Madhani et al. 2003, Br J Pharmacol. 139:1289-1296), venodilation (Chen and Burnett 1998, J Cardiovasc Pharmacol. 32 Suppl 3:S22-28, Wei et al. 1993, J Clin Invest. 92:2048-2052), and dilation of both, afferent and efferent arterioles in glomeruli, as shown in the hydronephrotic rat kidney (Endlich and Steinhausen 1997, Kidney Int. 52:202-207).
Glomerulopathies like glomerulonephritis are typically associated with mesangial cell proliferation, and leukocyte infiltration (Buschhausen et al. 2001, Cardiovasc Res. 51:463-469). The inhibitory effect of CNP on leukocyte infiltration via downregulation of ICAM-1 has been shown before (Qian et al. 2002, Circ Res 91:1063-1069, Buschhausen et al. 2001, Cardiovasc Res. 51:463-469). In addition, all NPs show anti-proliferative effects on mesangial cells in vitro on rat cells (Suganami et al. 2001, J Am Soc Nephrol 12:2652-2663). In vivo, CNP infusion improved immune mediated glomerulonephritis in a rat mesangioproliferative anti-Thy 1.1 model (Canaan-Kuhl et al. 1998, Kidney Int 53:1143-1151). In yet another study CNP inhibited glomerular mesangial cell proliferation, MCP-1 secretion, and reduced collagen IV production from mesangial cells (Osawa et al. 2000, Nephron. 86:467-472).
The inhibitory effect of CNP on the proliferation of glomerular mesangial cells (Suganami et al. 2001, J Am Soc Nephrol 12:2652-2663, Canaan-Kuhl et al. 1998, Kidney Int 53:1143-1151, Osawa et al. 2000, Nephron. 86:467-472) suggests its use in the treatment of kidney fibrosis.
11. Liver Diseases, Especially Portal Vein Hypertension, Liver Cirrhosis, Liver Ascites, Liver Fibrosis and Hepatorenal Syndrome
Evidence for a local natriuretic peptide system in the human liver comes from mRNA analysis; specific transcripts for all three NPRs, namely NPR-A, NPR-B, and NPR-C, could be detected, along with mRNA for ANP and CNP, but not BNP (Vollmar et al. 1997, Gut. 40:145-150). During chronic liver diseases, hepatic stellate cells, believed to play a role in the pathogenesis of liver fibrosis and portal hypertension (Friedman 1993, N Engl J Med. 328:1828-1835), acquire a myofibroblastic phenotype, proliferate, and synthetize components associated with fibrosis. Activation of NPR-B by CNP in myofibroblastic hepatic stellate cells was shown to inhibit both growth and contraction (Tao et al. 1999, J Biol Chem. 274:23761-23769), suggesting that during chronic liver diseases, CNP may counteract both liver fibrogenesis and associated portal hypertension.
Liver cirrhosis is the result of a chronic liver disease characterized by replacement of liver tissue by fibrous scar tissue. The presence of CNP in the human kidney and urine (Mattingly et al. 1994, Kidney Int. 46:744-747) suggests a role for CNP in fluid and electrolyte homeostasis, and thus possibly a role in renal function disturbances in patients with cirrhosis of the liver. CNP in the urine of cirrhotic patients with impaired renal function was increased, while plasma levels were normal (Gulberg et al. 2000, Gut. 47:852-857). In cirrhotic patients, ANP infusion reduced the portal pressure and increased the hepatic blood flow, indicative of a lowering of intra-hepatic resistance to portal flow (Brenard et al. 1992, J Hepatol. 14:347-356). Administration of pharmacological doses of CNP to cirrhotic rats significantly decreased portal pressure and peripheral vascular resistance, and increased cardiac output (Komeichi et al. 1995, J Hepatol. 22:319-325).
Many disorders can cause ascites, but cirrhosis is the most common. Hence, treatment of disorders such as liver cirrhosis will eventually help in the avoidance of ascites.
According to the vasodilation theory, the hepatorenal syndrome is the result of the effect of vasoconstrictor systems acting on the renal circulation. Due to this increased activity of the vasoconstrictor systems, renal perfusion and glomerular filtration rate are markedly reduced, while tubular function is preserved. Any substance that increases renal perfusion and/or glomerular filtration rate is thus suited to be used against the hepatorenal syndrome.
12. Lung Diseases, Especially Pulmonary Hypertension, Asthma and Pulmonary Fibrosis
CNP was shown to be locally synthesized in pulmonary tissues and therefore might have action on airway patency (Suga et al. 1992, Circ Res. 71:34-39). In vitro CNP was one order of magnitude more potent than ANP in cGMP production in cultured aortic smooth muscle cells.
Pulmonary hypertension is a progressive disease, characterized by an elevated pressure in the pulmonary arterial system. Common treatment is the use of vasodilatory substances. The ability to relax arteries, probably via direct interaction with the VSMCs, has been shown before in isolated pig coronary arteries (Marton et al. 2005, Vascul Pharmacol. 43:207-212). More specifically, CNP was able to ameliorate monocrotaline-induced pulmonary hypertension in rats and to improve survival (Itoh et al. 2004, Am J Respir Crit Care Med. 170:1204-1211), even if treatment with CNP started 3 weeks after the onset of symptoms.
In an ovalbumin-induced asthmatic guinea pig model CNP was able to significantly inhibit the bronchoconstriction and microvascular leakage in a dose-dependent manner (Ohbayashi et al. 1998, Eur J Pharmacol. 346:55-64). In vivo in asthmatics Fluge et al. could demonstrate dose-dependent bronchodilating properties of intravenous natriuretic peptide (Fluge et al. 1995, Regul Pept. 59:357-370).
In a model of bleomycin-induced pulmonary fibrosis in mice, infusion of CNP markedly attenuated the fibrosis, as indicated by significant decreases in Ashcroft score and lung hydroxyproline content (Murakami et al. 2004, Am J Physiol Lung Cell Mol Physiol. 287:L1172-1177). Immunohistochemistry on lung sections revealed a significantly reduced infiltration of macrophages into the alveolar and interstitial regions. The markedly decreased number of Ki-67-positive cells in fibrotic lesions of the lung further supports the notion of CNP's anti-proliferative effects on pulmonary fibrosis.
13. Male and Female Fertility Problems, Especially Erectile Dysfunction, Stimulation of Male Fertility and Stimulation of Female Fertility
Penile erection depends on relaxation of the smooth muscle of the corpus cavernosum, one of the sponge-like regions of erectile tissue. The presence of NPR-B in rat and rabbit cavernosal membrane was shown by Kim et al. (Kim et al. 1998, J Urol. 159:1741-1746). They also showed that CNP could trigger the production of cGMP in this tissue, and that CNP was much more potent than BNP and ANP in doing so. NPR-B was also shown to be located in the human corpus cavernosum penis; in organ bath studies with corpus cavernosum muscle strips CNP at concentrations of 0.1 nM to 1 μM led to smooth muscle relaxation from 5% to 40% (Kuthe et al. 2003, J Urol. 169:1918-1922); further support for a role of CNP in erectile dysfunction comes from a recent study, showing that CNP levels are associated with the presence, severity, and duration of erectile dysfunction (Vlachopoulos et al. 2008, Eur Urol. in press).
The rationale for using CNP to stimulate male fertility is based on its potential function in testicular blood supply, the modulation of germ cell development and spermatozoan motility, and its role in penile erection (as described above). CNP has been found in seminal plasma of several species (Hosang and Scheit 1994, DNA Cell Biol. 13:409-417, Chrisman et al. 1993, J Biol Chem. 268:3698-3703); human Leydig cells, located adjacent to the seminiferous tubules in the testicle, contain both, CNP and the NPR-B receptor (Middendorff et al. 1996, J Clin Endocrinol Metab. 81:4324-4328). CNP was able to increase testosterone levels in vitro in purified mouse Leydig cells (Khurana and Pandey 1993, Endocrinology. 133:2141-2149), as well as in vivo in the spermatic vein in men (Foresta et al. 1991, J Clin Endocrinol Metab. 72:392-395). Because testosterone activates the initiation, processing and maintenance of spermatogenesis, CNP has thus an immediate influence on spermatogenesis. Local injection of natriuretic peptides in vivo in rats caused a dose-related increase in testicular blood flow (Collin et al. 1997, Int J Androl. 20:55-60).
A function of CNP in fertilization, pregnancy and embryonic development was first proposed after the detection of CNP in porcine seminal plasma (Chrisman et al. 1993, J Biol Chem. 268:3698-3703). Further studies showed expression of NPR-A and -B receptors in human placenta (Itoh et al. 1994, Biochem Biophys Res Commun. 203:602-607), and their modulation in rat ovary and uterus by the estrous cycle (Huang et al. 1996, Am J Physiol. 271:H1565-1575, Dos Reis et al. 1995, Endocrinology. 136:4247-4253, Noubani et al. 2000, Endocrinology. 141:551-559). In mice, uterine CNP mRNA concentrations increased during pregnancy, whereas in the ovaries these levels decreased compared to non-pregnant controls (Stepan et al. 2001, Regul Pept. 102:9-13). In human placenta and myometrium CNP is expressed with no dependency on gestational age in the third trimester. Pregnancies with intra-uterine growth retardation showed an opposite regulation of CNP in placenta and myometrium, indicating an organ-specific function of the peptide in human reproductive tissue (Stepan et al. 2002, Fetal Diagn Ther. 17:37-41). This could be substantiated by studying NPR-B knock-out mice; female mice were infertile due to the failure of the female reproductive tract to develop (Tamura et al. 2004, Proc Natl Acad Sci USA. 101:17300-17305).
14. Pre-Eclampsia and/or Preterm Labor
Pre-eclampsia, a hypertensive disorder of pregnancy, is usually associated with raised blood pressure, and affects about 2-8% of pregnancies. Inadequate blood supply to the placenta leads to endothelial dysfunction, eventually resulting in damage to the maternal endothelium and kidney and liver. In severe pre-eclampsia BNP levels are elevated, which might reflect ventricular stress and/or subclinical cardiac dysfunction associated with the condition (Resnik et al. 2005, Am J Obstet Gynecol. 193:450-454). Pregnancies with intra-uterine growth retardation or pre-eclampsia showed an opposite regulation of CNP, with a decrease in the placenta and an increase in the myometrium compared with normal pregnancies (Stepan et al. 2002, Fetal Diagn Ther. 17:37-41), while maternal CNP plasma levels remained constant; this could indicate a compensatory or causative organ-specific function of the peptide in human reproductive tissue under these pathophysiological conditions, suggesting that application of CNP may have benefits.
15. Skeletal Growth Disturbances, Especially Decreased Body Height (Dwarfism)
Dwarfism can be caused by over 200 separate medical conditions. C-type natriuretic peptide, acting through its receptor, NPR-B, plays a critical role in longitudinal bone growth (Olney 2006, Growth Horm IGF Res. 16 Suppl A:S6-14), as it stimulates endochondrial ossification (Tamura et al. 2004, Proc Natl Acad Sci USA. 101:17300-17305, Miyazawa et al. 2002, Endocrinology. 143:3604-3610). A spontaneous autosomal recessive point mutation in the CNP gene, called long bone abnormality (lbab), causes severe dwarfism in mice (Yoder et al. 2008, Peptides. 29:1575-1581, Tsuji et al. 2008, Biochem Biophys Res Commun. 376:186-190). Complete absence of CNP in mice resulted in dwarfism and early death (Chusho et al. 2001, Proc Natl Acad Sci USA. 98:4016-4021).
16. Defects of FGF-R (Fibroblast Derived Growth Factor Receptor) Signalling, Especially Overactivity of FGF-R, or Deficiency of CNP or Osteocrin, or Reduced Level of CNP or Osteocrin in the Growth Plates of Long Bones
In vitro and ex vivo studies showed that CNP acts within the growth plate. CNP, most likely synthetised by proliferating chondrocytes (Chusho et al. 2001, Proc Natl Acad Sci US A. 98:4016-4021), acts locally to stimulate further proliferation. As opposing element, the FGF/FGFR-3 pathway is known to negatively regulate endochondral ossification via activation of the Erk MAP kinase pathway, thus inhibiting chondrocyte proliferation and cartilage matrix production (Krejci et al. 2005, J Cell Sci. 118:5089-5100). The targeted overexpression of CNP in chondrocytes offset dwarfism in a mouse model of achondroplasia with activated fibroblast growth factor receptor 3 in the cartilage, suggesting a direct interaction of their signaling pathways (Yasoda et al. 2004, Nat Med. 10:80-86). Moreover, Ozasa et al. found that CNP was able to antagonize the activation of the MAPK cascade by FGFs, making the CNP/NPR-B pathway attractive as a novel therapeutic target in the treatment of achondroplasia (Ozasa et al. 2005, Bone. 36:1056-1064). CNP also partially antagonized the FGF2-induced expression, release and activation of several matrix-remodeling molecules including several matrix metalloproteinases. Independent of FGF signaling, CNP stimulated the upregulation of matrix production (Krejci et al. 2005, J Cell Sci. 118:5089-5100).
Osteocrin is a specific ligand of the natriuretic peptide clearance receptor NPR-C that modulates bone growth (Thomas et al. 2003, J Biol Chem. 278:50563-50571). By blocking the clearance function of NPR-C, it causes the local elevation of CNP levels, resulting in the proliferation of chondrocytes (Moffatt et al. 2007, J Biol Chem. 282:36454-36462).
In summary, there is a strong rationale to use CNP in order to compensate for overactive FGF receptors, and for deficiencies or reduced levels of CNP or osteocrin.
17. Arthritis, Especially Degenerative Diseases of Cartilage Tissue, Osteoarthritis and Cartilage Degeneration and Arthritis in Response to Traumatic Cartilage Injury
The rationale for the use of natriuretic peptides for the treatment and/or prevention of arthritic diseases comes from the observation that CNP is involved in the skeletal growth, especially in the generation of cartilage extracellular matrix (Chusho et al. 2001, Proc Natl Acad Sci USA. 98:4016-4021, Yasoda et al. 2004, Nat Med. 10:80-86), which is able to stabilize damaged cartilage.
CNP depletion was shown to result in impaired bone growth, like that observed in achondroplastic bones, with a similar histological picture of decreased width in both the proliferative and hypertrophic chondrocyte layers of the growth plate (Chusho et al. 2001, Proc Natl Acad Sci USA. 98:4016-4021). The targeted overexpression of CNP in chondrocytes counteracted dwarfism in a mouse model of achondroplasia with activated fibroblast growth factor receptor 3 in the cartilage. CNP corrected the decreased extracellular matrix synthesis in the growth plate through inhibition of the MAPK pathway of FGF signaling, resulting in the stimulation of glucosaminoglycans and cartilage collagen (type II) synthesis (Yasoda et al. 2004, Nat Med. 10:80-86).
In rat chondrosarcoma chondrocytes, after FGF2-mediated growth arrest, CNP mediated the inhibition of MMP induction, and stimulated extracellular matrix synthesis (Krejci et al. 2005, J Cell Sci. 118:5089-5100, Ozasa et al. 2005, Bone. 36:1056-1064), both effects resulting in a net increase in cartilage extracellular matrix (Krejci et al. 2005, J Cell Sci. 118:5089-5100).
18. Tissue Engineering and Cartilage Regeneration, Especially for the Ex Vivo Expansion of Cartilage Cells to a Cell Number Sufficient to Transplant Cells Back into a Patient
CNP has stimulatory activity on glucosaminoglycan and cartilage collagen (type II) synthesis in chondrocytes (Krejci et al. 2005, J Cell Sci. 118:5089-5100, Yasoda et al. 2004, Nat Med. 10:80-86), a feature that is beneficial for in vivo regeneration of cartilage. To produce ex vivo tissue from the limited number of cells that can be extracted from an individual for therapeutic purposes, it is also necessary to have a stimulation of cell proliferation. In a key publication, Waldman et al. reported, that in high-density 3D cultures low doses of CNP (10 to 100 pM) elicited chondrocyte proliferation of up to 43% increase in cellularity at the highest dose. Higher doses of CNP (10 nM) predominantly stimulated matrix deposition without affecting tissue cellularity (Waldman et al. 2008, Tissue Eng Part A. 14:441-448). CNP is thus suitable as a modulator of both chondrocyte proliferation and ECM deposition during in vitro cartilage growth.
19. Tissue Engineering and Bone Regeneration, Especially for the Acceleration of Bone Healing or for the Improvement of Regenerating Bone Tissue
The role of the NPR-B/CNP system as an important regulator of bone growth has been established by several publications: NPR-B knock-out mice displayed reduced bone growth (Tamura et al. 2004, Proc Natl Acad Sci USA. 101:17300-17305, Pfeifer et al. 1996, Science. 274:2082-2086); mice with a deletion of the CNP gene also showed reduced bone growth, and this phenotype could be rescued by the overexpression of CNP in chondrocytes (Chusho et al. 2001, Proc Natl Acad Sci USA. 98:4016-4021); overexpression of BNP in mice resulted in skeletal overgrowth (Suda et al. 1998, Proc Natl Acad Sci USA. 95:2337-2342). More specifically, CNP was able to promote chondrocyte proliferation and matrix formation (Krejci et al. 2005, J Cell Sci. 118:5089-5100, Ozasa et al. 2005, Bone. 36:1056-1064). Using an organ culture of fetal mouse tibias, an in vitro model of endochondral ossification, longitudinal bone growth was stimulated by CNP (Yasoda et al. 1998, J Biol Chem. 273:11695-11700).
In summary, the experimental evidence strongly supports the use of CNP in bone regenerating applications.
20. Modulation of Neuronal Activity, Especially for Replacement of CNP in its “Central Nervous Function”
The extensive distribution of the NPR-C receptor in the brainstem suggests an involvement of NPR-C in the neuromodulatory effect of natriuretic peptides (Abdelalim et al. 2008, Neuroscience. 155:192-202), which were shown to evoke a variety of peripheral effects when applied to the brain (Puurunen and Ruskoaho 1987, Eur J Pharmacol. 141:493-495, Bianciotti et al. 2001, Regul Pept. 102:127-133). Intra-cerebroventricular administration of atrial natriuretic peptide in anaesthetized rats, for example, resulted in the stimulation of gastric acid secretion, that was totally abolished by vagotomy, suggesting vagus nerve involvement (Puurunen and Ruskoaho 1987, Eur J Pharmacol. 141:493-495). In two studies by Sabbatini et al., the cerebroventricular administration of CNP in rats dose-dependently enhanced the exocrine pancreatic fluid output through the activation of the NPR-C receptor and the vago-vagal reflex (Sabbatini et al. 2005, Eur J Pharmacol. 524:67-74, Sabbatini et al. 2007, Eur J Pharmacol. 577:192-202), thus mimicking the effect of endogenous CNP.
21. Cancer, Through Inhibition of Proliferation of Tumor Cells, Especially Glioma Cells, Neuroblastoma Cells, Adenocarcinoma Cells, Adenocarcinoma Cells in Breast Pancreas and Prostate, Melanoma Cells and Renal Carcinoma Cells
Several publications have shown the presence of natriuretic peptide receptors on tumor cells, suggesting a potential to affect the proliferation of those cells via application of CNP, as has been shown in a range of other cell types.
Early in vitro data from cultered rat glioma cells demonstrated the presence of receptors on those cells, that showed strongest activation by CNP, i.e. cGMP production (Eguchi et al. 1992, Eur J Pharmacol. 225:79-82). In another cell line, a AtT-20 pituitary tumor cell line, the only natriuretic receptor present on the cell surface was the NPR-B receptor. cGMP production in these AtT-20 cells was stimulated up to 200-fold by CNP (Gilkes et al. 1994, Biochem J. 299 (Pt 2):481-487).
Western immunoblotting identified NPR-A and NPR-C receptors in human colon adenocarcinoma cells. Application of 1 mM ANP to these cells resulted in a decrease of up to 97% in cell number within 24 h, suggesting an anti-proliferative activity (Gower et al. 2005, Int J Gastrointest Cancer. 36:77-87).
CNP caused a 39% decrease in the number of small-cell lung cancer cells at 100 μM. The mechanism of growth inhibition supposedly is based on the inhibition of DNA synthesis, mediated in part by cGMP (Vesely et al. 2005, Eur J Clin Invest. 35:388-398).
In yet another cell type, in human renal carcinoma cells, CNP also decreased the cell number, at a concentration of 100 μM by 10%. This effect was sustained without any proliferation of the cells occurring for three days after treatment with CNP. All three types of natriuretic peptide receptors, NPR-A, NPR-B, and NPR-C, were identified on renal cancer cells (Vesely et al. 2006, Eur J Clin Invest. 36:810-819).
22. Fibrosis, Especially Pulmonary Fibrosis, Renal Fibrosis, Cardiac Fibrosis, Hepatic Fibrosis or Systemic Fibrosis/Sclerosis
Several studies, investigating fibrotic events in different organ systems, have shown that the application of natriuretic peptides, in particular of CNP, has a beneficial effect on disease progression. A more general effect of CNP-mediated cGMP generation in fibroblasts is the block of the activation of the mitogen-activated protein kinase cascade (Chrisman and Garbers 1999, J Biol Chem. 274:4293-4299), which could be exploited to treat any kind of fibrosis, in particular the multiorgan systemic fibrosis/sclerosis; treatment of single organ fibrosis with CNP is supported by the following data:
In a model of bleomycin-induced pulmonary fibrosis in mice, infusion of CNP markedly reduced bronchoalveolar lavage fluid levels of inflammatory IL-10, inhibited infiltration of macrophages into the alveolar and interstitial regions, and markedly attenuated the fibrosis, as indicated by significant decreases in Ashcroft score and lung hydroxyproline content (Murakami et al. 2004, Am J Physiol Lung Cell Mol Physiol. 287:L1172-1177).
With regard to kidney fibrosis, it was described that CNP had an inhibitory effect on the proliferation of glomerular mesangial cells (Suganami et al. 2001, J Am Soc Nephrol 12:2652-2663, Canaan-Kuhl et al. 1998, Kidney Int 53:1143-1151, Osawa et al. 2000, Nephron. 86:467-472). In particular, CNP inhibited also MCP-1 secretion, and reduced collagen IV production from glomerular mesangial cells (Osawa et al. 2000, Nephron. 86:467-472).
Cardiac fibrosis, characterized by the proliferation of interstitial fibroblasts and the biosynthesis of extracellular matrix components in the ventricles of the heart, is a consequence of remodeling processes. Soeki et al. showed that the application of CNP improved cardiac function and protected against cardiac remodeling after myocardial infarct in rats (Soeki et al. 2005, J Am Coll Cardiol 45:608-616). In vitro, in cardiac fibroblasts, CNP had a suppressive effect on fibroblast proliferation and extracellular matrix production, the effect being stronger than by ANP or BNP (Horio et al. 2003, Endocrinology. 144:2279-2284).
During chronic liver diseases, hepatic stellate cells, believed to play a role in the pathogenesis of liver fibrosis and portal hypertension (Friedman 1993, N Engl J Med. 328:1828-1835), acquired a my ofibroblastic phenotype, proliferated, and synthesized components associated with fibrosis. The activation of NPR-B by CNP in myofibroblastic hepatic stellate cells was shown to inhibit both growth and contraction (Tao et al. 1999, J Biol Chem. 274:23761-23769), suggesting that during chronic liver diseases, CNP may counteract fibrogenesis.
C. Pharmaceutical Preparations
Other embodiments of the present invention are directed to pharmaceutical compositions, comprising at least one novel NPR-B agonist described herein, directed to the treatment or prevention of a disease in a subject that is associated with elevated IOP, glaucoma, ocular hypertension, and/or retinal ganglion cell loss.
1. Effective Amount
As used herein, the term “effective amount,” or “therapeutically effective amount,” refers to an amount of the agent that will activate the function and/or activity of a type B natriuretic peptide receptor. The novel NPR-B agonists described herein lower intraocular pressure or treat ocular hypertension in a patient having elevated IOP or ocular hypertension. Thus, an effective amount is an amount sufficient to detectably and repeatedly ameliorate, reduce, minimize or limit the extent of any disease associated with elevated intraocular pressure or ocular hypertension, such as any of those diseases discussed above.
Treatment and/or prevention methods will involve treating an individual with an effective amount of a composition containing a therapeutically effective amount of at least one NPR-B agonist of the invention. A therapeutically effective amount is described, generally, as that amount that is known to be or suspected to be of benefit in the reduction of the signs or symptoms of a disease. In some embodiments of the present invention, an effective amount is generally an amount that is known or suspected to be of benefit in reducing the signs or symptoms of glaucoma and associated optic nerve or retinal damage in a subject. It is envisioned that the treatment with the NPR-B agonists hereof will stabilize or improve visual function (as measured by visual acuity, visual field, or other method known to those of ordinary skill in the art).
In some embodiments, an effective amount of a NPR-B agonist that may be administered to a subject includes a dose from about 1 microgram/kg/body weight to about 500 microgram/kg/body weight or more per administration, and any range derivable therein.
2. Formulations
Regarding the methods set forth herein, a NPR-B agonist can be formulated in any manner known to those of ordinary skill in the art. In the compositions set forth herein, the concentration of a NPR-B agonist can be any concentration known or suspected by those of ordinary skill in the art to be of benefit in the treatment and/or prevention of ophthalmic disease associated with elevated intraocular pressure or ocular hypertension.
The actual dosage amount of a composition of the present invention administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
In certain non-limiting embodiments, the ophthalmic pharmaceutical compositions may comprise, for example, at least about 0.03%, by weight or volume, of an active ingredient. In other embodiments, the active ingredient may comprise between about 0.001% to about 75% of the weight or volume of the unit, or between about 0.01% to about 60%, and any range derivable therein. In more particular embodiments, the pharmaceutical composition may comprise between about 0.03% to about 2.0% by weight or volume, of an active ingredient. In more particular embodiments, the composition comprises between about 0.05% to about 1.5% by weight or volume of an active ingredient. In further embodiments, the composition comprises between about 0.05% to about 1.2% by weight or volume of an active ingredient.
A dose may be any amount of pharmaceutical composition that is known or suspected to be of therapeutic benefit. For example, a dose may be about 1 microgram/kg/body weight to about 500 microgram/kg/body weight or more per administration, and any range derivable therein. A dose may be repeated as necessary as determined by one of ordinary skill in the art to achieve a desired therapeutic effect. For example, a dose may be repeated once, twice, three times, and so forth. In some embodiments, a dose is administered twice a day, three times a day, four times a day, or more often. In further embodiments, a dose is administered every other day, twice a week, once a month, or at a longer interval.
In certain embodiments of the present invention, the compositions set forth herein can include more than one NPR-B agonist. One of ordinary skill in the art would be familiar with preparing and administering pharmaceutical compositions that include more than one therapeutic agent. In some embodiments, the composition includes one or more additional therapeutic agents that are not NPR-B agonists.
In addition to the NPR-B agonists, the compositions of the present invention optionally comprise one or more excipients. Excipients commonly used in pharmaceutical compositions include, but are not limited to, carriers, tonicity agents, preservatives, chelating agents, buffering agents, surfactants and antioxidants.
A person of ordinary skill will recognize that the compositions of the present invention can include any number of combinations of ingredients (e.g., active agent, polymers, excipients, etc.). It is also contemplated that that the concentrations of these ingredients can vary. In non-limiting aspects, the percentage of each ingredient in the composition can be calculated by weight or volume of the total composition. A person of ordinary skill in the art would understand that the concentrations can vary depending on the addition, substitution, and/or subtraction of ingredients in a given composition.
In some embodiments of the invention, a specific amount of a NPR-B agonist is administered via the compositions described herein.
The phrase “pharmaceutically acceptable carrier” is art-recognized, and refers to, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the supplement and not injurious to the patient.
Any of a variety of carriers may be used in the formulations of the present invention including water, mixtures of water and water-miscible solvents, such as C1-7-alkanols, vegetable oils or mineral oils comprising from 0.5 to 5% non-toxic water-soluble polymers, natural products, such as gelatin, alginates, pectins, tragacanth, karaya gum, xanthan gum, carrageenin, agar and acacia, starch derivatives, such as starch acetate and hydroxypropyl starch, and also other synthetic products, such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, preferably cross-linked polyacrylic acid, mixtures of those polymers. The concentration of the carrier is, typically, from 1 to 100000 times the concentration of the active ingredient.
Suitable tonicity-adjusting agents include mannitol, sodium chloride, glycerin, sorbitol and the like. Suitable preservatives include p-hydroxybenzoic acid ester, benzalkonium chloride, benzododecinium bromide, polyquatemium-1 and the like. Suitable chelating agents include sodium edetate and the like. Suitable buffering agents include phosphates, borates, citrates, acetates and the like. Suitable surfactants include ionic and nonionic surfactants, though nonionic surfactants are preferred, such as polysorbates, polyethoxylated castor oil derivatives and oxyethylated tertiary octylphenol formaldehyde polymer (tyloxapol). Suitable antioxidants include sulfites, ascorbates, BHA and BHT. The compositions of the present invention optionally comprise an additional active agent.
In particular embodiments, the compositions are suitable for application to mammalian eyes. For example, for ophthalmic administration, the formulation may be a solution, a suspension, a gel, or an ointment.
In preferred aspects, the compositions that include NPR-B agonists will be formulated for topical application to the eye in aqueous solution in the form of drops. The term “aqueous” typically denotes an aqueous composition wherein the carrier is to an extent of >50%, more preferably >75% and in particular >90% by weight water. These drops may be delivered from a single dose ampoule which may preferably be sterile and thus rendering bacteriostatic or bacteriocidal components of the formulation unnecessary. Alternatively, the drops may be delivered from a multi-dose bottle which may preferably comprise a device which extracts preservative from the formulation as it is delivered, such devices being known in the art.
In other aspects, components of the invention may be delivered to the eye as a concentrated gel or similar vehicle which forms dissolvable inserts that are placed beneath the eyelids.
The compositions of the present invention may also be formulated as solutions that undergo a phase transition to a gel upon administration to the eye.
In addition to the one or more NPR-B agonists, the compositions of the present invention may contain other ingredients as excipients. For example, the compositions may include one or more pharmaceutically acceptable buffering agents, preservatives (including preservative adjuncts), non-ionic tonicity-adjusting agents, surfactants, solubilizing agents, stabilizing agents, comfort-enhancing agents, polymers, emollients, pH-adjusting agents and/or lubricants.
For topical formulations to the eye, the formulations are preferably isotonic, or slightly hypotonic in order to combat any hypertonicity of tears caused by evaporation and/or disease. The compositions of the present invention generally have an osmolality in the range of 220-320 mOsm/kg, and preferably have an osmolality in the range of 235-260 mOsm/kg. The compositions of the invention have a pH in the range of 5-9, preferably 6.5-7.5, and most preferably 6.9-7.4.
The formulations set forth herein may comprise one or more preservatives. Examples of preservatives include quaternary ammonium compounds, such as benzalkonium chloride or benzoxonium chloride. Other examples of preservatives include alkyl-mercury salts of thiosalicylic acid, such as, for example, thiomersal, phenylmercuric nitrate, phenylmercuric acetate or phenylmercuric borate, sodium perborate, sodium chlorite, parabens, such as, for example, methylparaben or propylparaben, alcohols, such as, for example, chlorobutanol, benzyl alcohol or phenyl ethanol, guanidine derivatives, such as, for example, chlorohexidine or polyhexamethylene biguanide, sodium perborate, or sorbic acid.
In certain embodiments, the NPR-B agonists are formulated in a composition that comprises one or more tear substitutes. A variety of tear substitutes are known in the art and include, but are not limited to: monomeric polyols, such as, glycerol, propylene glycol, and ethylene glycol; polymeric polyols such as polyethylene glycol; cellulose esters such hydroxypropylmethyl cellulose, carboxy methylcellulose sodium and hydroxy propylcellulose; dextrans such as dextran 70; water soluble proteins such as gelatin; vinyl polymers, such as polyvinyl alcohol, polyvinylpyrrolidone, and povidone; and carbomers, such as carbomer 934P, carbomer 941, carbomer 940 and carbomer 974P. The formulation of the present invention may be used with contact lenses or other ophthalmic products.
In some embodiments, the compositions set forth herein have a viscosity of 0.5-10 cps, preferably 0.5-5 cps, and most preferably 1-2 cps. This relatively low viscosity insures that the product is comfortable, does not cause blurring, and is easily processed during manufacturing, transfer and filling operations.
3. Route of Administration
Administration of the compositions of the invention can be by any method known to those of ordinary skill in the art, however, local administration is preferred. It is contemplated that all local routes to the eye may be used including topical, subconjunctival, periocular, retrobulbar, subtenon, intracameral, intravitreal; intraocular, subretinal, juxtascleral and suprachoroidal administration. Systemic or parenteral administration may be feasible including but not limited to intravenous, subcutaneous, intramuscular and oral delivery. The most preferred method of administration will be intravitreal or subtenon injection of solutions or suspensions, or intravitreal or subtenon placement of bioerodible or non-bioerodible devices, or by topical ocular administration of solutions or suspensions, or posterior juxtascleral administration of a gel formulation.
Those of skill in the art, in light of the present disclosure, will appreciate that obvious modifications of the embodiments disclosed herein can be made without departing from the spirit and scope of the invention. All of the embodiments disclosed herein can be made and executed without undue experimentation in light of the present disclosure. The full scope of the invention is set out in the disclosure and equivalent embodiments thereof. The specification should not be construed to unduly narrow the full scope of protection to which the present invention is entitled.
While a particular embodiment of the invention has been shown and described, numerous variations and alternate embodiments will occur to those skilled in the art. Accordingly, the invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes to the claims that come within the meaning and range of equivalency of the claims are to be embraced within their scope. Further, all published documents, patents, and applications mentioned herein are hereby incorporated by reference, as if presented in their entirety.
D. Secondary Forms of Therapy
In certain embodiments of the present invention, the subject is receiving one or more secondary forms of therapy directed to treatment or prevention of a particular eye disease.
A NPR-B agonist-containing ophthalmic composition of the present invention may be administered along with another agent or therapeutic method. For example, administration of the NPR-B agonist-containing composition of the present invention to a human subject may precede, follow, or be concurrent with other therapies for glaucoma, elevated intraocular pressure or ocular hypertension. In some embodiments, the NPR-B agonist is formulated in the same composition as the secondary form of therapy. In other embodiments, the NPR-B agonist is formulated separately from the secondary form of therapy. One of ordinary skill in the art would be familiar with protocols for administering more than one form of pharmacological therapy to a subject with a disease, and would be familiar with methods of formulating more than one pharmacological agent in the same composition.
Examples of secondary therapeutic agents include, but are not limited to: anti-glaucoma agents, such as beta-blockers including timolol, betaxolol, levobetaxolol, carteolol, miotics including pilocarpine, carbonic anhydrase inhibitors, prostaglandins, seretonergics, muscarinics, dopaminergic agonists, adrenergic agonists including apraclonidine and brimonidine; anti-angiogenesis agents; anti-infective agents including quinolones such as ciprofloxacin, and aminoglycosides such as tobramycin and gentamicin; non-steroidal and steroidal anti-inflammatory agents, such as suprofen, diclofenac, ketorolac, rimexolone and tetrahydrocortisol; growth factors, such as nerve growth factor (NGF), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), ciliary neutrophic factor (CNTF); immunosuppressant agents; and anti-allergic agents including olopatadine. Information pertaining to olopatadine formulations can be found in U.S. Pat. No. 6,995,186, U.S. Patent App. Pub. No. 2005/0158387, and U.S. Patent App. Pub. No. 2003/0055102, each of which is hereby specifically incorporated by reference. The ophthalmic drug may be present in the form of a pharmaceutically acceptable salt, such as timolol maleate, brimonidine tartrate or sodium diclofenac.
Other examples of a secondary therapeutic agent include a receptor tyrosine kinase (RTK) inhibitor. Exemplary RTK inhibitors are described in U.S. Patent App. Pub. No. 2006/0189608, and U.S. Pat. No. 7,297,709, both of which are hereby specifically incorporated by reference. In preferred embodiments, the receptor tyrosine kinase inhibitor is N-[4-[3-amino-1H-indazol-4-yl]phenyl]-N′-(2-fluoro-5-methylphenyl)urea.
In other particular embodiments, the secondary therapeutic agent is a prostaglandin or a prostaglandin analog. For example, the prostaglandin analog may be latanoprost, bimatoprost, unoprostone or travoprost.
In particular embodiments, the secondary therapeutic agent is a steroid. For example, the steroid may be a glucocorticoid, a progestin, a mineralocorticoid, or a corticosteroid. Exemplary corticosteroids include cortisone, hydrocortisone, prednisone, prednisolone, methylprednisone, triamcinolone, fluoromethalone, dexamethasone, medrysone, betamethasone, loteprednol, fluocinolone, flumethasone, or mometasone. Other examples of steroids include androgens, such as testosterone, methyltestosterone, or danazol. The secondary therapeutic agent may also be a glucocorticoid that is devoid of typical glucocorticoid side-effects, such as a cortisene. Preferred cortisenes for use in the methods of the invention include anecortave acetate and anecortave desacetate. Often steroids are administered as ester, acetal, or ketal prodrugs, many of which are water-insoluble. The secondary therapeutic agents may be directed to treatment or prevention of a single disease, or can be directed to treatment or prevention of two or more diseases.
In addition to pharmacological agents, surgical procedures can be performed in combination with the administration of the NPR-B agonists. One such surgical procedure can include laser trabeculoplasty or trabeculectomy. In laser trabeculoplasty, energy from a laser is applied to a number of noncontiguous spots in the trabecular meshwork. It is believed that the laser energy stimulates the metabolism of the trabecular cells, and changes the extracellular material in the trabecular meshwork.
Another surgical procedure may include filtering surgery. With filtering surgery, a hole is made in the sclera near the angle. This hole allows the aqueous fluid to leave the eye through an alternate route. The most commonly performed filtering procedure is a trabeculectomy. In a trabeculectomy, a conjunctiva incision is made, the conjunctiva being the transparent tissue that covers the sclera. The conjunctiva is moved aside, exposing the sclera at the limbus. A partial thickness scleral flap is made and dissected half-thickness into the cornea. The anterior chamber is entered beneath the scleral flap and a section of deep sclera and/or trabecular meshwork is excised. The scleral flap is loosely sewn back into place. The conjunctival incision is tightly closed. Post-operatively, the aqueous fluid passes through the hole, beneath the scleral flap which offers some resistance and collects in an elevated space beneath the conjunctiva called a bleb. The fluid then is either absorbed through blood vessels in the conjunctiva or traverses across the conjunctiva into the tear film.
E. Examples
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
Material and Methods
The materials and methods as well as general methods are further illustrated by the following examples:
Solvents:
Solvents were used in the specified quality without further purification.
Acetonitrile (Gradient grade, J.T. Baker); dichloromethane (for synthesis, VWR); diethylether (for synthesis, VWR); N,N-dimethylformamide (LAB, VWR); dioxane (for synthesis, Aldrich); methanol (for synthesis, VWR).
Water: Milli-Q Plus, Millipore, demineralized.
Reagents:
The used reagents were purchased from Advanced ChemTech (Bamberg, Germany), Sigma-Aldrich-Fluka (Deisenhofen, Germany), Bachem (Heidelberg, Germany), J.T. Baker (Phillipsburg, USA), Iris Biotech (Marktredwitz, Germany), Lancaster (Griesheim, Germany), VWR (Darmstadt, Germany), NeoMPS (Strasbourg, France), Novabiochem (Bad Soden, Germany, from 2003 on Merck Biosciences, Darmstadt, Germany) and Acros (Geel, Belgium, distributor Fisher Scientific GmbH, Schwerte, Germany), Peptech (Cambridge, Mass., USA), Synthetech (Albany, Oreg., USA), Pharmacore (High Point, N.C., USA), Anaspec (San Jose, Calif., USA) and used in the specified quality without further purification.
Non-commercially available non-conventional amino acids were prepared according to standard protocols either as building blocks for solid phase synthesis or by derivatization of commercially available amino acids during solid phase synthesis.
If not stated differently, concentrations are given as percent by volume.
Analysis of Peptides According to the Present Invention:
The analyses of peptides were performed with analytical HPLC methods followed by either ESI-MS or MALDI-MS detection. For analytic chromatography a Hewlett Packard 1100-system together with an ESI-MS (Finnigan LCQ ion trap mass spectrometer) was used. Helium was used as impact gas in the ion trap. For chromatographic separation a RP-18-column (Vydac (Merck) at 30° C. was used. A binary gradient was applied for all chromatograms (5-95% B, linear, A: 0.1% TFA in water and B: 0.1% TFA in CH3CN). UV detection was at λ=220 nm.
Analyses by means of HPLC/MS was performed using a linear gradient from 95:5 to 5:95 (A: 0.1% TFA in water and B: 0.1% TFA in acetonitrile), RP columns were from the companies Phenomenex or Waters (Typ Luna C-18, 3 μm, 2.00×50 mm, Symmetry C18 Column MV Kit, 5 μm, 4.6×250 mm, respectively); For ESI-MS measurements a mass spectrometer ThermoFinnigan Advantage and/or LCQ Classic (both iontrap) was used. For ESI ionization helium served as impact gas in the ion trap. In case of MALDI-MS analyses an Applied Biosystems Voyager RP MALDI mass spectrometer was used with α-Cyano-4-hydroxycinnamic acid as internal calibration matrix.
Purification of Peptides with Preparative HPLC:
Preparative HPLC separations were performed using Varian PLRP-S (10 μm, 100 Å) columns (150×25 mm or 150×50 mm) with the following gradient solvents: A: 0.05% TFA in H2O and B: 0.05% TFA in CH3CN







TABLE 4





Abbreviations:


















AAV
general procedure



Ac
Acetyl



Acm
Acetamidomethyl



DCM
Dichloromethane



DIC
Diisopropylcarbodiimide



DIPEA
N,N-diisopropylethylamine



DMF
N,N-dimethylformamide



DMSO
Dimethylsulfoxide



eq.
Equivalent(s)



ESI
Electrospray ionisation



Fig.
Figure



Fmoc
9-fluorenylmethyloxycarbonyl



H
hour(s)



HATU
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium-




hexafluorophosphate



HBTU
O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-




hexafluorophosphate



HOBt
1-hydroxybenzotriazole



HPLC
high-pressure liquid chromatography



MALDI
Matrix Assisted Laser Desorption/Ionization



Me
Methyl



min
minute(s)



ml
Milliliter



MS
Mass spectrometry



MW
Molecular weight



NMP
N-methylpyrrolidone



Ph
Phenyl



RP
Reversed phase



tBu
tert-butyl



THF
Tetrahydrofuran



TIPS
Triisopropyl silane



TFA
trifluoroacetic acid



UV
Ultraviolet










Example 2
Synthesis of Peptides
Linear peptides were synthesized using the Fmoc-tBu-strategy. The synthesis was done either manually in polypropylene syringes or via an automatic synthesizer (Syro from Multisyntech, Witten or Sophas from Zinsser-Analytic, Frankfurt).
For the preparation of peptides carrying a C-terminal carboxylic acid, the C-terminal amino acid was either attached to a tritylchloride resin (approx. 100 mg resin; loading of reactive groups approx. 1.5 mmol/g; coupling with 0.8 eq. Fmoc-amino acid and 3.0 eq. DIPEA in DCM for 2 h; loading of the first amino acid approx. 0.2-0.4 mmol/g) or to Wang resin (100-200 mg resin; loading of reactive groups approx. 0.6 mmol/g; coupling with 4 eq. Fmoc-amino acid, 4 eq. DIC and 3 eq. NMI in DMF for 3 h; loading of the first amino acid approx. 0.2-0.6 mmol/g).
For the preparation of peptides carrying a C-terminal carboxylic amide, the first amino acid was attached to the resin via Fmoc deprotection of the Fmoc-Rink amide resin (ca. 100 mg resin, ca. 0.5 mmol/g loading; Fmoc deprotection with 20% piperidine in DMF for 20 min) and subsequent coupling of the Fmoc amino acid (reaction with 5 eq. Fmoc amino acid; 5 eq. HBTU or 5 eq. HATU and 10 eq. DIPEA in NMP for 30-60 min and this step was optionally repeated).
After the coupling of the first amino acid, the synthesis of the peptide was done via a repeated sequence of steps, as necessary, consisting of Fmoc deprotection and coupling of the corresponding Fmoc amino acid or carboxylic acid. For the Fmoc deprotection the resin was treated with 20% piperidine in DMF for 20 min. The coupling of the amino acids was carried out via reaction with 5 eq. of the amino acid, 5 eq. HBTU or 5 eq. HATU and 10 eq. DIPEA in DMF for 30-60 min. Each coupling step was optionally repeated.
For the introduction of the N-terminal acetyl group, the N-terminal free peptide, bound to the resin, was incubated with a solution of 10% acetic acid anhydride and 20% DIPEA in DMF for 20 min. For the introduction of the N-terminal sulfonyl group, the N-terminal free peptide, bound to the resin, was incubated with a solution of 2 eq. of the corresponding sulfonyl chloride and 4 eq. DIPEA in DMF or DCM for 30 min and this treatment was repeated once.
For the cleavage of the peptide from the resin and its side chain protecting groups, a mixture of 95% TFA, 2.5% H2O, 2.5% TIPS or a similar solution was added. Finally the crude peptide was isolated either by evaporation of TFA using a rotary evaporator or by precipitation with the aid of methyl-tbutyl-ether at 0° C.
Example 3
NPR-A Induced Production of Cyclic GMP in Stably Transfected Cell
To assess the specificity of compounds for NPR activation, human 293-T cells transfected with NPR-A (Potter and Garbers 1992, J Biol Chem. 267:14531-14534) are used in stimulation experiments.
In this homogenous assay, the cells are stimulated in suspension with the test compound and the production of cyclic GMP (cGMP) is determined, from which EC50 values are calculated. ANP, the naturally occurring ligand of NPR-A is used as an internal control and to determine the maximal cGMP production of the cells, which allows the calculation of activation values of the tested compounds relative to ANP.
Preparation of Cells:
NPR-A transfected 293-T cells are washed once with phosphate buffered saline (PBS) and detached from a 75 cm2 tissue culture flask by addition of 3 ml of non enzymatic cell dissociation solution (Sigma-Aldrich) and incubation for 10 min. at room temperature. Detached cells are harvested in 20 ml PBS and centrifuged for 10 min at 200×g at room temperature. The cells are resuspended in DMEM/Ham's F12 mix supplemented with 1 mM IBMX (Medium) and adjusted to a density of 1.25×105 cells/ml and incubated for 15 min. at room temperature.
Stimulation of Cells:
20 μl of cells (2.5×103 cells) are added to each well of a 96 well white optical bottom tissue culture plate (Nunc, Germany). 10 μl of compound dilution is added and the cells are stimulated for 25 min. at room temperature. The stimulation is stopped by addition of 20 μl of Lysis buffer (reagent included in cGMP Assay Kit).
Determination of cGMP:
The amount of produced cGMP in the cells is determined using HitHunterTM cGMP Assay kit (DiscoveRX) according to manufacturer's instructions.
Dilution of Compounds:
For EC50 determinations, duplicate wells are stimulated with a serial dilution of a 10 mM DMSO compound stock solution. Dilutions are prepared in Medium supplemented with IBMX (1 mM). The final compound concentration in the assay is in the range from 45 μM to 20 nM. The internal standard compound ANP is used at concentrations ranging from 5 μM to 310 pM.
Example 4
NPR-B Induced Production of Cyclic GMP in Human Glaucoma Trabecular Meshwork Cells (GTM-3)
The potency of compounds to activate NPR-B was evaluated in a functional assay using endogenously NPR-B expressing GTM-3 cells (Pang, Shade et al. 1994). In this assay the dose dependent production of cyclic GMP (cGMP) is determined and EC50 values are calculated. The natural occurring ligand for NPR-B, i.e. CNP is used as an internal control and to determine the maximal cGMP production of the cells, which allows the calculation of activation values of the tested compounds relative to CNP.
Preparation of Cells:
In a 96 well white optical bottom tissue culture plate (Nunc, Germany) 1.5×105 cells/well are seeded in Dulbecco's MEM (DMEM, Biochrom) supplemented with Gentamycin (0.056 mg/ml) and incubated for 18 h with 10% CO2 in a humidified atmosphere.
Stimulation of Cells:
The cell culture medium is aspirated and each well is washed with 200 μl DMEM/Ham's F12=Medium (Gibco). Then, 200 μl Medium supplemented with 1.5 mM IBMX (3-Isobutyl-1-methyl-Xanthin, Sigma) is added to each well and incubated for 15 min. at room temperature. 25 μl of compound dilution is added and the cells are stimulated for 15 min. at room temperature. The stimulation is stopped by aspiration of the medium and addition of 20 μl of Lysis buffer (reagent included in cGMP Assay Kit).
Determination of cGMP:
The amount of produced cGMP in the cells is determined using HitHunterTM cGMP Assay kit (DiscoveRX) according to manufacturer's instructions.
Dilution of Compounds:
For EC50 determinations, duplicate wells are stimulated with a serial dilution of a 10 mM DMSO compound stock solution. Dilutions are prepared in Medium supplemented with IBMX (1.5 mM). Final compound concentrations are in the range from 45 μM to 20 nM. Highly active compounds, e.g. CNP are used for stimulation at concentrations ranging from 5 μM to 6 nM.
Example 5
Efficacy in the Rabbit
A single 30 μL drop of a test item formulation was administered to rabbit eyes (n=8 to 10). Intraocular pressure (IOP) was assessed in each eye at 0 hr, just prior to dosing, and again hourly for up to 4 hr post dose. The efficacy of a given formulation was determined based on the difference between the pretreatment IOP readings at 0 hr and the post treatment readings. A maximum percent reduction in IOP greater than 15% was noted by the “+” symbol. A maximum IOP reduction of less than 15% was assigned the “−” symbol.
Results obtained with novel compounds of the invention in the above-described assays are provided in Table 5, below:







TABLE 5







In vivo results with novel compounds of the invention according to the methods


described in Example 5.













RIOP


SEQ


dose 300 ug


ID


- IOP reduction <15%


NO:
JAL
STRUCTURE
+ IOP reduction >15%





  3
CNP
CNP
−


 


 81
 781+
Occ-ala-ala-Phe-Gly-Leu-Pro-Leu-Asp-Arg-
-




Lle-NH2;



 


127
 955++
Occ-pro-Phe-Gly-Leu-Pro-Nml-Asp-Arg-Ile-
-




NH2;



 


130
 958++
Occ-Sni-Nmf-Gly-Leu-Pro-Nml-Asp-Arg-Ile-
-




NH2;



 


135
 967+
Occ-Sni-Nmf-Gly-Leu-Pro-Leu-Asp-Arg-Ile-
-




NH2;



 


182
1041+
Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
+




NH2;



 


203
1085+
Occ-ala-Nmf-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
+




NH2;



 


187
1047++
Occ-ala-Phe-arg-Leu-Hyp-Leu-Asp-Arg-Ile-
+




NH2;



 


204
1086++
Occ-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-
+




NH2;



 


183
1042+
Occ-ala-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
-




NH2;



 


195
1060++
Occ-ala-Phe-lys-Leu-Hyp-Nml-Asp-Arg-Ile-
+




NH2;



 


267
1287
Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-
+




Arg-Ile-NH2;



 


274
1295+
Occ-Sni-Phe-leu-Leu-Tap-Nml-Asp-Arg-Ile-
+




NH2;



 


355
1400+
Occ-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Val-Arg-
+




Ile-NH2;



 


292
1325+
Occ-Sni-Phe-dap(Me2)-Leu-Tap-Nml-Asp-Arg-
+




Ile-NH2;



 


332
1369+
Oct-Sni-Phe-dap(Me2)-Leu-Tap-Nml-Asp-Arg-
+




Ile-NH2;



 


372
1429++
Oct-Sni-Phe-dap(Me2)-Leu-Hyp-Nml-Asp-Arg-
+




Ile-NH2;



 


414
1496+
Occ-Sni-Eaa-leu-Leu-Hyp-Nml-Asp-Arg-Ile-
-




NH2;



 


421
1512++
Occ-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Pro-Che;
+


 


425
1555++
Occ-Sni-Phe-Apc-Leu-Hyp-Nml-Asp-Arg-Ile-
+




NH2;



 


481
1654+
Occ-Sni-Phe-leu-Leu-Tap-Nml-Val-Pro-Che;
-


 


506
1729+
Occ-Sni-Phe-leu-Leu-Tap-Nml-Val-Arg-Che;
+


 


507
1730+
Occ-Sni-Phe-leu-Leu-Hyp-Nml-Val-Arg-Che;
+


 


269
1289+
Occ-Sni-Phe-orn(Me2)-Leu-Hyp-Nml-Asp-
+




Arg-Ile-NH2





HCl salt except *TFA;


Dose is 300 μg topical ocular unless (##);


DB rabbits unless NZA, scores 1-4 (4 = IOP could not be taken);


“” indicates hypertensive phase;


(n = #R) means # responders out of 10-12 animals tested;


1% is +susp ++sol






All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.
All references cited herein, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

## Claims

1. A compound selected from:
6054-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:228);
Occ-Sni-Mpa-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:244);
Occ-Sni-Ppa-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:245);
(6071-OH)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:246);
5587-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:248);
(AR-201-48)-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:270);
(AR-201-48)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:272);
(AR-201-58)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:307);
(AR-201-59)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:308);
Sbt-Sni-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:311);
Sbt-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:313);
Nbt-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:314);
(AR-201-72)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:339);
(AR-201-72)-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:340);
Miy-Gab-Hgl-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:374);
Ac-Miy-Gab-Hgl-ala-Phe-arg-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:375);
Occ-Sni-Tyr-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:408);
Occ-Sni-Bmf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:409);
Occ-Sni-Phe-Ala-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:423);
Occ-Sni-Phe-nml-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:437);
2553-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:457);
6988-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:460);
1695-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:462);
Occ-Sni-Otf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:467);
Occ-Sni-NHfe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:474);
785-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:484);
3218-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:486);
1281-G-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:489);
1281-Bal-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:490);
Occ-Sni-Hfe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:504);
Occ-ala-Nmf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:505);
Gluc-Aoa-hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:533);
(1270)-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:535);
Occ-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:537);
(AR-314-170)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:555);
(AR-314-171)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:556);
(AR-385-008)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:557); and
Occ-Sni-Phe-thr-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:565).

2. The compound of claim 1 selected from:
2553-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:457);
6988-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:460);
1695-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:462);
785-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:484);
3218-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:486);
1281-G-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:489);
1281-Bal-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:490);
Gluc-Aoa-hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:533);
(1270)-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:535); and
Occ-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:537).

3. A compound of the formula:



wherein
B is selected from the group consisting of Rb1— and Rb2—C(O)—, wherein:
Rb1 is selected from the group consisting of C6-C10 alkyl and C6-C10 alkyl substituted by NRb4Rb5;
Rb2 is C6-C10 alkyl substituted by NRb4Rb5; and
Rb4 and Rb5 are, independently, selected from the group consisting of H and C1-C4 alkyl; and

R11b is selected from the group consisting of H, C1-C8 alkyl, C4-C8 cycloalkyl, C7-C12 bicycloalkyl, and C1-C4 alkyl-C4-C8 cycloalkyl.

4. The compound of claim 3, wherein:
R11b is selected from the group consisting of H and C1-C8 alkyl.

5. A compound of the formula:



wherein
B is selected from the group consisting of Rb1— and Rb2—C(O)—, wherein:
Rb1 is selected from the group consisting of C6-C10 alkyl and C6-C10 alkyl substituted by NRb4Rb5;
Rb2 is C6-C10 alkyl substituted by NRb4Rb5; and
Rb4 and Rb5 are, independently, selected from the group consisting of H and C1-C4 alkyl; and
R11b is selected from the group consisting of H, C1-C8 alkyl, C4-C8 cycloalkyl, C7-C12 bicycloalkyl, and C1-C4 alkyl-C4-C8 cycloalkyl.

6. The compound of claim 5, wherein:
R11b is selected from the group consisting of H and C1-C8 alkyl.

7. The compound of claim 1, wherein said compound is selected from:
Occ-Sni-Mpa-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:244);
Occ-Sni-Ppa-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:245);
(AR-201-48)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:272);
(AR-201-72)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:339);
Occ-Sni-Tyr-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:408);
Occ-Sni-Bmf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:409);
2553-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:457);
6988-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:460);
1695-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:462);
Occ-Sni-Otf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:467);
Occ-Sni-NHfe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:474);
785-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:484);
3218-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:486);
1281-G-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:489);
1281-Bal-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:490);
Occ-Sni-Hfe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:504);
Occ-ala-Nmf-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:505);
Gluc-Aoa-hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:533);
(1270)-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:535);
Occ-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:537);
(AR-314-170)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:555);
(AR-314-171)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:556); and
(AR-385-008)-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:557).

8. The compound of claim 7, wherein said compound selected from:
(AR-201-72)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:339);
2553-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:457);
6988-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:460);
1695-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:462);
785-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:484);
3218-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:486);
1281-G-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:489);
1281-Bal-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:490);
Gluc-Aoa-hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:533);
(1270)-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:535);
Occ-Hgl-Sni-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:537);
(AR-314-170)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:555); and
(AR-314-171)-Phe-leu-Leu-Hyp-Nml-Asp-Arg-Ile-NH2 (SEQ ID NO:556).