Guanylate Cyclase Receptor Agonists For The Treatment Of Tissue Inflammation And Carcinogenesis

Abstract

A method of treatment of inflamed, pre-cancerous or cancerous tissue or polyps in a mammalian subject is disclosed. The treatment involves administration of a composition of at least one peptide agonist of a guanylate cyclase receptor and/or other small molecules that enhance intracellular production of cGMP. The at least one peptide agonist of a guanylate cyclase receptor may be administered either alone or in combination with an inhibitor of cGMP-dependent phosphodiesterase. The inhibitor may be a small molecule, peptide, protein or other compound that inhibits the degradation of cGMP. Without requiring a particular mechanism of action, this treatment may restore a healthy balance between proliferation and apoptosis in the subject's population of epithelial cells, and also suppress carcinogenesis. Thus, the method may be used to treat, inter alia, inflammation, including gastrointestinal inflammatory disorders, general organ inflammation and asthma, and carcinogenesis of the lung, gastrointestinal tract, bladder, testis, prostate and pancreas, or polyps.

Claims

1. A guanylate cyclase receptor agonist (GCRA) peptide consisting of the amino acid sequence of SEQ ID NO: 21.

2. A composition in unit dose form comprising a guanylate cyclase receptor agonist (GCRA) peptide consisting of the amino acid sequence of SEQ ID NO: 21.

3. A composition in unit dose form comprising: a) a GCRA peptide consisting of the amino acid sequence of SEQ ID NO: 21; and b) at least one compound selected from the group consisting of a cGMP-dependent phosphodiesterase inhibitor, an anti-inflammatory agent, an antiviral agent, and an anticancer agent.

4. The composition of claim 2, wherein the unit dose form is selected from the group consisting of a tablet, a capsule, a solution, and an inhalation formulation.

5. The composition of claim 3, wherein the unit dose form is selected from the group consisting of a tablet, a capsule, a solution, and an inhalation formulation.

6. The composition of claim 2, further comprising at least one pharmaceutically acceptable excipient.

7. The composition of claim 3, further comprising at least one pharmaceutically acceptable excipient.

8. A peptide conjugate comprising polyethylene glycol (PEG) attached to a peptide consisting of the amino acid sequence of SEQ ID NO:21.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention is based upon several concepts. The first is that there is a cGMP-dependent mechanism which regulates the balance between cellular proliferation and apoptosis and that a reduction in cGMP levels, due to a deficiency of uroguanylin/guanylin and/or due to the activation of cGMP-specific phosphodiesterases, is an early and critical step in neoplastic transformation. A second concept is that the release of arachidonic acid from membrane phospholipids, which leads to the activation of cPLA.sub.2, COX-2 and possibly 5-lipoxygenase during the process of inflammation, is down-regulated by a cGMP-dependent mechanism, leading to reduced levels of prostaglandins and leukotrienes, and that increasing intracellular levels of cGMP may therefore produce an anti-inflammatory response. In addition, a cGMP-dependent mechanism, is thought to be involved in the control of proinflammatory processes. Therefore, elevating intracellular levels of cGMP may be used as a means of treating and controlling inflammatory bowel diseases such as ulcerative colitis and Crohn's disease and other organ inflammation (e.g., associated with asthma, nephritis, hepatitis, pancreatitis, bronchitis, cystic fibrosis).

[0024] Without intending to be bound by any theory, it is envisioned that ion transport across the plasma membrane may prove to be an important regulator of the balance between cell proliferation and apoptosis that will be affected by compositions altering cGMP concentrations. Uroguanylin has been shown to stimulate K.sup.+ efflux, Ca.sup.++ influx and water transport in the gastrointestinal tract (3). Moreover, atrial natriuretic peptide (ANP), a peptide that also binds to a specific guanylate cyclase receptor, has also been shown to induce apoptosis in rat mesangial cells, and to induce apoptosis in cardiac myocytes by a cGMP mechanism (26-29). It is believed that binding of the present agonists to a guanylate cyclase receptor stimulates production of cGMP. This ligand-receptor interaction, via activation of a cascade of cGMP-dependent protein kinases and CFTR, is then expected to induce apoptosis in target cells. Therefore, administration of the novel peptides defined by SEQ ID NOs:2-21, as shown in Tables 2 and 3, or uroguanylin, or guanylin or E. coli ST peptide is expected to eliminate or, at least retard, the onset of inflammatory diseases of the GI tract and general organ inflammation (e.g., asthma, nephritis, hepatitis, pancreatitis, bronchitis, cystic fibrosis).

[0025] In another aspect, the invention is directed to a method for preventing, treating or retarding the onset of cancer, particularly cancer of epithelial cells, in a subject by administering a composition comprising an effective amount of a guanylate cyclase receptor agonist, preferably a synthetic a guanylate cyclase receptor agonist. The term effective amount refers to sufficient agonist to measurably increase intracellular levels of cGMP. The term synthetic refers to a peptide created to bind a guanylate cyclase receptor, but containing certain amino acid sequence substitutions not present in known endogenous guanylate cyclase agonists, such as uroguanylin. The agonist should be a peptide selected from those defined by SEQ ID NOs:2-21 and which are listed in Tables 2 and 3. Also included in the invention are methods of treating primary and metastatic cancers, other than primary colon cancer, by administering an effective dosage of a peptide selected from the group consisting of: uroguanylin; guanylin; and E. coli ST peptide. Any known form of uroguanylin or guanylin can be used for this purpose, although the human peptides are preferred.

[0026] The cGMP-dependent mechanism that regulates the balance between cellular proliferation and apoptosis in metastatic tumor cells may serve as a mechanism for targeting and treating metastatic tumors. The liver is the most common site of metastasis from a primary colorectal cancer. Toward later stages of disease, colorectal metastatic cells may also invade other parts of the body. It is important to note that metastatic cells originating from the primary site in the gastrointestinal tract typically continue to express guanylate cyclase receptors and therefore, these cells should be sensitive to apoptosis therapy mediated by intestinal guanylate cyclase receptors. Peptides having uroguanylin activity, when used either alone or in combination with specific inhibitors of cGMP-phosphodiesterase, also retard the onset of carcinogenesis in gut epithelium by restoring a healthy balance between cell proliferation and apoptosis via a cGMP-mediated mechanism.

[0027] As used herein, the term guanylate cyclase receptor refers to the class of guanylate cyclase receptors on any cell type to which the inventive agonist peptides or natural agonists described herein bind.

[0028] As used herein, the term guanylate cyclase receptor-agonist refers to peptides and/or other compounds that bind to a guanylate cyclase receptor and stimulate cGMP production. The term also includes all peptides that have amino acid sequences substantially equivalent to at least a portion of the binding domain comprising amino acid residues 3-15 of SEQ ID NO: 1. This term also covers fragments and pro-peptides that bind to guanylate cyclase receptor and stimulate cGMP production. The term substantially equivalent refers to a peptide that has an amino acid sequence equivalent to that of the binding domain where certain residues may be deleted or replaced with other amino acids without impairing the peptide's ability to bind to a guanylate cyclase receptor and stimulate cGMP production.

[0029] Strategy and Design of Novel Guanylate Cyclase Receptor Agonists

[0030] Uroguanylin is a peptide secreted by the goblet and other epithelial cells lining the gastrointestinal mucosa as pro-uroguanylin, a functionally inactive form. The human pro-peptide is subsequently converted to the functionally active 16 amino acid peptide set forth in SEQ ID NO:1 (human uroguanylin sequence, see Table 2) in the lumen of the intestine by endogenous proteases. Since uroguanylin is a heat-resistant, acid-resistant, and proteolysis-resistant peptide, oral or systemic administration of this peptide and/or other peptides similar to the functionally active 16 amino acid peptide sequence of SEQ ID NO:1 may be effectively employed in treatment methods.

[0031] Peptides similar to, but distinct from, uroguanylin are described below, including some which produce superior cGMP enhancing properties and/or other beneficial characteristics (e.g., improved temperature stability, enhanced protease stability, or superior activity at preferred pH's) compared to previously known uroguanylin peptides. The peptides may be used to inhibit GI inflammation and for treating or preventing the onset of polyp formation associated with gut inflammation. Epithelial tissues susceptible to cancer cell formation may also be treated. The guanylate cyclase receptor agonists described have the amino acid sequences shown in Tables 2 and 3. The binding domain for agonist-receptor interaction includes the amino acid residues from 3-15 of SEQ ID NO:1.

[0032] Molecular modeling was applied to the design of novel guanylate cyclase receptor agonists using methods detailed in (30). It consisted of energy calculations for three compounds known to interact with guanylate cyclase receptors, namely for

TABLE-US-00002 humanuroguanylin,bicyclo[4,12;7,15] (UG,SEQIDNO:1) Asn.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val.sup.8-Asn.sup.9-Val.sup.10- Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cys.sup.15-Leu.sup.16; humanguanylin,bicyclo[4,12;7,15] (GU,SEQIDNO:22) Pro.sup.1-Gly.sup.2-Thr.sup.3-Cys.sup.4-Glu.sup.5-Ile.sup.6-Cys.sup.7-Ala.sup.8-Tyr.sup.9-Ala.sup.10- Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-.sup.15Cys; and E.colismallheat-stableenterotoxin,tricyclo [6,10;7,15;11-18] (ST,SEQIDNO:23) Asn.sup.1-Ser.sup.2-Ser.sup.3-Asn.sup.4-Tyr.sup.5-Cys.sup.6-Cys.sup.7-Glu.sup.8-Leu.sup.9-Cys.sup.10- Cys.sup.11-Asn.sup.12-Pro.sup.13-Ala.sup.14-Cys.sup.15-Thr.sup.16-Gly.sup.17-Cys.sup.18-Tyr.sup.19.
Geometrical comparisons of all possible low-energy conformations for these three compounds were used to reveal the common 3D structures that served as the templates for the bioactive conformation, i.e., for the conformation presumably adopted by GU, UG and ST during interaction with receptor. It allowed designing novel analogs with significantly increased conformational population of the bioactive conformation at the expense of other low-energy conformations by selecting individual substitutions for various amino acid residues.

[0033] Energy calculations were performed by use of build-up procedures (30). The ECEPP/2 potential field (31,32) was used assuming rigid valence geometry with planar trans-peptide bonds, including that for Pro.sup.13 in ST. The angle in Pro.sup.13 was allowed to vary. Aliphatic and aromatic hydrogens were generally included in united atomic centers of CH.sub.n type; H.sup.-atoms and amide hydrogens were described explicitly.

[0034] The main calculation scheme involved several successive steps. First, the sequences of the two monocyclic model fragments (three fragments for ST), Ac-cyclo (Cys.sup.i- . . . -Cys.sup.i)-NMe, were considered, where all residues except Cys, Gly and Pro were replaced by alanines; the i and j values corresponded to the sequences of GU, UG and ST. At this step, all possible combinations of local minima for the peptide backbone for each amino acid residue were considered, i.e., the minima in the Ramachandran map of E, F, C, D, A and A* types (according to the notation in (33)) for the Ala residue; of E*, F*, C*, D*, A, E, F, C D and A* types for the Gly residue; and of F, C and A types for Pro. For each backbone conformation, one optimal possibility to close a cycle employing the parabolic potential functions, intrinsic to the ECEPP force field, was found by checking an energy profile of rotation around the dihedral angle 1 for the D-Cys residue.

[0035] Totally, as many as ca. 180,000 conformations for each of the cyclic moieties were considered. Then, the conformers satisfying the EE.sub.min<E=15 kcal/mol criterion and differing by more than 40 in at least one value of any backbone dihedral angle were selected (from ca. 3,000 to 8,000 conformations for different model fragments). At the next step, the selected conformations of the matching monocyclic fragments were overlapped to create possible conformations of the bicyclic model fragments (the tricyclic fragments in the case of ST). Typically, this procedure yielded ca. 20,000-30,000 conformations. All these conformations were submitted for a new cycle of energy calculations, which resulted in 191 conformations satisfying the EE.sub.min<E=20 kcal/mol criterion for the ST model fragment and in 6,965 conformations satisfying the same criterion for the GU/UG model fragment. After that, the missing side chains in the model fragments were restored, and energy calculations were performed again, the dihedral angle values of side chain groups (except the 1 angle for the Cys residues) and of the terminal groups of the backbone being optimized before energy minimization to achieve their most favorable spatial arrangements, employing an algorithm previously described (34). For the UG 4-15 fragment, 632 conformations satisfied the criterion of E=20 kcal/mol; 164 of them satisfied the more stringent criterion of E=12 kcal/mol, which corresponds to the accepted criterion of 1 kcal/mol/residue (30). Subsequent elongation of the UG 4-15 fragment to 3-16, and then to the entire UG molecule was performed by the same build-up procedure. Finally, 31 backbone conformations of UG were found as satisfying the criterion of E=16 kcal/mol.

[0036] Geometrical comparison of conformers was performed in the following manner. The best fit in the superposition for the atomic centers in a pair of conformers was assessed to check the level of geometrical similarity between the two conformers, according to (35). The criterion for geometrical similarity was the rms value, which was calculated for a pair of conformations A and

[0037] B as follows:

[00001]$\mathrm{rms}.Math.=\left(1/N\right).Math.{.Math.}^N.Math.i={1\left[{\left(x^A.Math.i-x^B.Math.i\right)}^2+{\left(y^A.Math.i-y^B.Math.i\right)}^2+{\left(z^A.Math.i-z^B.Math.i\right)}^2\right]}^{1/2},$

where N is the number of the C.sup.-atom pairs chosen for superposition, and x, y and z are the Cartesian coordinates. By the criterion of geometrical similarity of rms <2.0 , low-energy conformations of the rigid conformational fragment UG 4-15 fell into seven conformational families. One of them consists of the same six conformers that are similar both to 1UYA and 1ETN; this family contains also the lowest-energy conformer of UG. (1UYA and 1ETN are the experimentally defined 3D structures of UG and ST, respectively, which are known to possess high biological activity (36,37); the 3D structures were available in the Protein Data Bank.)

TABLE-US-00003 TABLE 1 The values of dihedral angles (in degrees) for peptide backbone in the template conformation of UG Conformer's # Residue Angle 1 3 9 22 25 27 Cys.sup.4 37 41 40 55 38 54 Glu.sup.5 71 67 72 69 68 70 50 47 48 33 43 22 Leu.sup.6 86 86 85 81 88 91 163 165 160 153 160 156 Cys.sup.7 79 82 79 83 79 81 74 68 78 67 75 72 Val.sup.8 120 114 126 124 125 128 65 57 62 55 60 64 Asn.sup.9 83 95 82 88 89 82 119 113 134 118 111 116 Val.sup.10 84 82 97 90 82 82 21 13 16 4 15 16 Ala.sup.11 79 86 87 89 85 80 32 21 35 35 18 27 Cys.sup.12 86 92 78 79 95 90 52 53 55 57 53 54 Thr.sup.13 129 121 127 119 118 130 111 153 141 155 141 119 Gly.sup.14 64 78 78 80 78 68 83 64 68 62 67 78 Cys.sup.15 139 160 150 156 78 131

[0038] The dihedral angles and , values that determine the overall 3D shape of this UG fragment, are similar (Table 1). It allowed performing preliminary design of new analogs aimed at stabilizing this particular family of conformations employing the known local conformational limitations imposed by various types of amino acids.

[0039] For instance, it is known that Gly is more conformationally flexible compared to any other L-amino acid residue, since Gly may adopt conformations with any of the four combinations of signs for and , i.e., , +; , ; +, +; and +, . The last combination is sterically forbidden for the L-amino acids, as Ala. Therefore, substitution of Gly.sup.14 for Ala.sup.14 should limit conformational flexibility in position 14 preserving the conformations described in Table 1. Also, substitution for Aib (-Me-Ala, di--methyl-alanine) should limit the local conformational flexibility by two regions only, namely for , and +, +, the first one being compatible with conformers of Ala.sup.11 in Table 1. Therefore, one more desirable substitution is Aib.sup.11. In Pro, the 1 value is fixed at 75; this residue is also similar to valine by its hydrophobic properties. Therefore, Val.sup.10 may be replaced by Pro.sup.m, which adds more local conformational constraints to the UG conformers in Table 1. Replacement by Pro also requires that the preceding residue possesses only positive values; Asn.sup.9 in Table 1 fulfills this requirement. The Pro residue already exists in the corresponding position of ST. All suggested substitutions within SEQ ID NO:1 shown below (e.g., Pro.sup.10, Aib.sup.11 or Ala.sup.14) do not change the chemical nature of the non-aliphatic amino acids (such as Asn, Asp or Thr), which may be important for the actual interaction with receptor. The former substitutions should lead only to conformational limitations shifting conformational equilibrium in UG towards the suggested template 3-D shape.

[0040] Based on the 3D structures defined in Table 1, a three-dimensional pharmacophore for uroguanylin was defined, enabling the determination of distances between functional groups of uroguanylin thought to directly interact with the receptor. Those groups thought to directly interact with the receptor are side groups of residues in positions 3, 5, 9 and 13 of the backbone sequence. Preferably, the residues are Glu3, Glu5, Asn9, and Thr13, as shown in SEQ ID NO:2 and SEQ ID NO:20. Thus, a three dimensional pharmacophore of uroguanylin is described in which the spatial arrangement of the four side chains of the residues at positions 3, 5, 9 and 13 may be created such that the distances between these side chains enable optional biological activity. Those distances (measured as distances between C.beta. atoms of corresponding residues) are as follows: from 5.7 to 7.6 for the 3-5 distance, from 4.0 to 6.0 for 3-9; from 7.7 to 8.3 for 3-13, from 9.4 to 9.5 for 5-9, from 9.4 to 9.5 for 5-13, and from 5.8 to 6.3 for 9-13.

[0041] The distances above depend only on conformations of the peptide backbone. In some cases, however, conformations of side chains themselves are also important. For instance, calculations showed that there is no conformational difference between the backbones of UG (SP301), [Glu.sup.2]-UG (SP303), [Glu.sup.3]-UG (SP304) and [Glu.sup.2, Glu.sup.3]-UG (SP302) in terms of their low-energy conformations. However, there is a distinct difference in the spatial positions of the -carboxyls of Asp and -carboxyls of Glu in position 3. Namely, -carboxyls of the Glu residues in position 3 are clearly stretched outwards of the bulk of the molecules farther than the corresponding -carboxyls of the Asp residues. The above observation strongly suggests that the negatively charged carboxyl group of the side chain in position 3 specifically interacts with a positively charged binding site on the receptor; therefore, analogs containing Glu.sup.3 instead of Asp.sup.3 should be more active. At the same time, to ensure efficiency of this particular interaction, an entire system of the long-range electrostatic interactions between ligand and receptor should be well balanced. Since the Glu.sup.2 side chain presents more conformational possibilities compared to the Asp.sup.2 side chain, this balance may be slightly changed in SP302 (double substitution of Asp's for Glu's) compared to SP304 (single substitution of Asp.sup.3 for Glu.sup.3).

[0042] Compounds capable of adopting low-energy conformations described in Table 1 are listed in Table 2. All compounds are [4,12; 7,15] bicycles.

TABLE-US-00004 TABLE2 1. Parentcompound,uroguanylin SEQIDNO:1 Asn.sup.1-Asp.sup.2-Asp.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val.sup.8-Asn.sup.9-Val.sup.10-Ala.sup.11-Cys.sup.12-Thr.sup.13-Gly.sup.14-Cys.sup.15- Leu.sup.16 2. Compoundwithoutmodificationsofcysteines: Commonsequence(SEQIDNO:2): Asn.sup.1-Xaa.sup.2-Xaa.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Cys.sup.7-Val.sup.8-Asn.sup.9-Xaa.sup.10-Xaa.sup.11-Cys.sup.12-Thr.sup.13-Xaa.sup.14-Cys.sup.15- Leu.sup.16 whereXaa.sup.2=Asp,Glu;Xaa.sup.3=Asp,Glu withtheexceptionthatXaa.sup.2andXaa.sup.3arenotbothAspinsamemolecule AndwhereXaa.sup.10=Val,Pro;Xaa.sup.11=Ala,Aib;Xaa.sup.14=Gly,Ala 3. Compoundswithmercaptoproline(Mpt)substitutedforcysteineinposition7: Commonsequence(SEQIDNO:3): Asn.sup.1-Xaa.sup.2-Xaa.sup.3-Cys.sup.4-Glu.sup.5-Leu.sup.6-Xaa.sup.7-Val.sup.8-Asn.sup.9-Xaa.sup.10-Xaa.sup.11-Cys.sup.12-Thr.sup.13-Xaa.sup.14-Cys.sup.15- Leu.sup.16 whereXaa.sup.2=Asp,Glu;Xaa.sup.3=Asp,Glu whereXaa.sup.10=Val,Pro;Xaa.sup.11=Ala,Aib;Xaa.sup.l4=Gly,Ala 4. Compoundswithpenicillamines(,-dimethylcysteines,Pen)substitutedfor cysteines: Commonsequence(SEQIDNO:4): Asn.sup.1-Xaa.sup.2-Xaa.sup.3-Xaa.sup.4-Glu.sup.5-Leu.sup.6-Xaa.sup.7-Val.sup.8-Asn.sup.9-Xaa.sup.10-Xaa.sup.11-Xaa.sup.12-Thr.sup.13-Xaa.sup.14-Xaa.sup.15- Leu.sup.16 whereXaa.sup.2=Asp,Glu;Xaa.sup.3=Asp,Glu whereXaa.sup.10=Val,Pro;Xaa.sup.11=Ala,Aib;Xaa.sup.l4=Gly,Ala andXaa.sup.4,Xaa.sup.7,Xaa.sup.12,Xaa.sup.15areeitherCysorPen(exceptnotallareCysinthesame conformer) 5. Compoundswithlactambridgessubstitutedfordisulfidebridges: Commonsequence(SEQIDNO:5): Asn.sup.1-Xaa.sup.2-Xaa.sup.3-Xaa.sup.4-Glu.sup.5-Leu.sup.6-Xaa.sup.7-Val.sup.8-Asn.sup.9-Xaa.sup.10-Xaa.sup.11-Xaa.sup.12-Thr.sup.13-Xaa.sup.14-Xaa.sup.15- Leu.sup.16 whereXaa.sup.2=Asp,Glu;Xaa.sup.3=Asp,Glu whereXaa.sup.10=Val,Pro;Xaa.sup.11=Ala,Aib;Xaa.sup.14=Gly,Ala andallcombinationsofthefollowing(Dprisdiaminopropionicacid): Xaa.sup.4iseitherAsporGlu,andXaa.sup.12isDpr; Xaa.sup.7iseitherCysorPen; Xaa.sup.15iseitherCysorPen; or: Xaa.sup.7isDprandXaa.sup.15iseitherAsporGlu; Xaa.sup.7iseitherAsporGlu,andXaa.sup.15isDpr; Xaa.sup.4iseitherCysorPen; Xaa.sup.12iseitherCysorPen;

[0043] Some of the peptides shown in Table 2 contain 16 amino acid residues in which cysteine residues form disulfide bridges between Cys.sup.4 and Cys.sup.12, and Cys' and Cys.sup.15, respectively. These peptides differ from the peptide sequences described in WO 01/25266, and are designed on the basis of peptide conformation and energy calculations.

[0044] In addition, peptides, varying in length from 13 to 16 amino acids, shown in Table 3, are designed, based on energy calculations and three-dimensional structures, to promote stabilization of the biologically active conformer and minimize or eliminate interconversion to biologically inactive conformers. These peptides are also designed to promote stability against proteolysis and higher temperatures. The design of these peptides involves modifications of amino acid residues that contain ionic charges at lower pH values, such as glutamic and aspartic acids.

TABLE-US-00005 TABLE3 SEQIDNO:6 X1 GluGluCysX2X3CysX4AsnX5X6CysX7X8CysX9 SEQIDNO:7 X1 GluAspCysX2X3CysX4AsnX5X6CysX7X8CysX9 SEQIDNO:8 X1 AspGluCysX2X3CysX4AsnX5X6CysX7X8CysX9 SEQIDNO:9 X1 AspAspCysX2X3CysX4TyrX5X6CysX7X8CysX9 SEQIDNO:10 X1 GluGluCysX2X3CysX4TyrX5X6CysX7X8CysX9 SEQIDNO:11 X1 AspGluCysX2X3CysX4TyrX5X6CysX7X8CysX9 SEQIDNO:12 Xl GluAspCysX2X3CysX4TyrX5X6CysX7X8CysX9 SEQIDNO:13 Xl AspAspCysX2X3CysX4GlnX5X6CysX7X8CysX9 SEQIDNO:14 X1 GluGluCysX2X3CysX4GlnX5X6CysX7X8CysX9 SEQIDNO:15 X1 AspGluCysX2X3CysX4GlnX5X6CysX7X8CysX9 SEQIDNO:16 X1 GluAspCysX2X3CysX4GlnX5X6CysX7X8CysX9 SEQIDNO:17 GluCysX2X3CysX4AsnX5X6CysX7X8CysX9 SEQIDNO:18 GluCysX2X3CysX4AsnX5X6CysX7X8Cys SEQIDNO:19 X1GluCysX2X3CysX4AsnX5X6CysX7X8CysX9 1 2345678910111213141516 SEQIDNO:20 Asn AspGluCysGluLeuCysValAsnValAlaCysThrGlyCysLeu SEQIDNO:21 GluCysGluLeuCysValAsnValAlaCysThrGlyCysLeu
X1 to X9 can be any amino acid. The disulfide bridges are formed between Cys residues at 4 and 12 and between 7 and 15, respectively. SEQ ID NO:18 represents the minimum length requirement for these peptides to bind a guanylate cyclase receptor.

[0045] Pharmaceutical Compositions and Formulations

[0046] The guanylate cyclase receptor agonists of the present invention (Table 2; SEQ ID NOs:2-5 and Table 3; SEQ ID NOs:6-21), as well as uroguanylin, guanylin and/or bacterial enterotoxin ST, may be combined or formulated with various excipients, vehicles or adjuvants for oral, local or systemic administration. Peptide compositions may be administered in solutions, powders, suspensions, emulsions, tablets, capsules, transdermal patches, ointments, or other formulations. Formulations and dosage forms may be made using methods well known in the art (see, e.g., Remington's Pharmaceutical Sciences, 16.sup.th ed., A. Oslo ed., Easton, Pa. (1980)).

[0047] Inhibitors of cGMP-dependent phosphodiesterase may be small molecules, peptides, proteins or other compounds that specifically prevent the degradation of cGMP. Inhibitory compounds include suldinac sulfone, zaprinast, motapizone and other compounds that block the enzymatic activity of cGMP-specific phosphodiesterases. One or more of these compounds may be combined with a guanylate cyclase receptor agonist exemplified in SEQ ID NOs:2-21, uroguanylin, guanylin and E. Coli ST peptide.

[0048] The selection of carriers (e.g., phosphate-buffered saline or PBS) and other components suitable for use in compositions is well within the level of skill in this art. In addition to containing one or more guanylate cyclase receptor agonists, such compositions may incorporate pharmaceutically acceptable carriers and other ingredients known to facilitate administration and/or enhance uptake. Other formulations, such as microspheres, nanoparticles, liposomes, pegylated protein or peptide, and immunologically-based systems may also be used. Examples include formulations employing polymers (e.g., 20% w/v polyethylene glycol) or cellulose, or enteric formulations and pegylated peptide analogs for increasing systemic half-life and stability.

[0049] Treatment Methods

[0050] The term treatment refers to reducing or alleviating symptoms in a subject, preventing symptoms from worsening or progressing, or preventing disease development. For a given subject, improvement in a symptom, its worsening, regression, or progression may be determined by any objective or subjective measure typically employed by one of skill in the art. Efficacy of the treatment in the case of cancer may be measured as an improvement in morbidity or mortality (e.g., lengthening of the survival curve for a selected population). Thus, effective treatment would include therapy of existing disease, control of disease by slowing or stopping its progression, prevention of disease occurrence, reduction in the number or severity of symptoms, or a combination thereof. The effect may be shown in a controlled study using one or more statistically significant criteria.

[0051] Combination therapy with one or more medical/surgical procedures and/or at least one other chemotherapeutic agent may be practiced with the invention. Other suitable agents useful in combination therapy include anti-inflammatory drugs such as, for example, steroids or non-steroidal anti-inflammatory drugs (NSAIDS), such as aspirin and the like. Prophylactic methods for preventing or reducing the incidence of relapse are also considered treatment.

[0052] Cancers expected to be responsive to compositions include breast, colorectal, lung, ovarian, pancreatic, prostatic, renal, stomach, bladder, liver, esophageal and testicular carcinoma. Further examples of diseases involving cancerous or precancerous tissues that should be responsive to a therapeutic comprising at least one guanylate cyclase receptor agonist include: carcinoma (e.g., basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs, Merkel cell, small or non-small cell lung, oat cell, papillary, bronchiolar, squamous cell, transitional cell, Walker), leukemia (e.g., B-cell, T-cell, HTLV, acute or chronic lymphocytic, mast cell, myeloid), histiocytoma, histiocytosis, Hodgkin disease, non-Hodgkin lymphoma, plasmacytoma, reticuloendotheliosis, adenoma, adeno-carcinoma, adenofibroma, adenolymphoma, ameloblastoma, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, sclerosing angioma, angiomatosis, apudoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinosarcoma, cementoma, cholangioma, cholesteatoma, chondrosarcoma, chondroblastoma, chondrosarcoma, chordoma, choristoma, craniopharyngioma, chrondroma, cylindroma, cystadenocarcinoma, cystadenoma, cystosarcoma phyllodes, dysgerminoma, ependymoma, Ewing sarcoma, fibroma, fibro-sarcoma, giant cell tumor, ganglioneuroma, glioblastoma, glomangioma, granulosa cell tumor, gynandroblastoma, hamartoma, hemangioendothelioma, hemangioma, hemangio-pericytoma, hemangiosarcoma, hepatoma, islet cell tumor, Kaposi sarcoma, leiomyoma, leiomyosarcoma, leukosarcoma, Leydig cell tumor, lipoma, liposarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, medulloblastoma, meningioma, mesenchymoma, mesonephroma, mesothelioma, myoblastoma, myoma, myosarcoma, myxoma, myxosarcoma, neurilemmoma, neuroma, neuroblastoma, neuroepithelioma, neurofibroma, neurofibromatosis, odontoma, osteoma, osteosarcoma, papilloma, paraganglioma, paraganglioma nonchromaffin, pinealoma, rhabdomyoma, rhabdomyosarcoma, Sertoli cell tumor, teratoma, theca cell tumor, and other diseases in which cells have become dysplastic, immortalized, or transformed.

[0053] A bolus of the inventive composition may be administered over a short time. Once a day is a convenient dosing schedule to treat, inter alia, one of the above-mentioned disease states. Alternatively, the effective daily dose may be divided into multiple doses for purposes of administration, for example, two to twelve doses per day. The dose level selected for use will depend on the bioavailability, activity, and stability of the compound, the route of administration, the severity of the disease being treated, and the condition of the subject in need of treatment. It is contemplated that a daily dosage will typically be between about 10 g and about 2 mg (e.g., about 100 g to 1 mg) of the compound per kilogram body weight. The amount of compound administered is dependent upon factors known to a person skilled in this art such as, for example, chemical properties of the compound, route of administration, location and type of cancer, and the like.

[0054] The subject mammal may be any animal or human patient. Thus, both veterinary and medical treatments are envisioned according to the invention.

[0055] The invention will be further described by the following non-limiting example.

Examples

Materials and Methods

[0056] Cell Culture: Human T84 colon carcinoma cells were obtained from the American Type Culture Collection at passage 52. Cells were grown in a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U penicillin/ml, and 100 g/ml streptomycin. The cells were fed fresh medium every third day and split at a confluence of approximately 80%.

[0057] T84 cell-based assay for determining the intracellular levels of cGMP: Peptide analogs were custom synthesized by Multiple Peptide Systems, San Diego, Calif., and by Princeton Biomolecules, Langhorne, Pa. Biological activity of the synthetic peptides was assayed as previously reported (15). Briefly, the confluent monolayers of T-84 cells in 24-well plates were washed twice with 250 l of DMEM containing 50 mM HEPES (pH 7.4), pre-incubated at 37 C. for 10 min with 250 l of DMEM containing 50 mM HEPES (pH 7.4) and 1 mM isobutylmethylxanthine (IBMX), followed by incubation with peptide analogs (0.1 nM to 10 M) for 30 min. The medium was aspirated, and the reaction was terminated by the addition of 3% perchloric acid. Following centrifugation, and neutralization with 0.1 N NaOH, the supernatant was used directly for measurements of cGMP using an ELISA kit (Caymen Chemical, Ann Arbor, Mich.).

[0058] Results

[0059] Peptides shown in Table 4 were custom synthesized and purified (>95% purity) using a published procedure (38). Peptide analogs were evaluated in the T84 cell-based assay for their ability to enhance intracellular levels of cGMP. As shown in Table 4, SP304 (SEQ ID NO:20) gave the greatest enhancement of intracellular cGMP of all the analogs tested. SP316 (SEQ ID NO:21) was second in effectiveness, whereas the biological activities of SP301, SP302 and SP303 were all somewhat weaker. The peptide analogs SP306 and SP310 were not active in this assay. These results indicate that SP304 is the most potent peptide for enhancing cGMP. These results also suggest that the cysteine residue at position 7 cannot be substituted with penicillamine as a component of the [7,15] disulfide linkage, and that the Asn residue at position 9 cannot be changed to a Gln.

TABLE-US-00006 TABLE 4 Peptide agonists evaluated for biological activity in the T84 cell bioassay. Compound cGMP Level** SEQ ID NO.* Code (pmol/well) 1 SP301 205 6 SP302 225 7 SP303 195 20 SP304 315 14 SP306 0 4 SP310 0 21 SP316 275 *SEQ ID's for SP301, SP304 and SP316 are the precise amino acid sequences for these analogs as given in the text. **Intracellular cGMP level observed in T84 cells following treatment with 1 micromolar solution of the respective peptide agonist for 30 minutes. The value observed for SP304 was statistically significant with a p > 0.5.

[0060] To examine heat stability, 10 micromolar solutions of peptide analogs were heated at 95 C. for up to 90 minutes. At specific times during the treatment, samples were tested for their biological activity in the T84 cell-based assay. Biological activity of SP301, SP302, SP303 and SP304 did not change significantly after 60 minutes of heating. After 90 minutes, the activities of SP301, SP302 and SP303 were reduced to about 80% of their original values, whereas the biological activity of SP304 remained unaltered. This indicates that SP304 is more stable to heat denaturation compared to the other peptides tested. Based on energy calculations and 3D structure, we expected that the negatively charged carboxyl group of the side chain in position 3 of SEQ ID NO:1 specifically interacts with a positively charged binding site on the receptor. In the case where this interaction can be enhanced, analogs containing Glu.sup.3 instead of Asp.sup.3 should be more active, as was found to be the case with SP304. At the same time, to ensure efficiency of this particular interaction, an entire system of the long-range electrostatic interactions between ligand and receptor should be well balanced. Since the Glu.sup.2 side chain presents more conformational possibilities compared to the Asp.sup.2 side chain, this balance may be slightly changed in SP302 (double substitution of Asp's for Glu's) compared to SP304 (single substitution of Asp.sup.3 for Glu.sup.3). Indeed, biological activity of SP 304 is the best amongst the analogs evaluated.

[0061] Synthetic peptides SP301, SP302, SP303 and SP304 were also tested for their activities at different pH values of the T84 cell-based assay. Whereas all of these peptides showed enhanced intracellular production of cGMP at pH's ranging from 5 to 7, SP304 showed the greatest enhancement in the range between 6.5 and 7. It is important to note that the physiological pH of the large intestine is in a similar range, and, therefore, SP304 would be expected to be especially efficacious for colon cancer treatment.

[0062] We also evaluated peptides used either alone or in combination with inhibitors of cGMP dependent phosphodiesterase (e.g., zaprinast or sulindac sulfone) in T84 cell-based assays for enhancement of intracellular levels of cGMP. Combinations of an inhibitor of cGMP dependent phosphodiesterase with SP304 displayed a dramatic effect in enhancing cGMP levels in these experiments. Synthetic peptide SP304 substantially increased the cGMP level over the level reached in the presence of either zaprinast or sulindac sulfone alone. Treatment of wells with SP304 in combination with either Zaprinast or sulindac sulfone resulted in synergistic increases in intracellular cGMP levels. These increases were statistically significant, with p values of <0.5. These data indicate that treatments combining a peptide agonist of a guanylate cyclase receptor with one or more inhibitors of cGMP dependent phosphodiesterase result in a greater than additive increase in cGMP concentrations.

[0063] While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to those of ordinary skill in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

REFERENCES

[0064] 1. Currie, et al., Proc. Nat'l Acad. Sci. USA 89:947-951 (1992). [0065] 2. Hamra, et al., Proc. Nat'l Acad. Sci. USA 90:10464-10468 (1993). [0066] 3. Forte, L., Reg. Pep. 81:25-39 (1999). [0067] 4. Schulz, et al., Cell 63:941-948 (1990). [0068] 5. Guba, et al., Gastroenterology 111:1558-1568 (1996). [0069] 6. Joo, et al., Am. J. Physiol. 274:G633-G644 (1998). [0070] 7. Evan, et al., Nature (London) 411:342-348 (2001). 8. Eastwood, G., J Clin. Gastroenterol. 14:S29-33 (1992). [0071] 9. Lipkin, M. Arch. Fr. Mal. Appl Dig. 61:691-693 (1972). [0072] 10. Wong, et al., Gut 50:212-217 (2002). [0073] 11. Potten, et al., Stem Cells 15:82-93. [0074] 12. Basoglu, et al., in: Proceedings of the Second FEPS Congress, June 29-Jul. 4, 1999, Prague, Czech Republic., http://f2.cuni.cz/physiolres/feps/basoglu.htm. [0075] 13. Sindic, et al., J. Biol. Chem. Mar. 11, 2002, manuscript M110627200 (in press). [0076] 14. Zhang, et al., Science 276:1268-1272 (1997). [0077] 15. Shailubhai, et al., Cancer Res. 60:5151-5157 (2000). [0078] 16. Shailubhai, et al., In: Proceedings of the 1999 AACR.NCI.EORTC International Conference. November 1999, Abstract #0734. [0079] 17. Cohen, et al., Lab. Invest. 78:101-108 (1998). [0080] 18. Sciaky, et al., Genomics 26:427-429 (1995). [0081] 19. Whitaker, et al., Genomics 45:348-354 (1997). [0082] 20. Leister, et al., Cancer Res. 50:7232-7235 (1990). [0083] 21. Cheng, et al., Cell 63:827-834 (1990). [0084] 22. Welsh, et al., Cell 73:1251-1254 (1993). [0085] 23. Weber, et al., Am. J. Physiol. Lung Cell Mol. Physiol. 281(1):L71-78 (2001). [0086] 24. Venkatakrishnan, et al., Am. J. Respir. Cell Mol. Biol. 23(3):396-403 (2000). [0087] 25. Hudson, et al., Free Radic. Biol. Med. 30:1440-1461 (2001). [0088] 26. Bhakdi, et al., Infect. Immun. 57:3512-3519 (1989). [0089] 27. Hughes, et al, J. Biol. Chem. 272:30567-30576 (1997). [0090] 28. Cermak, et al., Pflugers Arch. 43:571-577 (1996). [0091] 29. Wu, et al., J. Biol. Chem. 272:14860-14866 (1997). [0092] 30. Nikiforovich, G., Int. J. Pept. Prot. Res. 44:513-531 (1994). [0093] 31. Dunfield, et al., J. Phys. Chem. 82:2609-2616 (1978). [0094] 32. Nemethy, et al., J. Phys. Chem. 87:1883-1887 (1983). [0095] 33. Zimmerman, et al., Biopolymers 16:811-843 (1977). [0096] 34. Nikiforovich, et al., Biopolymers 31:941-955 (1991). [0097] 35. Nyburg, S., Acta Crystallographica B30 (part 1):251-253 (1974). [0098] 36. Chino, et al., FEBS Letters 421:27-31 (1998). [0099] 37. Schulz, et al., J. Peptide Res. 52:518-525 (1998). [0100] 38. Klodt, et al., J. Peptide Res. 50:222-230 (1997). [0101] 39. Shailubhai, I., Curr. Opin. Drug Discov. Devel. 5:261-268 (2002)